The use of a decremental dose regimen in patients treated with a chronic low-dose step-up protocol for WHO Group II anovulation: a prospective randomized multicentre study

BACKGROUND: In women with chronic anovulation, the choice of the FSH starting dose and the modality of subsequent dose adjustments are critical in controlling the risk of overstimulation. The aim of this prospective randomized study was to assess the efficacy and safety of a decremental FSH dose regimen applied once the leading follicle was 10-13 mm in diameter in women treated for WHO Group II anovulation according to a chronic low-dose (CLD; 75 IU FSH for 14 days with 37.5 IU increment) step-up protocol. METHODS: Two hundred and nine subfertile women were treated with recombinant human FSH (r-hFSH) (Gonal-f) for ovulation induction according to a CLD step-up regimen. When the leading follicle reached a diameter of 10-13 mm, 158 participants were randomized by means of a computer-generated list to receive either the same FSH dose required to achieve the threshold for follicular development (CLD regimen) or half of this FSH dose [sequential (SQ) regimen]. HCG was administered only if not more than three follicles >or=16 mm in diameter were present and/or serum estradiol (E(2)) values were <1200 pg/ml. The primary outcome measure was the number of follicles >or=16 mm in size at the time of hCG administration. RESULTS: Clinical characteristics and ovarian parameters at the time of randomization were similar in the two groups. Both CLD and SQ protocols achieved similar follicular growth as regards the total number of follicles and medium-sized or mature follicles (>/=16 mm: 1.5 +/- 0.9 versus 1.4 +/- 0.7, respectively). Furthermore, serum E(2) levels were equivalent in the two groups at the time of hCG administration (441 +/- 360 versus 425 +/- 480 pg/ml for CLD and SQ protocols, respectively). The rate of mono-follicular development was identical as well as the percentage of patients who ovulated and achieved pregnancy. CONCLUSIONS: The results show that the CLD step-up regimen for FSH administration is efficacious and safe for promoting mono-follicular ovulation in women with WHO Group II anovulation. This study confirms that maintaining the same FSH starting dose for 14 days before increasing the dose in step-up regimen is critical to adequately control the risk of over-response. Strict application of CLD regimen should be recommended in women with WHO Group II anovulation.     

Antibacterial properties of dermaseptin S4 derivatives under extreme incubation conditions

Antibacterial properties of the frog-derived peptide dermaseptin S4 and a series of synthetic derivatives against the food pathogen Escherichia coli O157:H7 were investigated under extreme incubation conditions. The 28-mer analog K4K20S4 (P28) displayed an MIC of 8 microM and rapid bactericidal kinetics under standard culture conditions. Potent bactericidal properties were maintained at high salt concentrations, under acidic or basic conditions, and at extreme temperatures. The N-terminal 14-mer sequence (P14) displayed higher potency (MIC, 4 microM) but only within a narrow range of incubation conditions, pointing to the importance of the C-terminal domain of P28. The potency range was reextended upon conjugation of aminododecanoic acid to P14. The resulting lipopeptide was even more potent (MIC, 2 microM) and affected bacterial viability under most of the conditions tested, including in commercial apple juice. The mechanistic implications of peptides' hydrophobicity, charge, structure, and binding to an idealized membrane were probed and are discussed here. Collectively, the data indicate interest in simple peptide-based compounds for design of antimicrobials that affect pathogens under a variable range of incubation conditions.     

Decreased sternal vascularity after internal thoracic artery harvesting resolves with time: an assessment with single photon emission computed tomography

OBJECTIVE: We sought to prospectively evaluate the long-term effect of left internal thoracic artery harvesting on sternal vascularity. METHODS: Twelve consecutive patients undergoing primary coronary artery bypass grafting were studied. In all patients a pedicled left internal thoracic artery was harvested. Each patient underwent a preoperative technetium-99m methylene diphosphonate bone scan with single photon emission computed tomography. The ratio of the mean counts per pixel for each side of the sternum was obtained. All patients had early repeat bone scans 6 plus minus 2.4 days postoperatively and late repeat bone scans 18.6 plus minus 3.5 months after the operation. Ratios of unilateral sternal uptakes were compared between the different scans. One patient died during follow-up and was excluded from the study. RESULTS: There was a significant decrease in flow to the left hemisternum in the early postoperative scan compared with that in the preoperative scan (P <.001). At late follow-up scans, flow to the left hemisternum had returned to normal (P =.119). Midterm clinical follow-up demonstrated 3 superficial wound infections. No musculoskeletal pain existed at the time of follow-up, but 3 patients had numbness or tingling at the skin area corresponding to the site from which the left internal thoracic artery was harvested. CONCLUSIONS: Acute postoperative sternal ischemia caused by harvesting of a pedicled left internal thoracic artery is temporary and resolves with time.     

C-reactive protein and orosomucoid determinations in a neonatal pathology unit

We report the result of CRP and ORM assays performed at the admission in the unit, in 157 neonates (NN). The NN were divided into 3 groups: 122 "controls" (group I), 21 having septicemia with or without meningitis but with symptoms of infection (group II), 16 having a positive blood culture without symptom of infection (group III). In group I, the CRP level does not depend on gestational age at birth, neither on the age at the assay. The level of ORM depends on both factors. Neonatal infection leads to a very significant rise of the CRP rate (often above 100 mg/l). The rise of ORM is not so dramatic. These data suggest that the assay of these two proteins are useful in the diagnosis of neonatal infection if they are performed together and at several times.     

In Vitro Antibacterial Activity of Acyl-Lysyl Oligomers against Helicobacter pylori

The gastric pathogen Helicobacter pylori has developed resistance to virtually all current antibiotics; thus, there is a pressing need to develop new anti-H. pylori therapies. The goal of this work was to evaluate the antibacterial effect of oligo-acyl-lysyl (OAK) antimicrobial peptidomimetics to determine if they might represent alternatives to conventional antibiotic treatment of H. pylori infection. A total of five OAK sequences were screened for growth-inhibitory and/or bactericidal effects against H. pylori strain G27; four of these sequences had growth-inhibitory and bactericidal effects. The peptide with the highest efficacy against strain G27, C₁₂K-2β₁₂, was selected for further characterization against five additional H. pylori strains (26695, J99, 7.13, SS1, and HPAG1). C₁₂K-2β₁₂ displayed MIC and minimum bactericidal concentration (MBC) ranges of 6.5 to 26 μM and 14.5 to 90 μM, respectively, across the six strains after 24 h of exposure. G27 was the most sensitive H. pylori strain (MIC = 6.5 to 7 μM; MBC = 15 to 20 μM), whereas 26695 was the least susceptible strain (MIC = 25 to 26 μM; MBC = 70 to 90 μM). H. pylori was completely killed after 6 to 8 h of incubation in liquid cultures containing two times the MBC of C₁₂K-2β₁₂. The OAK demonstrated strong in vitro stability, since efficacy was maintained after incubation at extreme temperatures (4°C, 37°C, 42°C, 50°C, 55°C, 60°C, and 95°C) and at low pH, although reduced killing kinetics were observed at pH 4.5. Additionally, upon transient exposure to the bacteria, C₁₂K-2β₁₂ showed irreversible and significant antibacterial effects and was also nonhemolytic. Our results show a significant in vitro effect of C₁₂K-2β₁₂ against H. pylori and suggest that OAKs may be a valuable resource for the treatment of H. pylori infection.Helicobacter pylori is a microaerophilic gram-negative bacterium that colonizes the gastric mucosa. It is known to be a principal gastric pathogen of humans and is associated with the development of gastritis, gastric ulcers, duodenal ulcers, and gastric cancer (46, 55, 56, 60). Approximately half of the world''s population is infected with H. pylori (79). Thus, the bacterium poses a significant public health problem, which is further compounded by the fact that H. pylori has developed antimicrobial resistance to virtually all current antibiotics, a phenomenon that is hampering efforts to treat the infection (40, 51).Since the original isolation of H. pylori in the early 1980s, treatment of the bacterial infection has undergone a significant evolutionary development from initial monotherapy to dual, triple, and in more recent trials quadruple therapy (8, 18). Current treatment strategies employ combination therapy, since single-antibiotic therapy often results in failure to eradicate the infection (21). The highest H. pylori eradication rates have been reported with triple therapy, which involves the utilization of two antibiotics in combination with bismuth or a proton pump inhibitor, PPI (34, 44). Amoxicillin (amoxicilline) with either clarithromycin or metronidazole is often the antibiotic combination of choice as a first- or second-line treatment regimen, respectively. However, in recent years the efficacy of the standard first-line triple therapy has also been decreasing dramatically, mainly due to development of resistance to the drugs (35, 59). Failure to cure H. pylori infection has been noted for more than 20 to 30% of patients (37). In addition, several studies have found an eradication rate lower than 75% (6, 11, 59), and values as low as 25 to 45% have also been recently reported (22, 24). Thus, prolonged standard triple therapy for up to 2 weeks has been recommended (9, 23, 34), and in cases of eradication failure, a quadruple therapy with a proton pump inhibitor, bismuth salt, tetracycline, and metronidazole has been advised as a second-line therapy (8, 13, 44). More recently, sequential therapy (PPI and amoxicillin for 5 days, followed by PPI, clarithromycin, and tinidazole for 5 days) has become very attractive for clinical practice since impressive efficacy was seen (36, 73). However, broad adoption of this strategy as standard first-line therapy for H. pylori infection is still debatable because of impending validation in other geographic locations and studies to demonstrate efficacy superior to that of quadruple therapy, which is still considered a simpler regimen than sequential therapy (74). Of note, all the aforementioned therapies including sequential therapies employ multiple drugs and relatively complex regimens for the treatment of H. pylori infection, hence the search for new/better antibiotics.The bactericidal activity of amoxicillin results from interference with the interpeptide linkage of peptidoglycan by binding to penicillin binding proteins and blocking their function as transporters during cell wall synthesis. Clarithromycin, like other macrolides, binds to the 50S subunit of bacterial ribosomes, thus inhibiting translocation of tRNA during translation. Binding of clarithromycin to H. pylori ribosomes has been shown to be very strong and is irreversible (27). Finally, metronidazole is a 5-nitroimidazole drug whose mode of action is mediated by nitro metabolites, such as the radical anion (NO₂·⁻) and perhaps nitroso (RNO) and hydroxylamine (RNHOH) derivatives (39). Such metabolites have been demonstrated to cause DNA damage that results in cell death.H. pylori resistance to amoxicillin is very rare, while resistance to clarithromycin varies significantly and may range from 10 to 25% (14). However, in a recent study, it was reported that the first-line anti-H. pylori triple therapies containing clarithromycin failed in 7 to 49% of patients (19, 26), indicating the underlying significant increase in antimicrobial resistance and occurrence of refractory H. pylori infections (32, 50, 78). Currently, PPI-amoxicillin-metronidazole triple therapy is highly effective as a second-line regimen for the treatment of H. pylori infection in patients showing failure of the first-line regimen (PPI-amoxicillin-clarithromycin) (47). However, high rates of resistance have been reported for people with a history of metronidazole treatment (49). Given the immense challenge in rising antimicrobial resistance (38), there is an enormous need for new antibiotics for the treatment of H. pylori infection.One of the pharmacodynamic parameters most studied for antibiotics is the postantibiotic effect (PAE), which describes the suppression of bacterial growth after a short exposure of bacteria to an antimicrobial agent (29). From a clinical standpoint, PAE provides a rationale for the modification of the dosing interval of antimicrobials and could be significant for the optimization of a treatment regimen and the minimization of drug-induced adverse effects. Similarly, the success of intermittent dosing with drugs that exhibit short half-lives has been attributed to the presence of significant PAE. A long and/or positive PAE is considered an attractive characteristic for an effective new antibiotic.In the last decade, antimicrobial peptides (AMPs) have attracted attention as potential therapeutic agents mainly due to their ability to be promptly synthesized by the host upon induction and their capacity to subsequently lyse cell membranes of pathogens through direct interaction with them. Hence, AMPs are recognized as a cell-free host defense mechanism and are important component of the innate immune systems of living organisms, including plants (76), insects (30), amphibians (75), and mammals (80). These natural membrane-lytic peptides display immense diversity in terms of sequence, secondary structural motifs, charge (cationic and anionic), and/or the abundance of certain specific amino acids (16, 66). Despite the immense diversity, a common feature for cationic AMPs is that they all form amphipathic structures that allow them to bind to the membrane interface of microbes (5, 69). Peptides which are not cationic are known to exhibit less selectivity toward microbes than toward mammalian cells, since electrostatic interactions are critical for initial binding of the peptide to membrane containing anionic lipids (45).Oligomers of acylated lysines (OAKs) constitute a novel class of synthetic AMP mimics that consists of alternating amino acyl chains and cationic amino acids arranged to create an optimal molecular charge and hydrophobicity for enhanced potency (61, 65). This design has been reported to be advantageous over conventional AMPs by allowing the capacity for fine-tuning of the OAK structure to enhance potency against a broad spectrum of organisms while being devoid of apparent toxicity against mammalian cells (62, 64). This selective activity has been attributed to a design that lacks the secondary structures present in natural peptides (63) and to a mode of action that appears to target multiple sites, such as membranes and DNA (64). Circular dichroism studies of OAKs have demonstrated that they lack secondary structure in the presence of liposomes or hydrophobic media such as trifluoroethanol and sodium dodecyl sulfate (63). Additional characterization of OAKs with microbial pathogens other than H. pylori has demonstrated significant stability in the presence of serum and serum components and has shown no hemolysis of host erythrocytes (64). Two recent in vivo studies have also shown that administration of OAKs protected mice from an Escherichia coli lethal challenge (63, 64). Therefore, OAKs display characteristic features that are attractive for the development of a potent therapeutic drug.Given the increasing antibiotic resistance rates of H. pylori and the current complicated treatment regime, the need for new potent antibiotics has never been greater. Given the potent effect of OAKs on other pathogens, we investigated the in vitro antibacterial activities of five representative OAKs against H. pylori. The selected sequences belong to two distinct groups: one group consisted of C₁₂K-7α₈ and its shorter analog, C₁₂K-5α₈, two-well characterized compounds (15, 63, 64) both of which are known for potent activity against gram-negative bacteria (25); the second group consisted of the less-characterized OAKs C₁₂K-2β₁₂ and two shorter analogs, C₁₂-2β₁₂ and 2β₁₂, for which a preliminary study (62) predicted broad-spectrum activities at least for the longer analogs. Together, these representative OAKs were anticipated to provide a preliminary structure-activity assessment on the potential activity of OAKs against H. pylori. Our results indicate that four of the tested peptides show efficacy against the pathogen. Of these, C₁₂K-2β₁₂ demonstrated the most potent activity, was active against a spectrum of strains, and was remarkably stable at low pH and after exposure to extreme temperatures.     

Oxidative stress in Cohen diabetic rat model by high-sucrose, low-copper diet: inducing pancreatic damage and diabetes

Increased oxidative stress contributes to the development and progression of both types of diabetes mellitus (DM) and its complications. In the Cohen diabetic (CD) rats, a known genetic model of nutritionally induced type 2 DM, a high-sucrose, low-copper diet (HSD) induces within 4 weeks DM in the sensitive (CDs) rats but not in the resistant (CDr) rats. To assess the possible involvement of oxidative stress in the induction of DM, we studied the effect of HSD on the tissue levels of antioxidants and the extent of oxidative injuries in these animals in comparison with the regular outbred strain of nondiabetic Sabra rats. The specific aims were to investigate, at the onset of HSD-induced DM, (1) the extent of oxidative injury, as reflected by levels of malondialdehyde and protein carbonyl groups; (2) the overall antioxidant capacities to cope with increased oxidative stress; and (3) the modification of oxidative damage biomarkers in various tissues of CDr, CDs, and Sabra rats. Female CDs, CDr, and Sabra rats were fed regular diet or HSD for 4 to 5 weeks; and several parameters of oxidative injuries and antioxidant levels were determined. Changes in the levels of nonenzymatic low-molecular weight antioxidants (LMWAs) were measured by cyclic voltammetry and oxygen radical absorbance capacity. The activities of the antioxidant enzymes superoxide dismutase and catalase were measured. Oxidative damage was evaluated by measuring lipid peroxidation and protein oxidation. (1) In all animals fed HSD, the levels of LMWAs were decreased in most organs, although not plasma. (2) A significant difference was consistently found in antioxidant enzymes' activities in the pancreas of HSD-fed CDs rats, but not in other tissues. (3) The activities of superoxide dismutase and catalase and the levels of malondialdehyde and protein carbonyl group increased, whereas the levels of LMWAs decreased, in the pancreas of HSD-fed CDs rats. In the CD rats that develop DM when fed HSD, the pancreas showed susceptibility to oxidative stress-induced injuries. Thus, enhanced oxidative stress seems to play a role in the pathogenesis of DM in this strain.     

In vitro antiplasmodium effects of dermaseptin S4 derivatives

The 13-residue dermaseptin S4 derivative K(4)S4(1-13)a (P) was previously shown to kill intraerythrocytic malaria parasites through the lysis of the host cells. In this study, we have sought peptides that will kill the parasite without lysing the erythrocyte. To produce such peptides, 26 compounds of variable structure and size were attached to the N terminus of P and screened for antiplasmodium and hemolytic activities in cultures of Plasmodium falciparum. Results from this screen indicated that increased hydrophobicity results in amplified antiplasmodium effect, irrespective of the linearity or bulkiness of the additive. However, increased hydrophobicity also was generally associated with increased hemolysis, with the exception of two derivatives: propionyl-P (C3-P) and isobutyryl-P (iC4-P). Both acyl-peptides were more effective than P, with 50% growth inhibition at 3.8, 4.3, and 7.7 microM, respectively. The antiparasitic effect was time dependent and totally irreversible, implying a cytotoxic effect. The peptides were also investigated in parallel for their ability to inhibit parasite growth and to induce hemolysis in infected and uninfected erythrocytes. Whereas the dose dependence of growth inhibition and hemolysis of infected cells overlapped when cells were treated with P, the acyl-peptides exerted 50% growth inhibition at concentrations that did not cause hemolysis. Noticeably, the acyl derivatives, but not P, were able to dissipate the parasite plasma membrane potential and cause depletion of intraparasite potassium under nonhemolytic conditions. These results clearly demonstrate that the acyl-peptides can affect parasite viability in a manner that is dissociated from lysis of the host cell. Overall, the data indicate the potential usefulness of this strategy for development of selective peptides as investigative tools and eventually as antimalarial agents.     

Mechanisms mediating bactericidal properties and conditions that enhance the potency of a broad-spectrum oligo-acyl-lysyl

Previous studies have established the potential of the oligo-acyl-lysyl (OAK) concept in generating simple chemical mimics of host defense peptides (HDPs) with improved antimicrobial properties. We investigated the antibacterial properties of such an OAK, C(16(ω7))-KK-C(12)-K(amide), to obtain a better understanding of the complex mode(s) of action of cationic antibacterial peptides. The average MIC, determined against a multispecies panel of 50 strains, was 6 ± 5 μg/ml. However, although the OAK exerted an essentially dose-dependent bactericidal effect (time-kill curves typically exhibited 99% death within 2 h), marked differences in the killing rates occurred among inter- and intraspecies strains. Mechanistic comparison between equally sensitive and related strains revealed death of one strain to stem from the OAK's capacity to breach the cell membrane permeability barrier, whereas the death of the related strain resulted from the OAK's direct interference with DNA functions in vivo, without detectable membrane damage. These findings therefore support the notion that the antibacterial mechanism of action of a single HDP can vary among inter- and intraspecies strains. In addition, we present data illustrating the differential effects of environmental conditions (pH, ionic strength and temperature), on the OAK's antibacterial properties, and ultimately demonstrate potency enhancement (by orders of magnitude) through selection of optimal incubation conditions. Such attributes might be useful in a variety of antibacterial applications.