Small molecule FGF receptor inhibitors block FGFR-dependent urothelial carcinoma growth in vitro and in vivo.

BACKGROUND: Activating mutations of FGFR3 are frequently identified in superficial urothelial carcinoma (UC) and increased expression of FGFR1 and FGFR3 are common in both superficial and invasive UC. METHODS: The effects of inhibition of receptor activity by three small molecule inhibitors (PD173074, TKI-258 and SU5402) were investigated in a panel of bladder tumour cell lines with known FGFR expression levels and FGFR3 mutation status. RESULTS: All inhibitors prevented activation of FGFR3, and inhibited downstream MAPK pathway signalling. Response was related to FGFR3 and/or FGFR1 expression levels. Cell lines with the highest levels of FGFR expression showed the greatest response and little or no effect was measured in normal human urothelial cells or in UC cell lines with activating RAS gene mutations. In sensitive cell lines, the drugs induced cell cycle arrest and/or apoptosis. IC(50) values for PD173074 and TKI-258 were in the nanomolar concentration range compared with micromolar concentrations for SU5402. PD173074 showed the greatest effects in vitro and in vivo significantly delayed the growth of subcutaneous bladder tumour xenografts. CONCLUSION: These results indicate that inhibition of FGFR1 and wild-type or mutant FGFR3 may represent a useful therapeutic approach in patients with both non-muscle invasive and muscle invasive UC.

Retargeted adenoviral cancer gene therapy for tumour cells overexpressing epidermal growth factor receptor or urokinase-type plasminogen activator receptor.

We have assessed the ability of bispecific fusion proteins to improve adenovirus-mediated transfer of therapeutic and marker transgenes. We constructed an expression vector that can be easily modified to synthesize a variety of fusion proteins for retargeting adenoviral gene therapy vectors to cell surface markers, which are differentially expressed between normal and cancer cells. Adenoviral transduction can be improved in a number of tumour cell lines which overexpress EGFR (epidermal growth factor receptor) or uPAR (urokinase-type plasminogen activator receptor), but which have only low levels of endogenous hCAR (human coxsackie B and adenovirus receptor) expression. Up to 40-fold improvement in beta-galactosidase transgene expression was seen using an EGFR retargeting protein, and up to 16-fold using a second fusion protein targeting uPAR. In vitro, our uPAR retargeting fusion protein improved the sensitivity to adenoviral herpes simplex virus thymidine kinase/ganciclovir by an order of magnitude, whereas in vivo, our EGFR retargeting protein is able to significantly delay tumour growth in rodent animal models in a dose-dependent manner. The 'cassette' design of our fusion protein constructs offers a flexible method for the straightforward synthesis of multiple adenoviral retargeting proteins, directed against a variety of tumour-associated antigens, for use in clinical trials.

A prospective observational study of developmental outcomes in survivors of neonatal hypoxic ischaemic encephalopathy in South Africa.

BACKGROUND: Neonatal hypoxic ischaemic encephalopathy (NHIE) is an important cause of long-term handicap in survivors. There is limited information on the burden of handicap from NHIE in sub-Saharan Africa. OBJECTIVES: To determine the developmental outcomes in survivors of NHIE in South Africa (SA). METHODS: In this prospective observational study, the developmental outcomes in 84 infants who had survived hypoxic ischaemic encephalopathy (the NHIE group) were compared with those in 64 unaffected infants (the control group). The Bayley Scales of Infant Development version III were used for assessment of developmental outcomes. RESULTS: Significant differences were found between the developmental outcomes of the two groups, with a significantly lower composite language score and higher proportions with language, motor and cognitive developmental delays in the NHIE group than in the control group. Cerebral palsy (CP) was present in 13 of the infants with NHIE (15.5%) and none in the control group (p<0.001). CP was associated with developmental delay, and also with the severity of NHIE. Therapeutic hypothermia (TH) was administered in 58.3% of the study group, but although it was associated with lower rates of CP and developmental delay than in the group without TH, the only significant difference was for delay on the language subscale. CONCLUSIONS: Survivors of NHIE in SA are at risk of poor developmental outcomes.

Reducing the cost of screening novel agents using the hollow fibre assay.

Although the in vivo hollow fibre assay (HFA) as utilised by the National Cancer Institute is a highly effective screening tool, it has not been adopted en masse in the cancer pharmacology field. However, in laboratories which have adopted it, the effectiveness of HFA has also been confirmed. If immunocompetent mice could be used with the HFA, thereby reducing the cost of the assay, accessibility would increase and reductions in the cost of selecting appropriate agents for early clinical trials would result. It was demonstrated here that there was no difference in terms of cell growth and response to chemotherapy for cancer cells in hollow fibres in immunocompetent compared with immunodeficient mice. The HFA can thus be performed in these less expensive and more easily available mice with the implication of considerable savings to the preclinical cancer pharmacology community.

Effect of carbon dioxide injection on production of wood cement composites from waste medium density fiberboard (MDF).

The possibility of recycling waste medium density fiberboard (MDF) into wood-cement composites was evaluated. Both new fibers and recycled steam exploded MDF fibers had poor compatibility with cement if no treatment was applied, due to interference of the hydration process by the water soluble components of the fiber. However, this issue was resolved when a rapid hardening process with carbon dioxide injection was adopted. It appears that the rapid carbonation allowed the board to develop considerable strength before the adverse effects of the wood extractives could take effect. After 3-5 min of carbon dioxide injection, the composites reached 22-27% of total carbonation and developed 50-70% of their final (28-day) strength. Composites containing recycled MDF fibers had slightly lower splitting tensile strength and lower tensile toughness properties than those containing new fibers especially at a high fiber/cement ratio. Composites containing recycled MDF fibers also showed lower values of water absorption. Unlike composites cured conventionally, composites cured under CO(2) injection developed higher strength and toughness with increased fiber content. Incorporation of recycled MDF fibers into wood cement composites with CO(2) injection during the production stage presents a viable option for recycling of this difficult to manage waste material.

Angiogenesis in the hollow fiber tumor model influences drug delivery to tumor cells: implications for anticancer drug screening programs.

The National Cancer Institute uses the hollow fiber assay as part of its screening program for anticancer drug discovery. Angiogenesis to hollow fibers implanted s.c. has not been reported, thereby raising concerns about the efficiency of drug delivery and its subsequent effects on chemosensitivity. By extending postimplantation times beyond the 6-day period presently used, extensive vascular networks develop, resulting in both increased delivery and chemosensitivity to doxorubicin. This study suggests that present protocols used to evaluate compounds may produce false negative results, and additional studies to determine the predictive value of the assay are required.

Development of a modified hollow fibre assay for studying agents targeting the tumour neovasculature.

BACKGROUND: Previous studies have shown extensive vascularisation surrounding subcutaneously implanted fibres when the duration of the US National Cancer Institute (NCI) hollow fibre assay was prolonged. MATERIALS AND METHODS: The feasibility of adapting the NCI assay for evaluating agents targeting the tumour vasculature was investigated in vitro and in vivo. Finally, in the optimised assay, changes in neovasculature formation around the fibres following treatment with the anti-vascular agent paclitaxel were quantified by immunohistochemistry. RESULTS: Correlations between cell number seeded, time in culture and vascular endothelial growth factor (VEGF) secretion were seen. In vivo studies showed that transplanting single rather than 3 fibres at a site reduced inflammation, reducing the length of the fibre transplanted, as did without any significant loss in cell growth over 21 days. A statistically significant reduction in neovascularisation surrounding the fibres was seen accompanying paclitaxel treatment. CONCLUSION: Modifications made here to the NCI hollow fibre assay demonstrate its potential for analysing anti-tumour vasculature agents.

Evaluation of transgenic approaches for controlling tuber moth in potatoes.

A wide spectrum of strategies to genetically engineer potato plants resistant to potato tuber moth, Phthorimaea operculella (Zeller), have been investigated. The potato cv Iwa was transformed with a range of genes under the transcriptional control of the CaMV 35S promoter using Agrobacterium-mediated gene transfer. The transferred genes encode protease inhibitors (spleen inhibitor and alpha1-antitrypsin inhibitor), biotin-binding proteins (avidin and streptavidin) and Cry proteins (crylAc9, cry1Ba1, crylCa5 and cry9Aa2). Of these three transgenic approaches, cry genes have proved the most useful. In order to control the expression of the cry genes in foliage and not in the tubers a light-inducible Lhca3 promoter from potato was also used. The interaction of different cry genes was investigated using an experimental approach to simulate gene pyramiding in potato. Potato plants transgenic for both the crylAc9 and cryAa2 genes were developed and evaluated to help provide a more durable resistance to potato tuber moth.

The intrauterine environment is a strong determinant of glucose tolerance during the neonatal period, even in prematurity.

The aim of this study was to determine the contribution of birth weight and gestational age to glucose tolerance in premature neonates. The study group consisted of 100 premature and/or small-for-gestational age infants. Anthropometric measurements were performed both at birth and at the time of a standardized milk feed carried out at 19.6 +/- 12.1 d (range, 1-65 d) after birth. Fasting and postprandial glucose and insulin levels were measured. Birth weight, as a proxy mirror of the intrauterine environment, was found to influence the glucose concentration following a standardized milk feed (beta = -0.46; P = 0.01 for birth weight z-score with 60-min glucose level), whereas gestational age did not. Small-for-gestational age neonates had higher 60-min insulin levels than appropriate-for-gestational age neonates (115.4 +/- 9.5 vs. 68.4 +/- 14.2; P < 0.05) despite similar glucose levels. Neonates born of mothers who were on antihypertensive treatment were smaller and had a higher insulin secretory response than neonates from normotensive mothers. Postnatal growth velocity (kilograms per day) correlated with birth weight (beta = -0.65; P < 0.0001) and insulin resistance (beta = -0.31; P = 0.0004), independently of each other. This study shows that glucose tolerance of the neonate is determined by weight attained at birth irrespective of gestational age and that maternal blood pressure may influence insulin sensitivity of the newborn. Furthermore, catch-up growth in neonates is determined by birth weight and insulin sensitivity.

Chemokines induce the cellular migration of MCF-7 human breast carcinoma cells: subpopulations of tumour cells display positive and negative chemotaxis and differential in vivo growth potentials.

We previously reported that chemotactic cytokines (chemokines) induce the directional migration of cells derived from the breast carcinoma cell line MCF-7 in vitro, however it was apparent that only a small percentage of cells displayed the ability to migrate upon stimulation. In the present study three sub-lines derived from the parental MCF-7 cell line were selected for their ability to migrate in response to MIP-1alpha, MIP-1beta or RANTES across Transwell filters of 8 microm pore size. The first round selection of migratory cells resulted in sub-populations which demonstrated an increased chemotactic response compared with parental cells. Cells migrating to MIP-1beta were subjected to four further rounds of positive or negative selection, resulting in two sub-lines, MCF-7L4 and MCF-7U4 which displayed an increased and decreased chemotactic response respectively to MIP-1alpha MIP-1beta and RANTES. No difference in chemokine receptor RNA message expression between these sub-lines and the parental MCF-7 line were detected, although increased levels of alpha3, alpha6 and alphav integrin sub-units were shown for MCF-7L4 (positively selected sub-line) compared with MCF-7U4 cells. Moreover, the in vivo growth of cells derived from the two MCF-7 sub-lines was inversely correlated with their chemotactic response. The results of this study depict further the inherent heterogeneity in cancer, suggesting that the chemotactic response may influence the migratory traits of sub-populations within the tumour and potentially contribute to their in vivo behavior, growth and survival.

Six-year follow-up of early physiotherapy intervention in very low birth weight infants.

Eighty very low birth weight infants assigned to neurologically normal or at-risk groups on the basis of a neurodevelopmental score were previously described. Infants were assigned to physiotherapy or control groups, and the effect of physiotherapy was assessed at 1 year. At-risk infants had a significantly lower developmental quotient than the normal group and no beneficial effect of physiotherapy was shown. Of the original 80 subjects, 49 were reassessed at a mean age of 74.7 months. As observed previously, physiotherapy until 1 year did not influence subsequent outcome in either normal or at-risk children. At-risk and normal children had similar mean developmental quotients at 6 years, but the locomotor score of at-risk children was significantly below that of normal children. Cerebral palsy occurred in 6 of 24 at-risk vs 0 of 25 normal subjects (P less than .01) and remedial therapy was recommended in 17 of 24 at-risk subjects vs 6 of 25 normal subjects (P less than .001). These results confirm that the neurodevelopmental score predicts a risk for either cerebral palsy or soft neurological problems, and early physiotherapy is of questionable benefit in preventing such problems.

Prospective study of fluconazole therapy in systemic neonatal fungal infection.

BACKGROUND: Standard neonatal systemic antifungal therapy with amphotericin B and flucytosine can be associated with toxicity, drug resistance and the need for prolonged venous access. There is consequently a need for alternative treatment options. OBJECTIVES: To assess the efficacy and safety of fluconazole in the treatment of systemic neonatal fungal infections. METHOD: Open, nonrandomized evaluation of fluconazole treatment in 20 consecutively enrolled neonates with systemic fungal infection. RESULTS: Clinical and microbiologic cure was achieved in 12 of 19 (63%) of infants treated. One additional infant received prior amphotericin B therapy and is included for assessment of side effects. One infant with Torulopsis glabrata infection failed treatment. Six infants died of Gram-negative bacterial infection and other intercurrent medical problems. CONCLUSION: Fluconazole appeared to be safe and effective for treatment of systemic candidal infection in the neonate although more data are required in very low birth weight infants.

Prophylactic miconazole oral gel for the prevention of neonatal fungal rectal colonization and systemic infection.

Prior rectal colonization with fungi may be an important risk factor for development of systemic fungal infection in the neonate. This placebo-controlled study evaluated the benefits of miconazole oral gel in the prevention of fungal rectal colonization and systemic infection in high risk neonates admitted to the Neonatal Intensive Care Unit. Repeated oral application of miconazole gel reduced the overall prevalence of postnatally acquired rectal colonization; a yeast was grown in 19.5% of the weekly rectal swabs in the miconazole-treated group compared with 36.2% in the control group (69 of 354 vs. 146 of 403, P < 0.0001). There was no reduction in the incidence of systemic fungal infection in the two groups although the overall incidence of the infection was low in both groups, at 2.0% vs. 2.6% (6 of 298 vs. 8 of 302, P not significant). No relationship was shown between prior rectal colonization and subsequent systemic fungal infections in either of the two groups. This study does not support the use of prophylactic miconazole oral gel for the prevention of neonatal systemic fungal infections.

Use of C-reactive protein to guide duration of empiric antibiotic therapy in suspected early neonatal sepsis.

BACKGROUND: Serial C-reactive protein (CRP) measurements have been shown to be useful for guiding duration of antibiotic therapy in neonates. This study sought to determine whether this is a safe and practical approach in a developing country. METHODS: The study was conducted at the Johannesburg Hospital between September 15, 1998, and January 15, 1999. Subjects included all neonates evaluated for suspected sepsis in the first 24 h of life who had negative initial and repeat CRP values (< or = 10 mg/l) [corrected]. Repeat CRP measurements were performed between 24 and 48 h after birth. Antibiotic therapy was stopped in these infants at 24 to 48 h, and they were observed until 72 h, when the final blood culture results were available. The number of positive blood cultures in this group was determined. RESULTS: The repeat CRP estimation correctly identified 99 of 100 infants in the study as not requiring further antibiotic therapy (negative predictive value, 99%; 95% confidence intervals, 95.6 to 99.97%). The 1 infant with a positive blood culture was premature with a gestational age of 31 weeks. Eight babies required repeat evaluation for suspected sepsis, 4 presented on Day 3 to 4 and one of these babies died. All these neonates were of < or =33 weeks gestation. CONCLUSION: The use of serial CRP measurements to guide antibiotic therapy is a safe and practical approach in neonates with suspected sepsis in a developing country.

Fluconazole vs. amphotericin B for the treatment of neonatal fungal septicemia: a prospective randomized trial.

OBJECTIVE: Fungal septicemia is a devastating disease in the neonate, especially in the low birth weight preterm infant who is especially vulnerable to disseminated fungal sepsis. The objective of this study was to compare the efficacy, safety and overall convenience of fluconazole vs. amphotericin B for the treatment of disseminated fungal sepsis in neonates. DESIGN: A prospective, randomized, collaborative study conducted at two South African neonatal units. SUBJECTS: Twenty-four infants with proven fungal septicemia were treated from June, 1992, to June, 1993. Twelve received fluconazole, 11 received amphotericin B and 1 was excluded. Assessment of hepatic, renal and hematologic functions were performed before, during and after treatment. The two groups were comparable at the time of enrollment into the study. RESULTS: Infants receiving amphotericin B had significantly higher values of total and direct bilirubin and alkaline phosphatase values at the end of treatment, while the fluconazole group showed a significant increase in the platelet count. The cumulative total numbers of days receiving intravenous therapy for the administration of antifungal drugs were 57 for the fluconazole group and 162 for the amphotericin group; no central lines were needed in the fluconazole group, whereas 3 babies given amphotericin B had central catheters for a cumulative total of 27 days. The case fatality rate was 33% in the fluconazole group and 45% in the amphotericin B group; there was still proof of fungal septicemia at the time of death in 1 patient given amphotericin B and 2 given fluconazole. CONCLUSION: Fluconazole showed fewer side effects than amphotericin B and was more convenient to use.

HIV-1 co-infection in children hospitalised with tuberculosis in South Africa.

SETTING: Hospitals associated with the Department of Paediatrics at the University of the Witwatersrand, Johannesburg, South Africa. OBJECTIVES: To define the prevalence of human immunodeficiency virus (HIV) co-infection and differences in clinical presentation between HIV-infected and non-infected hospitalised children with tuberculosis. DESIGN: Children were prospectively enrolled between August 1996 and January 1997. RESULTS: Of 161 children enrolled, 42% were HIV-infected, including 67/137 with pulmonary tuberculosis (PTB) and 1/24 with extra-pulmonary disease (EPTB). Positive microscopy or bacteriology did not differ by HIV status for children with either PTB or EPTB. Although age did not differ between HIV-infected and non-infected children with PTB, non-HIV-infected children with EPTB were significantly older than those with PTB only (median age 32 months vs 14.5 months, P = 0.004). Chronic weight loss, malnutrition and the absence of BCG scarring were more common in HIV-infected children with PTB. HIV-infected children were also more likely to show cavitation (P = 0.001) and miliary TB (P = 0.01) on chest X-ray. Reactivity to tuberculin (> or = 5 mm and > or = 10 mm in HIV-infected and non-infected children, respectively) was significantly lower in HIV-infected children, as were CD4+ lymphocyte levels. The mortality rate during the study was 13.4% in HIV-infected children compared with 1.5% in non-HIV-infected children (P = 0.03). CONCLUSIONS: There is a high prevalence of HIV co-infection in children with TB. Progressive PTB and death are more common in HIV-infected children. Tuberculin skin testing is of limited use in screening for TB in HIV-infected children even when using a cut-point of > or = 5 mm.

Comparison of capillary electrochromatography with high-performance liquid chromatography for the analysis of pirimicarb and related compounds.

The applicability of capillary electrochromatography (CEC) to the analysis of pirimicarb and structurally related pyrimidines has been investigated. Methods were developed to improve the separation of closely related compounds. Resolution was achieved both by the use of running buffers containing a mixture of two organic modifiers to increase selectivity and reduce retention times. Solvent composition step gradients were used to separate compounds of widely differing retention factors. A comparison has been made between HPLC and CEC using identical separation parameters and the same stationary phase, from which two important conclusions are drawn. First, it has been shown that values of k' for the compounds analyzed were the same in both techniques. Secondly, although it is evident that CEC produces higher efficiencies than HPLC when running buffers with high organic solvent content are used, as the aqueous content of the running buffer is increased the efficiencies achieved in CEC and HPLC converge until they become equivalent. This is contrary to the theoretical model which predicts efficiencies are inherently higher using electrically rather than pressure driven flow. Disadvantages of the limited control of flow-rate in CEC in comparison with HPLC, are shown.

Combretastatin A-1 phosphate potentiates the antitumour activity of cisplatin in a murine adenocarcinoma model.

BACKGROUND: The tubulin depolymerizing drug Combretastatin A-1 phosphate (CA1P), a water-soluble derivative of combretastatin A-1, has been recently shown to have a better efficacy in experimental models than the clinically active, close structural analogue, combretastatin A-4 phosphate (CA4P). Previous studies with CA4P in combination with standard anti-cancer agents have demonstrated improved efficacy relative to the standard agents. MATERIALS AND METHODS: In this study the synergistic effects of administering CA1P in combination with cisplatin (CPL) in a well-differentiated transplantable murine colon model (MAC 29) was evaluated. RESULTS: CA1P at 100 mgkg-1 significantly potentiated the anti-tumour effects of CPL. The effect with CPL was similar to that seen for CA1P at its maximum tolerated dose (MTD) alone. CONCLUSION: These data demonstrate that the combination of CA1P and CPL has significant preclinical antitumour activity against a transplantable murine adenocarcinoma model that is related to the antivascular effects of CA1P.

Combretastatin A-1 phosphate a novel tubulin-binding agent with in vivo anti vascular effects in experimental tumours.

In view of the clinical potential of a number of natural products, Combretastatin A-1 phosphate was developed as a water-soluble derivative of combretastatin A-1. This study examined the anti-tumour activity of this compound against an experimental colon tumour (MAC29) in mice. A comparison was made with the clinically active combretastatin A-4 phosphate. The new compound was well-tolerated up to a dose of 250 mg/kg and was more effective at producing tumour growth delays than the A-4 analogue. Significant growth delays were seen at a dose of 50 mg/kg whereas the A-4 phosphate produced no measurable growth delay until a dose of 150 mg/kg was administered. Histological examination of treated tumours indicated that combretastatin A-1 phosphate caused very severe haemorrhagic necrosis in the tumour tissue and analysis of the sections indicated that almost 94 percent of the tumour was dead within 24 hours of treatment. The mechanism of action of combretastatin A-1 phosphate appears to be similar to the A-4 phosphate in that tumour vascular shutdown occurs within 4 hours of treatment. In summary combretastatin A-1 phosphate, the water-soluble analogue of combretastatin A-1, is more potent against a well-vascularised murine colon tumour than its predecessor, combretastatin A-4 phosphate. These data suggest this compound may have potential for clinical development.

Anti-tumor and anti-vascular effects of the novel tubulin-binding agent combretastatin A-1 phosphate.

The combretastatins are derived from an African medicinal plant Combretum caffrum (Combretaceae). They have previously been shown to be potent inhibitors of microtubule assembly that cause marked haemorrhagic necrosis in murine subcutaneous tumors. Promising clinical trial results with combretastatin A-4 phosphate led to this investigation of the anti-tumor and anti-vascular effects of a close structural analog, combretastatin A-1 phosphate. This compound caused identical disruption of the tubulin cytoskeleton in HUVECs in vitro at similar concentrations and duration of exposure as combretastatin A-4 phosphate. Treatment of a well-vascularised murine colon adenocarcinoma (MAC 29) with an effective dose (150 mg/kg) of combretastatin A-1 phosphate resulted in a dramatic decrease in functional vascular volume 2 hours after administration. Vascular shutdown was complete within 4 hours after treatment apart from in small areas of the tumor periphery. Morphological examination of hepatic deposits of HT29 and DLD-1 human colon tumors in nude mice demonstrated that combretastatin A-1 phosphate displays greater anti-tumor effects than the A-4 analog at the same dose and this order of activity (A-1 > A-4) is mirrored in the subcutaneous site with the same tumor type. In summary, combretastatin A-1 phosphate can exert its anti-tumor action via an anti-vascular mechanism. The results indicate that, despite having similar in vitro anti-tubulin properties, combretastatin A-1 phosphate seems to have greater in vivo anti-tumor activity than combretastatin A-4 phosphate at the same doses and may therefore be more successful in the clinic.