[Should we screen for colonization to control the spread of multidrug resistant bacteria?]. / Quelles stratégies découlent de la surveillance des bactéries multirésistantes aux antibiotiques afin de mieux maîtriser leur diffusion?

Should we screen for colonization to control the spread of multidrug-resistant bacteria? A multidrug-resistant bacteria surveillance program was performed in 1999 at Laënnec Hospital (Nantes, France). After a 3-year period, the results permit us to determine the strategy to strengthen their spread. In 2001, Staphylococcus aureus resistant to methicillin represented 45% of the 202 multidrug-resistant bacteria isolated. The global incidence rate per 100 admissions remained stable between 1999 and 2001 (0.42%), but those of infections acquired in our institution decreased significantly from 0.27% in 1999 to 0.18% in 2001 (P < 0.05), particularly in medical care units (P < 0.04). In spite of this surveillance program and hygiene trainings, the global incidence remained stable during the study period, even if our action contributed to decrease the incidence of S. aureus resistant to methicillin acquired in our institution. Isolation precautions and screening for colonization policy in intensive care units are not sufficient to control the spread of MRB at hospital level. They should be strengthened by procedures for the transfer of infected or colonized patients and by antibiotic use control.

Thermoreversible gel as a candidate barrier to prevent the transmission of HIV-1 and herpes simplex virus type 2.

BACKGROUND: Sexually transmitted diseases (STDs) caused by HIV, herpes simplex virus (HSV), and other pathogens are spreading dramatically. The need to develop active products and vehicles to reduce this epidemic is urgent. GOAL: The efficacy of a thermoreversible gel formulation as a possible barrier to prevent the transmission of pathogens causing STDs was evaluated. STUDY DESIGN: This evaluation investigated the ability of the gel formulation to prevent infection of susceptible cells by HIV-1 and HSV-2 in vitro, the diffusion of radiolabeled herpes virus and micelles of polymer through an insertion membrane, and the electron microscopic appearance of herpes virus and gel alone or mixed together. RESULTS: The gel formulation prevents infection of susceptible cells by HIV-1 and HSV-2. It acts as an effective artificial physical barrier against the herpes virus within the first 4 hours of incubation. Herpes virus is coated by the gel or entrapped within micelles of the gel, which could hinder its attachment to target cells and inhibit its infectivity. CONCLUSION: This thermoreversible gel formulation represents an attractive matrix for the incorporation of microbicides to prevent the spread of STDs.

Classification of kinin receptors.

This minireview is divided into three parts: the first part refers to the characterization and classification of kinin receptors using agonists and antagonists in isolated tissues (classical pharmacology). Two kinin receptors have been considered on the basis of their distinct pharmacology, namely the B1 receptor of the rabbit aorta (rank order of potency of agonists: LysdesArg9BK > desArg9BK > or = LysBK > BK; apparent affinities of antagonists Lys[Leu8]desArg9BK (pIC50 8.4) > [Leu8]desArg9BK (pIC50 7.4) >>> HOE 140, a B2 receptor antagonist, pIC50<5.0), and the B2 receptor of the rabbit jugular vein (potency of agonists: LysBK = BK >>> LysdesArg9BK = desArg9BK and HOE 140 (pIC50 9.0) >>> Lys[Leu8]desArg9BK, pIC50<5.0). The second part describes species-related B1 receptor subtypes, demonstrated by different pharmacological profiles of agonists and antagonists: human, rabbit and pig subtypes (LysdesArg9BK >> desArg9BK and Lys[Leu8]desArg9BK > [Leu8]desArg9BK) and dog, rat, mouse and hamster B1 receptors (desArg9BK = LysdesArg9BK and [Leus]desArg9BK = Lys[Leu8]desArg9BK). Affinities of agonists and antagonists in some species (man, rabbit, pig) are significantly increased (at least 10-fold) by the presence of a Lys at their N-terminus. The last part describes species-related B2 receptor subtypes supported by results obtained with non-peptide receptor agonists (FR 190997) and antagonists (FR 173657). While BK acts as a full agonist in man, rabbit and pig, FR 190997 behaves as a full agonist on human, as partial agonist on rabbit, and as pure antagonist on pig B2 receptors. Various hypotheses are considered to interpret these findings.

Evidence for a role of endogenous neurotensin in the initiation of amphetamine sensitization.

This study was aimed at testing the hypothesis that endogenous neurotensin plays a role in the initiation of sensitization to the locomotor activating effect of amphetamine. During an initial training phase, different groups of male rats were injected on four occasions (every second day: Days 1, 3, 5 and 7) with one of three doses (40, 80 or 160 microg/kg, ip) of the neurotensin antagonist, SR-48692, or its vehicle, followed 30 min later by amphetamine (1.5 mg/kg, ip), or saline. Ambulatory, non-ambulatory, and vertical movements were measured for 2 h in photocell cages immediately following the second injection. One week after the training phase, sensitivity to amphetamine (0.75 mg/kg, ip) was tested in all the rats (sensitization test). The results show that SR-48692, when given alone, produced levels of locomotor activity that were not statistically different from control. At the low dose, it potentiated amphetamine-induced ambulatory and non-ambulatory movements, an effect observed on Day 7 but not on Day 1. On the day of the sensitization test, rats pre-exposed to amphetamine alone displayed stronger ambulatory and non-ambulatory movements than vehicle pre-exposed rats, a sensitization effect that was attenuated and prevented by SR-48692 at 80 and 160 microg/kg, respectively. The present results demonstrate that activation of neurotensin receptors by endogenous neurotensin is required for the initiation of amphetamine sensitization. They provide additional evidence that an increase in central neurotensinergic neurotransmission may lead to a lasting increased sensitivity to psychostimulant drugs.

Synthesis and antibacterial activity of HMR 3647 a new ketolide highly potent against erythromycin-resistant and susceptible pathogens.

In the search for new ketolides with improved activities against erythromycin-resistant S. pneumoniae and H. influenzae we synthesized a new 11,12 carbamate ketolide substituted by an imidazo-pyridyl side chain: HMR 3647. This compound demonstrated a potent activity against erythromycin susceptible and resistant pathogens, including penicillin G/erythromycin A-resistant S. pneumoniae and H. influenzae. In vivo, HMR 3647 displayed good pharmacokinetic parameters (Cmax = 2.9 microg/ml, bioavailability=49%, AUC0.8 = 17.2 microg.h/l, t1/2=1h) and was shown to have a high therapeutic efficacy in mice infected by various respiratory pathogens, including multi-resistant S. pneumoniae and Gram negative bacteria such as H. influenzae. HMR 3647 appears to be a very promising agent for the treatment of respiratory infections and is currently in clinical trials.

Kinin B1 receptor antagonists containing alpha-methyl-L-phenylalanine: in vitro and in vivo antagonistic activities.

-To protect from metabolism and to improve potency of the AcLys-[D-betaNal7,Ile8]desArg9-bradykinin (BK) (R 715), we prepared and tested 3 analogues containing alpha-methyl-L-Phe ([alphaMe]Phe) in position 5: these are the AcLys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 892), Lys-Lys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 913), and AcLys-Lys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 914). The new compounds were tested against the contractile effect induced by desArg9BK on 2 B1 receptor bioassays, the human umbilical vein, and the rabbit aorta. Their antagonistic activities were compared with those of the early prototypes (Lys-[Leu8]desArg9BK and [Leu8]desArg9BK) and with other recently described peptide antagonists. The 3 (alphaMe)Phe analogues showed high antagonistic potencies (pA2) at both the human (8.8, 7.7, and 8. 7, respectively) and rabbit (8.6, 7.8, and 8.6, respectively) B1 receptors. No antagonistic effects (pA2<5) were observed on the B2 receptors that mediate the contractile effects of BK on the human umbilical vein, the rabbit jugular vein, and the guinea pig ileum. Moreover, these new B1 antagonists were found to be resistant to in vitro degradation by purified angiotensin-converting enzyme from rabbit lung. The Nalpha-acetylated forms, R 892 and R 914, were resistant to aminopeptidases from human plasma. In vivo antagonistic potencies (ID50) of B1 receptor antagonists were evaluated in anesthetized lipopolysaccharide-treated (for B1 receptor) and nontreated (for B2 receptor) rabbits against the hypotensive effects of exogenous desArg9BK and BK. R 892 efficiently inhibited (ID50 2.8 nmol/kg IV) hypotension induced by desArg9BK without affecting that evoked by BK (ID50 >600 nmol/kg IV). Conversely, the peptide antagonists Lys-Lys-[Hyp3,Igl5,D-Igl7,Oic8]desArg9BK (B 9858) and DArg-[Hyp3,Thi5,D-Tic7,Oic8] desArg9BK (S 0765) showed dual B1/B2 receptor antagonism in vitro and in vivo. It is concluded that R 892 and congeners provide selective, highly potent, and metabolically stable B1 kinin receptor antagonists that can be useful for the assessment of the physiological and pathological roles of kinin B1 receptors.

Effects of kinins on isolated stomachs of control and transgenic knockout B2 receptor mice.

The aim of this study was to investigate the pharmacological profile of the kinin B1 and B2 receptors in isolated stomachs from wild-type control and B2 receptor knockout mice. Isometric contractions evoked by bradykinin (BK) (9 nM) and desArg9BK (28 nM) were shown to be different. The contraction induced by desArg9BK had a longer duration than that evoked by BK and increased during incubation in vitro in stomachs of wild-type controls, while in the transgenic B2 receptor knockout mice, the contractions evoked by desArg9BK and BK were similar and followed the B1 receptor agonist pattern. BK but not the carboxypeptidase-resistant analog, [Phe8psi(CH2-NH)Arg9]BK, was found to be active in the stomach of B2 receptor knockout mice. BK-induced contractions were prevented by mergetpa (a carboxypeptidase M inhibitor) (10 microM) and by a the B receptor antagonist, AcLys[DbetaNal7,Ile8]desArg9BK (R 715) (0.88 microM), while not being influenced by the B2 receptor antagonist HOE 140 (0.38 microM). BK and [Phe8psi(CH2-NH)Arg9]BK were potent contractants of the wild-type mice stomach and their effects were not influenced by mergetpa or by the B receptor antagonist: they were reduced by HOE 140. After incubation in vitro for 3-4 hours, the tissues were treated with HOE 140 (4 microM) and FR-173657 (17 microM) to eliminate B2 receptor function. In these tissues, BK evoked a B1-like contraction which was inhibited by mergetpa (10 microM) and antagonized by R 715 (8 microM). The results indicate that BK acts primarily on B2 receptors. However, after intramural conversion to desArg9BK, activation of B1 receptors of the mice stomach occurs. In the tissues of B2 receptor knockout mice, BK behaves as a pure B1 receptor agonist while in stomachs of control animals, the B2 receptor contribution is overwhelming. After complete blockade of the B2 receptor, BK is able to evoke B1-mediated responses similar to those observed in tissues of B2 receptor knockout mice. It is concluded that the disruption of the B2 receptor gene eliminates the B2 receptor without influencing the B1 receptor system.

Encapsulation of foscarnet in liposomes modifies drug intracellular accumulation, in vitro anti-HIV-1 activity, tissue distribution and pharmacokinetics.

OBJECTIVE: To improve the in vitro anti-HIV-1 activity, intracellular accumulation in macrophages and in vivo pharmacokinetics and tissue distribution of foscarnet (trisodium phosphonoformate; PFA) by encapsulation in liposomes. METHODS: The accumulation of free and liposome-encapsulated PFA was determined in monocyte-macrophage RAW 264.7 cells and human premonocytoid U937 cells. The antiviral activity was evaluated in U937 cells infected with HIV-1IIIB. Tissue distribution and pharmacokinetics of free and liposomal PFA were determined in female Sprague-Dawley rats following the administration of an intravenous bolus dose (10 mg PFA/kg). RESULTS: The entrapment of PFA in liposomes resulted in a higher drug accumulation in both U937 and RAW 264.7 cells. A slightly greater efficacy against HIV-1IIIB replication into U937 cells was observed upon encapsulation of PFA into liposomes. Improved pharmacokinetics was observed upon entrapment of PFA in liposomes. Much higher drug levels were found in plasma for the liposomal formulation. The systemic clearance of the liposomal drug was 77 times lower than that of free drug. The encapsulation of PFA in liposomes greatly enhanced the drug accumulation in organs of the reticuloendothelial system. CONCLUSION: The encapsulation of PFA in liposomes modified the tissue distribution and plasma pharmacokinetics of the antiviral agent, resulting in a marked improvement of drug accumulation in organs involved in HIV immunopathogenesis and in a greater PFA bioavailability. The antiviral activity of liposomal PFA was slightly greater than that of free drug in HIV-1IIIB-infected U937 cells.

Regulation of guinea pig adrenal P450c21 messenger RNA, protein and activity by RU486.

The role of ACTH, forskolin and 8Br-cAMP on the regulation of mRNA abundance, protein levels and enzymatic activity of cytochrome P450 21-hydroxylase (P450c21, CYP21) were investigated in guinea pig adrenal cell cultures. In untreated cells, 21-hydroxylase activity was diminished throughout a 48 h period of incubation. Although incubation with forskolin and 8Br-cAMP restored 21-hydroxylase activity to normal levels, the addition of ACTH did not prevent the decrease of 21-hydroxylase activity. Treatment of cells with RU486 for 24 h inhibited 21-hydroxylase activity by 93%; however, after removal of the drug a slight increase of enzyme activity was observed; this rise was enhanced by the addition of ACTH. Forskolin and 8Br-cAMP increased the levels of 21-hydroxylase activity to the same range as seen in untreated cells. In cells that were not pretreated with RU486, incubation with cycloheximide for 1 h had no effect on 21-hydroxylase activity and could not prevent the modest increase of 21-hydroxylase activity induced by forskolin or 8Br-cAMP after 48 h of incubation. In RU486-treated cells, cycloheximide blocks the stimulation of enzyme activity induced by ACTH, forskolin and 8Br-cAMP. Our findings indicate that 21-hydroxylase activity can be stimulated by ACTH, forskolin or 8Br-cAMP solely in the presence of reduced enzymatic activity. Western immunoblot analysis of P450c21 protein levels in untreated or RU486-treated adrenal cells indicate that P450c21 protein levels were in the same range and further incubation with ACTH caused a similar elevation of P450c21 protein levels in both the untreated and RU486-treated cells. Northern blot analysis on RNA isolated from adrenal cells showed that RU486 did not alter the basal steady state levels of P450c21 mRNA. As well, incubation with ACTH or 8Br-cAMP increased the levels of P450c21 transcript to the same extent in both untreated and RU486-treated cells. These results taken together provide additional evidence for the presence of an adrenal specific protein factor(s) modulating 21-hydroxylase activity.

Regulation of 21-hydroxylase activity by steroids.

In this study, we investigated the effect of steroids on guinea pig and bovine adrenal steroidogenesis, especially 21-hydroxylase activity. Analysis of guinea pig adrenal steroids indicated the presence of high concentrations of androstenedione in the guinea pig adrenal; furthermore, in vitro studies using guinea pig adrenal cortex cells in primary culture confirmed that androstenedione is one of the major C19 steroids produced and secreted. The direct action of steroids on steroid production by adrenal cells was investigated. Our data indicate that steroids themselves increase C19 steroid synthesis and inhibit glucocorticoid production by guinea pig adrenal cells without affecting gene expression for steroidogenic enzymes. Incubation of a series of C19 steroids, namely, androstenedione, with guinea pig adrenal cell cultures demonstrated that the decrease in 21-hydroxylase activity is largely independent of the androgenic activity of C19 steroids. RU38486, a synthetic C18 steroid possessing a 4-ene-3-ketosteroid with an aryl group at position 11 and a very low affinity for the androgen receptor, also irreversibly altered 21-hydroxylase activity. An effect of RU38486 on 21-hydroxylase activity was also demonstrated in bovine adrenal cells. Further studies with bovine adrenal cells showed that the decrease in 21-hydroxylase activity induced by RU38486 was accompanied by a small but significant inhibition of P450c21 protein levels at both basal and ACTH-stimulated levels. In summary, our data indicate that alteration of 21-hydroxylase activity by steroids is likely due to a direct action on P540c21 protein, and the levels of androstenedione in the adrenal are high enough to inhibit 21-hydroxylase activity.

Antiviral efficacy, intracellular uptake and pharmacokinetics of free and liposome-encapsulated 2',3'-dideoxyinosine.

OBJECTIVE: To evaluate the effect of liposome encapsulation on the in vitro antiviral efficacy, intracellular uptake and in vivo pharmacokinetics of 2',3'-dideoxyinosine (ddl). METHODS: The accumulation of free and liposome-encapsulated ddl was determined in murine monocyte-macrophage RAW 264.7 cells and human premonocytoid U937 cells. The antiviral efficacy was evaluated in U937 cells infected with HIVIIIB. Tissue distribution and pharmacokinetics of free and liposomal ddl were determined in female Sprague-Dawley rats following the administration of a single intravenous bolus dose (3 mg ddl/kg). RESULTS: The entrapment of ddl in liposomes results in a lower drug accumulation in both U937 and RAW 264.7 cells. A lower antiviral efficacy against HIVIIIB replication in U937 cells was observed on encapsulation of ddl in liposomes. Improved pharmacokinetics were observed on entrapment of ddl in liposomes. Higher drug levels were found in plasma for the liposomal formulation. The systemic clearance of the liposomal drug was 120 times lower than that of free drug. Liposome encapsulation of ddl greatly enhanced the drug accumulation in organs of the reticuloendothelial system. CONCLUSION: The encapsulation of ddl in liposomes modified the tissue distribution and plasma pharmacokinetics of the antiviral agent resulting in a marked improvement of drug biodisponibility. The antiviral efficacy of liposomal ddl was lower than that of free drug in HIVIIIB-infected U937 cells.

Comparison of cellular accumulation, tissue distribution, and anti-HIV activity of free and liposomal 2',3'-dideoxycytidine.

We have investigated the cellular accumulation, tissue distribution, and antihuman immunodeficiency virus activity of free dideoxycytidine (ddC) and liposomal ddC (L-ddC). We have found that L-ddC was more efficiently taken up than its free form by RAW 264.7 cells (a monocyte-macrophage cell line) (p < 0.01) while a comparable uptake was seen in U937 cells (a promonocytic cell line). In the rat, L-ddC accumulated preferentially in liver and spleen when injected intravenously (p < 0.01), and mostly in spleen when given intraperitoneally (p < 0.01). In contrast, free ddC was rapidly eliminated out of the body. Liposomal ddC showed a similar anti-HIV activity in comparison with free ddC in U937 cells. Given the fact that encapsulation of ddC in liposomes does not affect its anti-HIV activity but enhances its in vitro cellular accumulation and its in vivo distribution in reticuloendothelial system (RES) tissues, we conclude that ddC in liposomal formulation is a promising anti-HIV agent with a targeted action on the RES, which is considered a reservoir for dissemination of virus to other cells, tissues, and organs.

Effects of 4-MA, a potent inhibitor of 5 alpha-reductase, on 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase activity in guinea pig adrenals.

Previous reports have shown that 17 beta-N,N-Diethylcarbamoyl-4-methyl-4-aza- 5 alpha-androstan-3-one (4-MA), a synthetic inhibitor of 5 alpha-reductase, exerts an inhibitory effect on 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) activity. To characterize further the effects of 4-MA on steroidogenesis, guinea pig fasciculata-glomerulosa cells in primary culture were treated for 24 h with 4-MA. Our data indicate that 4-MA reduced 3 beta-HSD activity in cultured adrenal cells but had no effect on the activities of 11-hydroxylase, 21-hydroxylase, 17-hydroxylase, and 17,20-lyase. Be decreasing the conversion of pregnenolone into progesterone or 17-hydroxypregnenolone into 17-hydroxyprogesterone, 4-MA caused the steroidogenic pathway to shift toward the production of dehydroepiandrosterone. Despite the presence of 4-MA, androstenedione and 11 beta-hydroxyandrostenedione were produced at levels exceeding the control levels. In the presence of ACTH and 4-MA, cortisol production was inhibited by 90% whereas androstenedione and 11 beta-hydroxyandrostenedione were reduced by only 40%. The effect of the compound was reversed by washing the adrenal cells with medium, thus suggesting a direct action of 4-MA on the enzyme itself. In summary, our data indicate that 4-MA markedly reduces the production of cortisol in the adrenals and partially alters the formation of C-19 steroids. It is important to consider this finding in the use of 4-azasteroids in the treatment of prostate cancer, which was previously found to be sensitive to secretion of adrenal C-19 steroids.

Ceftriaxone protects against tobramycin nephrotoxicity.

The effect of ceftriaxone on tobramycin-induced nephrotoxicity was investigated. Female Sprague-Dawley rats were treated during 4 and 10 days with saline (NaCl, 0.9%), ceftriaxone at a dose of 100 mg/kg of body weight/12 h subcutaneously, tobramycin at doses of 40 and 60 mg/kg/12 h intraperitoneally, or the combination ceftriaxone-tobramycin. Creatinine levels in serum were significantly higher in animals treated with tobramycin alone given at 60 mg/kg/12 h during 10 days, compared with control animals (P < 0.01) or animals receiving the combination tobramycin-ceftriaxone (P < 0.01). After 10 days of treatment, ceftriaxone did not accumulate in renal tissue but did reduce the renal intracortical accumulation of tobramycin (P < 0.05). Tobramycin given alone at either 40 or 60 mg/kg/12 h induced a significant inhibition of sphingomyelinase activity compared with control animals (P < 0.05). However, this enzyme activity was significantly less inhibited when tobramycin was injected in combination with ceftriaxone (P < 0.05). Ceftriaxone alone had no effect on the activity of this enzyme. The [3H]thymidine incorporation into the DNA of renal cortex was also significantly lower in animals treated with tobramycin-ceftriaxone compared with animals receiving tobramycin alone (P < 0.05). The 24-h urinary excretion of beta-galactosidase was significantly reduced in animals treated with the combination tobramycin-ceftriaxone compared with the administration of tobramycin alone at 40 and 60 mg/kg/12 h after 5 and 10 days (P < 0.05). Histologically, ceftriazone induced very few cellular alterations and reduced considerably the presence of typical signs of tobramycin nephrotoxicity. This investigation demonstrated that ceftriaxone protects animals against tobramycin-induced nephrotoxicity.

Direct agglutination of weak D red cells by tetramolecular complexes containing monoclonal IgG anti-D.

The capacity of blood group antibodies to agglutinate red cells suspended in saline is largely dependent on the antibody isotype. The immunological cross-linking of IgG antibodies has previously been described as a means to increase the reactivity of IgG in many situations. We have prepared anti-D-containing complexes by blending a human IgG anti-D monoclonal antibody (mAb) and a murine anti-human IgG mAb. In standard red cell serology assays, the anti-D complexes exhibited a very high avidity and could agglutinate weak D-positive red cells in direct saline testing. These results indicate that potent saline hem-agglutinating reagents of RhD and eventually of other blood group specificities can be prepared from IgG mAbs.