Combination therapy with fluconazole and flucytosine in the murine model of cryptococcal meningitis

This study elucidates the role of combined fluconazole and flucytosine as therapy for cryptococcosis in the murine model of meningitis. Three strains of Cryptococcus neoformans for which the range of fluconazole MICs was wide--2 microg/ml (susceptible strain), 8 microg/ml (moderately susceptible strain), and 32 microg/ml (resistant strain)--were used for infection. One day postinfection, the mice were randomized into eight treatment groups: placebo; flucytosine (40 mg/kg of body weight/day); fluconazole at 3 mg/kg/day (low dosage), 10 mg/kg/day (moderate dosage), or 20 mg/kg/day (high dosage); and combined flucytosine and fluconazole at low, moderate, or high doses of fluconazole. Three major findings were demonstrated: (i) correlation between the MICs for the isolates and the in vivo effectiveness of fluconazole as assessed by the reduction in cryptococcal brain burden, (ii) a dose-response curve (a higher dose of fluconazole was significantly more efficacious than a lower dose [P < 0.001]), and (iii) synergism between fluconazole and flucytosine (therapy with a combination of fluconazole and flucytosine was superior to therapy with either agent alone [P < 0.01]).     

Specific interaction between tetrandrine and Quillaja saponins in promoting permeabilization of plasma membrane in human leukemic HL-60 cells

Spontaneous Ni2+ entry (leak), measured as fluorescence quench in fura-2-loaded HL-60 cells at the excitation wavelength of 360 nm, was strongly inhibited by tetrandrine (TET, 100 microM), a Ca2+ antagonist of Chinese herbal origin. Exposure of the cells for 5 min to saponins from Quillaja saponaria (QS, 30 microg/ml), surfactants well known to permeabilize the plasma membrane by complexing with cholesterol, promoted Ni2+ entry without causing fura-2 leak-out. Unexpectedly, TET caused an immediate (within 2.5 min) augmentation of QS-promoted Ni2+ entry; and a 5-min treatment with both TET and QS resulted not only in an enhanced Ni2+ entry, but also a fura-2 leak-out. Ginseng saponins (100 microg/ml) alone or together with TET did not cause such a permeabilization. Permeabilization induced by 1-3 microM digitonin, another cholesterol-complexing glycoside, could not be enhanced by TET. TET did not affect permeabilization induced by Triton X-100 (0.01%), a detergent which non-specifically disrupts the hydrophobic interaction at the plasma membrane. TET also did not enhance Ni2+ entry triggered by ionomycin (0.35 microM) or SK&F 96365 (20 microM). Further, it did not augment Ni2+ entry when the plasma membrane fluidity was modulated by changes of temperature (27-47 degrees C) or treatment with 5% ethanol. This QS-promoted Ni2+ entry could not be amplified by other lipophilic Ca2+ antagonists, such as diltiazem (100 microM) and verapamil (100 microM). The results hence indicate that TET enhanced Ni2+ entry (or permeabilization) elicited by QS treatment, but not other perturbations of the plasma membrane. We suggest that pore formation at the plasma membrane, a consequence of QS-cholesterol interaction, can be specifically enhanced by TET. Also, a comparative study of the effects of TET and its very close analogues, hernandezine and berbamine, reveals that the methoxyl group at the R2 position of TET appears to be crucial in enhancing QS-promoted Ni2+ entry.     

Constitutive expression of mature transforming growth factor beta1 in the liver accelerates hepatocarcinogenesis in transgenic mice

Transforming growth factor beta-1 (TGF-beta1) is a potent inhibitor of hepatocyte growth both in vivo and in vitro. In this study, we analyzed the effects of TGF-beta1 on both naturally occurring and diethylnitrosamine-induced hepatocarcinogenesis using single transgenic TGF-beta1 and double transgenic c-myc/TGF-beta1 mice in which the expression of both transgenes was targeted to the liver. Hepatocellular tumors developed spontaneously in 59% (10 of 17) of the TGF-beta1 mice by 16-18 months of age. Coexpression of TGF-beta1 and c-myc transgenes in the liver accelerated hepatic tumor growth in both the presence and absence of carcinogenic treatment. Moreover, diethylnitrosamine-initiated tumors in the c-myc/TGF-beta1 mice showed a high rate of malignant conversion associated with a reduced expression or lack of TGF-beta receptor type II. The results suggest that overexpression of TGF-beta1 may contribute to liver carcinogenesis and that loss of TGF-beta receptor type II transduced inhibitory growth signals and up-regulation of c-myc are critical steps in liver tumor progression.     

In vitro and in vivo antipseudomonal activity, acute toxicity, and mode of action of a newly synthesized fluoroquinolonyl ampicillin derivative

Compounds N-(6,7-difluoroquinolonyl)-ampicillin (AU-1) and N-(6-fluoroquinolonyl)-ampicillin (FQ-1), synthesized by coupling of the carboxyl group of 6,7-difluoroquinolone (FP-3) and 6-fluoroquinolone (FP4), respectively, with the alpha-amino-group of ampicillin side chain, exhibit antipseudomonal activity similar to and lower acute toxicity than that of norfloxacin, whereas neither ampicillin nor the fluoroquinolone moieties, compound FP-3 or FP4, alone have such activity. Also, AU-1 and FQ-1 are active against tested clinical isolates of Pseudomonas aeruginosa that are highly resistant to norfloxacin, gentamicin, or both. The therapeutic efficacies of FQ-1 and norfloxacin were assessed and compared in neutropenic mice infected with a 90% lethal dose of P aeruginosa. Mice intraperitoneally administered FQ-1 (10 mg/kg) 4, 8, 24, and 48 hours after infection had survival rates as high as 80%, comparable to those of mice treated with norfloxacin at the same dosage and dosing schedule. The study of protoplast formation revealed that FQ-1 did not inhibit cell-wall biosynthesis but did induce cell filamentation of Bacillus subtilis at a level close to its minimal inhibition concentration. Both AU-1 and FQ-1 were able to intercalate into the double-stranded DNA. However, that FQ-1 lost such activity after it was treated with penicillinase suggests that the lactam-ring structure in ampicillin moiety of FQ-1 was hydrolyzed by penicillinase and that the hydrolyzed structure of FQ-1 does not own DNA-intercalation activity.     

Assessment of height as an indicator of population nutritional status

BACKGROUND: Chilean girls of middle and low socioeconomic levels are shorter than their European or North American counterparts. AIM: To challenge the hypothesis that undernutrition is the cause of the shorter stature of Chilean girls. MATERIAL AND METHODS: Height, weight, sexual maturation, biacromial and bi-iliac diameters of low and middle socioeconomic level Chilean children aged 0 to 20 years old, were compared with data from French, English and North American children in the same age range. RESULTS: Chilean girls had an earlier sexual maturation, had bigger biacromial and bi-iliac diameters and had a higher weight for height than European girls. Weight for height of these girls was over the values proposed by the Chilean Ministry of Health, that are based in NCHS/WHO tables. Chilean girls with recent European ancestry were taller than girls without this ancestry. Chilean boys were smaller and had lower weights than their foreign counterparts. Bi-iliac diameters were similar to those of French boys and sexual maturation was similar to English boys. CONCLUSIONS: These results indicate that genetics and not undernutrition is involved in the shorter height of Chilean girls, when compared with their European or North American counterparts.     

Immunolocalization of interstitial collagenase (MMP-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in radicular cysts

To investigate the mechanisms involved in expansion of radicular cysts, monoclonal antibodies against interstitial collagenase (MMP-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) were used to localize the sites of MMP-1 and TIMP-1 expression in 30 radicular cysts. Positive MMP-1 staining was detected in the lining epithelium and subepithelial fibroblasts, macrophages, endothelial cells and osteoblasts/osteocytes in all specimens. Positive TIMP-1 staining was identified in osteoblasts/osteocytes and endothelial cells of all specimens, and in the lining epithelium and subepithelial fibrous connective tissue wall of five radicular cysts with an intense inflammatory cell infiltrate. The number and distribution of positive cells for MMP-1 or TIMP-1 varied widely among individual specimens, but strong immunostaining was constantly detected at sites with prominent subepithelial inflammation. Results here support the hypothesis that MMP-1 may play an important role in the expansion of radicular cysts. The absence of TIMP-1 expression in lining epithelium and subepithelial fibroblasts and macrophages in most cases studied indicated that an imbalance between MMP-1 and TIMP-1 production may lead to radicular cyst expansion.     

Insulin-like growth factor-I (IGF-I) concentration in 150-kilodalton complexes containing human IGF-binding protein-3 (hIGFBP-3) after intravenous injection of adult rats with hIGFBP-3

After i.v. injection into adult rats, human insulin-like growth factor-binding protein-3 (hIGFBP-3) forms 150-kDa complexes with excess endogenous rat acid-labile subunit (ALS) within 2 min (Lewitt et al., 1993, Endocrinology 133:1797). Because their previous in vitro studies indicated that hIGFBP-3 only bound to ALS in the presence of IGF-I, and because little free IGF-I is present in plasma, the authors postulated that IGF-I had been mobilized to the circulation to saturate the 150-kDa hIGFBP-3 complexes. We examined this hypothesis by determining whether the hIGFBP-3 that appears in 150-kDa complexes 2 min after i.v. injection is accompanied by an increase in IGF-I. Within 2 min, some of the injected hIGFBP-3 (approximately 30% as much as endogenous intact rat IGFBP-3) is present in complexes that are cleared slowly from the circulation and presumed to be 150-kDa complexes. Gel filtration and immunoprecipitation studies performed on blood collected 2 min after injection confirmed that the injected hIGFBP-3 (46-82% as much as rat IGFBP-3) was associated with ALS in 150-kDa complexes. The formation of 150-kDa complexes containing hIGFBP-3 was not accompanied by a corresponding change in the IGF-I content (determined by RIA) of whole serum or 150-kDa serum fractions, suggesting that the hIGFBP-3 had rapidly associated with rALS in vivo without mobilizing IGF-I. Surprisingly, however, hIGFBP-3 was cleared much more rapidly from 150-kDa complexes formed after injection of hIGFBP-3 than after injection of hIGFBP-3:IGF-I complexes, suggesting that the complexes observed after hIGFBP-3 injection might not have formed in vivo. In fact, 150-kDa complexes formed to a similar extent when hIGFBP-3 was added ex vivo to blood collected from rats that had not received hIGFBP-3. We conclude that hIGFBP-3 can rapidly associate with rALS to form 150-kDa complexes in vivo without the mobilization of IGF-I. Because 150-kDa complexes also are formed ex vivo, however, we are unable to resolve whether the complexes that formed in vivo formed as binary or ternary complexes.