Discovery and development of a synthetic peptide derived from lactoferrin for clinical use

There is an urgent need to develop new antimicrobial drugs especially for combating the rise of infections caused by multi-resistant pathogens such as MRSA and VRSA. The problem of antibiotic resistant micro-organisms is expected to increase disproportionally and controlling of infections is becoming difficult because of the rapid spread of those micro-organisms. Primary therapy with classical antibiotics is becoming more ineffective. Combinational therapy of antibiotics with antimicrobial peptides (AMP's) has been suggested as an alternative approach to improve treatment outcome. Their unique mechanism of action and safety profile makes AMP's appealing candidates for simultaneous or sequential use in different cases of infections. In this review, for antimicrobial treatment the application of synthetic antimicrobial peptide hLF(1-11), derived from the first 11 amino acids of human lactoferrin is evaluated in both pre-clinical and clinical settings. Present information indicates that this derivate from lactoferrin is well tolerated in pre-clinical tests and clinical trials and thus hLF(1-11) is an interesting candidate for further exploration in various clinical indications of obscure infections, including meningitis. Another approach of using AMP's is their use in prevention of infections e.g. as coating for dental or bone implants or in biosensing applications or useful as infection specific radiopharmaceutical.     

The human lactoferrin-derived peptide hLF1-11 exerts immunomodulatory effects by specific inhibition of myeloperoxidase activity

Because of their ability to eliminate pathogens and to modulate various host immune responses, antimicrobial peptides are considered as candidate agents to fight infections by (antibiotic-resistant) pathogens. We recently reported that hLF1-11 (GRRRRSVQWCA), an antimicrobial peptide derived from the N terminus of human lactoferrin, displays diverse modulatory activities on monocytes, thereby enhancing their actions in innate immune responses. The aim of this study was to identify the cellular target of hLF1-11 that mediates these effects. Results revealed that hLF1-11 binds and subsequently penetrates human monocytes, after which it inhibits the enzymatic activities of myeloperoxidase (MPO). Moreover, a chemical inhibitor of MPO (aminobenzoic acid hydrazide) mimicked the effects of hLF1-11 on the inflammatory response by monocytes and on monocyte-macrophage differentiation. Computer-assisted molecular modeling predicted that hLF1-11 can bind to the edge of and within the crevice of the active site of MPO. Experiments with a set of hLF1-11 peptides with amino acid substitutions identified the stretch of arginines and the cysteine at position 10 as pivotal in these immunomodulatory properties of hLF1-11. We conclude that hLF1-11 may exert its modulatory effects on human monocytes by specific inhibition of MPO activity.     

Anti‐infectious activity of synbiotics in a novel mouse model of methicillin‐resistant Staphylococcus aureus infection

The anti‐infectious activity of synbiotics against methicillin‐resistant Staphylococcus aureus (MRSA) infection was evaluated using a novel lethal mouse model. Groups of 12 mice treated with multiple antibiotics were infected orally with a clinical isolate of MRSA at an inoculum of 10⁸ CFU on day 7 after starting the antibiotics. A dose of 400 mg/kg 5‐fluorouracil (5‐FU) was injected intraperitoneally on day 7 after the infection. A dose of 10⁸ CFU Bifidobacterium breve strain Yakult and 10 mg of galactooligosaccharides (GOS) were given orally to mice daily with the antibiotic treatment until day 28. The intestinal population levels of MRSA in the mice on multiple antibiotics were maintained stably at 10⁸ CFU/g of intestinal contents after oral MRSA infection and the subsequent 5‐FU treatment killed all the mice in the group within 14 days. B. breve administration saved most of the mice, but the synbiotic treatment saved all of the mice from lethal MRSA infection. The synbiotic treatment was effective for the treatment of intestinal infection caused by four MRSA strains with different toxin productions. There was a large difference among the six Bifidobacteria strains that were naturally resistant to the antibacterial drugs used. B. breve in combination with GOS is demonstrated to have valuable preventive and curative effects against even fatal MRSA infections.     

Echinococcus granulosus: development of an intermediate host mouse model for use in vaccination studies.

A mouse model has been developed to evaluate potential protective antigens which could render intermediate hosts resistant to a challenge infection with Echinococcus granulosus eggs. DBA/2J, CBA/J, Balb/cJ, C57/B16J and CF-1 mice were initially infected orally and parenterally with eggs, hatched eggs or activated oncospheres. Generally less than 1% of the oral dose established as cysts. Mean cysts counts were increased when Balb/cJ mice were injected intraperitoneally or intravenously with activated oncospheres. A challenge regime using 600 activated oncospheres injected intraperitoneally into adult Balb/cJ mice was subsequently adopted yielding means of 15-51 cysts per mouse. When activated oncospheres were injected intraperitoneally into Balb/cJ, DBA/2J and CF-1 mice, cysts were restricted to the peritoneal cavity. Activated oncospheres injected intravenously, however, lodged almost exclusively in the lung and thoracic cavity, except in DBA/2J mice where 55% lodged in the liver. This anatomical localization enabled the outcome of prior infection and challenge to be monitored separately. Prior infection rendered Balb/cJ mice fully resistant to subsequent challenge.     

Alternative stabilities of a proline-rich antibacterial peptide in vitro and in vivo

The proline-rich designer antibacterial peptide dimer A3-APO is currently under preclinical development for the treatment of systemic infections caused by antibiotic-resistant Gram-negative bacteria. The peptide showed remarkable stability in 25% mouse serum in vitro, exhibiting a half-life of approximately 100 min as documented by reversed-phase chromatography. Indeed, after a 30-min incubation period in undiluted mouse serum ex vivo, mass spectrometry failed to identify any degradation product. The peptide was still a major peak in full blood ex vivo, however, with degradation products present corresponding to amino-terminal cleavage. When injected into mice intravenously, very little, if any unmodified peptide could be detected after 30 min. Nevertheless, the major early metabolite, a full single-chain fragment, was detectable until 90 min, and this fragment exhibited equal or slightly better activity in the broth microdilution antimicrobial assay against a panel of resistant Enterobactericeae strains. The Chex1-Arg20 metabolite, when administered three times at 20 mg/kg to mice infected with a sublethal dose (over LD(50)) of an extended spectrum beta-lactamase-producing Escherichia coli strain, completely sterilized the mouse blood, similar to imipenem added at a higher dose. The longer and presumably more immunogenic prodrug A3-APO, injected subcutaneously twice over a 3-wk period, did not induce any antibody production, indicating the suitability of this peptide or its active metabolite for clinical development.     

The human lactoferrin-derived peptide hLF1-11 primes monocytes for an enhanced TLR-mediated immune response

Earlier we reported that the peptide corresponding to the first eleven N-terminal amino acids of human lactoferrin (hLF1-11) is active against multi-drug resistant pathogens in mice. The mechanisms underlying this anti-infective activity remain unclear. Since hLF1-11 is ineffective against pathogens at physiological salt concentrations and hLF1-11 directs differentiation of monocytes toward a macrophage subset with enhanced effector functions, we investigated the effects of hLF1-11 on human and murine monocytes. Results revealed that human and murine monocytes exposed for 1 h to hLF1-11 and then stimulated with the Toll-like receptor (TLR)-ligand LPS for 18 h, displayed enhanced cytokine and chemokine production as compared to control (peptide-treated) monocytes. We also found that expression of mRNA, cell-surface receptor expression, and NF-κB activation by hLF1-11-exposed human monocytes were enhanced as compared to control (peptide-treated) monocytes. Furthermore, the kinetics of the cytokine production was unchanged as mRNA levels and protein levels paralleled the enhanced response of hLF1-11-exposed monocytes to LPS. The cytokine production by human monocytes in response to TLR4, TLR5, and TLR7 stimulation, but not to TLR2 stimulation, was elevated by hLF1-11. In concordance, translocation of NF-κB subunits to the nucleus was enhanced in hLF1-11-exposed monocytes after TLR stimulation, except for TLR2, as compared to control (peptide-exposed) monocytes. In conclusion, monocytes were primed by hLF1-11 for an enhanced inflammatory response upon TLR4, TLR5, and TLR7 stimulation, but not TLR2 stimulation. Such effects of hLF1-11 on monocyte reactivity should be taken into account when considering the clinical development of this peptide for a therapeutic intervention in patients.     

Histatin and lactoferrin derived peptides: antimicrobial properties and effects on mammalian cells

In order to analyze the clinical potential of two antimicrobial peptides, human lactoferrin 1-11 (hLF1-11) and synthetic histatin analogue Dhvar-5, we measured the killing effect on bacteria, and the potential toxicity on erythrocytes and bone cells. The antimicrobial activity was determined in a killing assay on six strains, including methicillin resistant Staphylococcus Aureus. The effect on human erythrocytes and MC3T3 mouse bone cells was measured with a hemolysis assay and a viability assay, respectively. Both hLF1-11 and Dhvar-5 dose-dependently killed all bacterial strains, starting at concentrations of 6 μg/mL. hLF1-11 had no effect on mammalian cells at concentrations up to 400 μg/mL, but Dhvar-5 induced significant hemolysis (37% at 200 μg/mL) and bone cell death (70% at 400 μg/mL). This indicates that both peptides are able to kill various resistant and non-resistant bacteria, but Dhvar-5 may exert a cytotoxic effect on host cells at higher concentrations.     

Passive immunity modulates genetic reassortment between rotaviruses in mixedly infected mice.

Genetic reassortment between simian rotavirus SA11 and rhesus rotavirus (RRV) occurs with high frequency following mixed infection of nonimmune suckling mice (J. L. Gombold and R. F. Ramig, J. Virol. 57:110-116, 1986). We examined the effects of passively acquired homotypic or heterotypic immunity on reassortment in vivo. Passively immune suckling mice obtained from dams immune to either serotype 3 simian rotavirus (SA11) or serotype 6 bovine rotavirus (NCDV) were infected orally with either SA11 or RRV or a mixture of SA11 and RRV (both serotype 3 viruses). At various times postinfection, signs of disease were noted and the intestines of individual mice were removed and homogenized for titration of infectious virus and isolation of progeny plaques. Electrophoresis of genomic RNA was used to identify reassortants among the viral progeny isolated from infected animals. No reassortants (less than 0.45%) were detected among 224 clones examined from mixedly infected, homotypically immune mice. Twenty-nine reassortants (10.66%) were identified among 272 progeny clones from mixedly infected, heterotypically immune mice. Thus, reassortment was reduced more than 50-fold by homotypic immunity and approximately threefold by heterotypic immunity compared with prior data obtained from mixed infections of nonimmune mice. In addition, reassortment between SA11 and RRV in nonimmune mice was shown to be dependent on the virus dose. Taken together, these results suggest that immune responses may modulate the frequency of reassortment by reducing the effective multiplicity of infection (by neutralization or other immune mechanisms), thereby preventing efficient mixed infection of enterocytes.     

Dramatic effect of the tridentate ligand on the stability of 99mTC "3 + 1" oxo complexes bearing arylpiperazine derivatives

Mixed-ligand model complexes of general formula [(99m)Tc(O)(kappa(3)-PNX)(kappa(1)-SPh))] [X = O (1a), S (2a)] were prepared in a one-step procedure from [(99m)TcO(4)(-)] using stannous chloride as reducing agent. Stability studies and challenge experiments with glutathione showed that complex 2a presented promising features for pursuing animal studies. The activity in the brain (% dose injected/organ) at 5 min (0.14% +/- 0.03) and 120 min (0.11% +/- 0.02) pi encouraged the synthesis of several mixed-ligand "3 + 1" oxo complexes of general formula [M(O)(kappa(3)-PNS)(kappa(1)-SL))] (M = (99m)Tc, 3a-6a, Re, 3-6), in which the tridentate ligand is the heterofunctionalized phosphine 2-(diphenylphosphanyl)-N-(2-thioethyl)benzamide (PNS) and the co-ligands are different arylpiperazine derivatives (HSL1-HSL4). The (99m)Tc complexes have been characterized by comparison of their retention times in the HPLC chromatogram (gamma-detection) with the retention times of the analogous Re complexes (UV detection at 254 nm). The (99m)Tc complexes, obtained with radiochemical purity higher than 95%, after HPLC purification, are stable in saline, 0.01 M PBS (pH 7.4), rat plasma (4 h, 37 degrees C), and glutathione (10 mM solutions, 2h, 37 degrees C). Binding affinity and selectivity for 5-HT(1A) receptors (relative to the 5-HT(2A) receptor) were determined, complex 5 demonstrating the best values (IC(50) for the 5-HT(1A) 2.35 +/- 0.02 nM; competitor 5-HT(2A) 372 +/- 11 nM). Biodistribution and stability studies in mice indicated a preferred hepatobiliary excretion, a high in vivo stability, but a poor brain uptake.     

Synthetic peptides derived from human antimicrobial peptide ubiquicidin accumulate at sites of infections and eradicate (multi-drug resistant) Staphylococcus aureus in mice

The presence and antimicrobial activity of antimicrobial peptides (AMPs) has been widely recognized as an evolutionary preserved part of the innate immune system. Based on evidence in animal models and humans, AMPs are now positioned as novel anti-infective agents. The current study aimed to evaluate the potential antimicrobial activity of ubiquicidin and small synthetic fragments thereof towards methicillin resistant Staphylococcus aureus (MRSA), as a high priority target for novel antibiotics. In vitro killing of MRSA by synthetic peptides derived from the alpha-helix or beta-sheet domains of the human cationic peptide ubiquicidin (UBI 1-59), allowed selection of AMPs for possible treatment of MRSA infections. The strongest antibacterial activity was observed for the entire peptide UBI 1-59 and for synthetic fragments comprising amino acids 31-38. The availability, chemical synthesis opportunities, and size of these small peptides, combined with their strong antimicrobial activity towards MRSA make these compounds promising candidates for antimicrobial therapy and detection of infections in man.     

Efficacy of enterocin S760 in treatment of mice with anthrax infection due to Bacillus anthracis M-71

The therapeutic efficacy of enterocin S760, a broad spectrum antimicrobial peptide produced by Enterococcus faecium LWP760 was tested on mice infected with Bacillus anthracis M-71 to induce anthrax (second Tsenkovsky's vaccine). Intraperitoneal four-, two- or one-fold administration of the peptide in a dose of 25 mg/kg for 10 days for prophylactic (1 hour after the contamination) and therapeutic (24 hours after the contamination) purposes prevented or cured the infection in 90-100% of the mice versus the 100-percent lethality in the control (untreated animals). The antimicrobial activity of enterocin S760 against B. anthracis M-71 in vivo correlated with activity in vitro. Enterocin S760 is considered a novel promising antimicrobial for the treatment of grampositive and gramnegative infections.     

The tissue distribution and the pattern of excretion of [14C]-13-labeled 12, 13-epoxytrichothec-9-ene in mice and rats

The distribution in the mouse tissues of 13-[14C]-12,13-epoxtrichothec-9-ene administered intravenously was determined by whole-body autoradiography and by tracing the radioactivity of the tissues oxidized in an Auto Sample Oxidizer. The appearance of the label in urine and feces was also followed by the tracer technique. The distributions of radioactivity in tissues as determined by the two methods were almost identical. On the autoradiograms of mice killed 10 min after the injection, marked blackening of the film was observed at the sites corresponding to the liver, kidney, and bladder with urine, and much less darkening at other sites. The radioactivities contained in the liver, kidney, urine and small intestine were 13.3, 2.3, 2.6 and 10.2% of the dose, respectively. The labeled toxin was rapidly excreted into urine and feces, 56.0 and 4.9% in 6 hr and 66.7 and 28.0% in 24 hr after injection, respectively. Oral administration of the labeled toxin to mother mice resulted in the appearance of radioactivity in the stomach contents of 7-day suckling mice, thus demonstrating indirectly the secretion of the toxin into the milk. An attempt to show a respiratory route of excretion in rats given the radioactive compound orally or intravenously failed to detect any radioactivity in the expired CO2 collected for 6 hr, suggesting that the 14C in the epoxy ring was intact.     

Substitution of Gly with Ala enhanced the melanoma uptake of technetium-99m-labeled Arg-Ala-Asp-conjugated alpha-melanocyte stimulating hormone peptide

The purpose of this study was to determine the melanoma targeting property of (99m)Tc-RAD-Lys-(Arg(11))CCMSH in B16/F1 melanoma-bearing C57 mice and compare with (99m)Tc-RGD-Lys-(Arg(11))CCMSH we previously reported. (99m)Tc-RAD-Lys-(Arg(11))CCMSH exhibited rapid and high tumor uptake (19.91±4.02% ID/g at 2h post-injection) in B16/F1 melanoma-bearing C57 mice. The tumor uptake of (99m)Tc-RAD-Lys-(Arg(11))CCMSH was 1.51, 1.34 and 1.43 times the tumor uptake of (99m)Tc-RGD-Lys-(Arg(11))CCMSH at 0.5, 2 and 4h post-injection, respectively. Flank B16/F1 melanoma lesions were clearly imaged at 2h post-injection using (99m)Tc-RAD-Lys-(Arg(11))CCMSH as an imaging probe. The substitution of Gly with Ala significantly enhanced the melanoma uptake of (99m)Tc-RAD-Lys-(Arg(11))CCMSH compared to (99m)Tc-RGD-Lys-(Arg(11))CCMSH in B16/F1 melanoma-bearing C57 mice, providing a new insight into the design of α-MSH peptides for melanoma targeting.     

Patterns of immunoenhancement and suppression induced by Chlamydia trachomatis in vivo and in vitro

We investigated the cycle of immune enhancement and suppression seen in mice infected with Chlamydia trachomatis by using in vivo and in vitro model systems. BALB/c mice injected intravenously with chlamydia reveal a three- to seven-fold increase in numbers of plaque-forming cells producing antibodies against sheep red blood cells (SRBC), when immunized with SRBC 0 to 5 days after chlamydia infection. When mice are injected with SRBC 10 to 15 days after initial chlamydia infection, the specific anti-SRBC plaque-forming cell response is suppressed two- to three-fold. In vitro, low numbers (2 to 5 X 10(6) bacteria/ml) of chlamydia stimulate potent proliferative responses by B lymphocytes while high numbers (25 X 10(6) bacteria/ml) of bacteria generate strong, general T suppressor activity. This model has important implications for regulation of immune responses that arise at different times during chlamydial infections, as well as for the potential effectiveness of chlamydial vaccines.     

Targeted photodynamic therapy of established soft-tissue infections in mice.

The worldwide rise in antibiotic resistance necessitates the development of novel antimicrobial strategies. Although many workers have used photodynamic therapy (PDT) to kill bacteria in vitro, the use of this approach has seldom been reported in vivo in animal models of infection. We have previously described the first use of PDT to treat excisional wound infections by Gram-(-) bacteria in living mice. However, these infected wound models involved a short timespan between infection (30 min) and treatment by PDT. We now report on the use of PDT to treat an established soft-tissue infection in mice. We used Staphylococcus aureus stably transformed with a Photorhabdus luminescenslux operon (luxABCDE) that was genetically modified to be functional in Gram-(+) bacteria. These engineered bacteria emitted bioluminescence, allowing the progress of the infection to be monitored in both space and time with a low light imaging charge-coupled device (CCD) camera. One million cells were injected into one or both thigh muscles of mice that had previously been rendered neutropenic by cyclophosphamide administration. Twenty-four hours later, the bacteria had multiplied more than one hundredfold; poly-L-lysine chlorin e6 conjugate or free chlorin e6 was injected into one area of infected muscle and imaged with the CCD camera. Thirty minutes later, red light from a diode laser was delivered as a surface spot or by interstitial fiber into the infection. There was a light dose dependent loss of bioluminescence (to <5% of that seen in control infections) not seen in untreated infections or those treated with light alone, but in some cases, the infection recurred. Treatment with conjugate alone led to a lesser reduction in bioluminescence. Infections treated with free chlorin e6 responded less well and the infection subsequently increased over the succeeding days, probably due to PDT-mediated tissue damage. PDT-treated infected legs healed better than legs with untreated infections. This data shows that PDT may have applications in drug-resistant soft-tissue infections.     

ENHANCEMENT OF SALMONELLOSIS AND EMERGENCE OF SECONDARY INFECTION IN MICE EXPOSED TO COLD.

Miraglia, Gennaro J. (Bryn Mawr College, Bryn Mawr, Pa.) and L. Joe Berry. Enhancement of salmonellosis and emergence of secondary infection in mice exposed to cold. J. Bacteriol. 84:1173-1180. 1962.-The ld(50) dose for mice of Salmonella typhimurium, strain RIA, is 4.1 x 10(5) for animals individually housed without bedding and maintained at 25 C. It is 3.8 x 10(3) for animals similarly housed but kept at 5 C. An intravenous injection of 0.1 ml of Proferrin 2 hr prior to infection with RIA lowers the ld(50) to 4.9 x 10(3) and to 4.0 x 10(1) for mice kept, respectively, at 25 and at 5 C. Low environmental temperature and "blockade" of the reticuloendothelial system (RES) lower the resistance of mice to about the same degree, but low temperature and RES impairment together lower resistance as if each were acting independently. No effect of cold could be detected in mice infected with the highly virulent SR-11 strain of S. typhimurium, since all animals died after infection with only a few cells. Mice that were natural carriers of salmonellae, as judged by fecal discharge, were highly resistant to challenge and responded to cold in a manner similar to normal mice infected with RIA. Strain RIA could be isolated from the tissues of infected animals with greater frequency and persisted longer in mice maintained at 5 C than those at 25 C. Staphylococci were isolated from livers of animals that survived salmonella infection for 14 days at 5 C, and the incidence of staphylococci was proportional to the number of salmonellae injected. At 25 C, only a small percentage of mice had staphylococci in tissues and these occurred independent of the infectious dose of salmonellae. These observations were made on normal mice infected with RIA and on carrier mice infected with SR-11.     

Purification and biochemical characterization of an 11 000-dalton beta-bungarotoxin

The chromatographic separation and biochemical characterization of a beta-bungarotoxin is described. This toxin is isolated as the most basic eluting protein of Bungarus multicinctus venom when separated by column chromatography on CM-Sephadex C-25. The protein migrated as a single band on pH 4.3 and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight of this toxin was estimated to be 10 000 +/- 1000 by analytical sedimentation analysis. This value was consistent with the electrophoretic mobility of the toxin in SDS-polyacrylamide gels. The amino acid composition of this 11 000-dalton beta-bungarotoxin was similar to that of the 22 000-dalton beta-bungarotoxin previously reported (Lee et al. (1972) J. Chromatogr. 72, 71--82; Kelly, R.B. and Brown, III, F.R. (1974) J. Neurobiol. 5, 135--150; Kondo et al. (1978) J. Biochem. Tokyo 83, 91--99), suggesting that the 11 000-dalton toxin may be one of the polypeptide chains of the larger toxin. The 11 000-dalton beta-bungarotoxin was toxic to mice when injected intravenously. Animals that received lethal doses exhibited hyperexcitability followed by ataxia, convulsions, and death. The minimum lethal dose was 0.12 microgram/g body weight. This beta-bungarotoxin exhibited Ca2+-dependent phospholipase A activity comparable to that of the 22 000-dalton beta-bungarotoxin. The enzyme exhibited phospholipid substrate specificity in the rank order of phosphatidyl-choline, phosphatidylserine, phosphatidylethanolamine, and phosphatidyl-inositol. The enzyme activity was destroyed by boiling for 3 min at pH 8.6. In addition, an enzymatically inactive quantity of the 11 000-dalton toxin, equivalent to five times the minimum lethal dose of enzymatically active toxin, was not lethal when injected into mice. To test whether phospholipase A activity is responsible for lethality, bee venom phospholipase A2 was injected into mice at similar and greater concentrations with no toxic effect. Thus, while phospholipase A activity may be required for the lethal effect of the 11 000-dalton beta-bungarotoxin, the specificity of action of the toxin is not determined by its enzyme activity.     

Deletion of selenoprotein P upregulates urinary selenium excretion and depresses whole-body selenium content

Deletion of the mouse selenoprotein P gene (Sepp1) lowers selenium concentrations in many tissues. We examined selenium homeostasis in Sepp1(-/-) and Sepp1(+/+) mice to assess the mechanism of this. The liver produces and exports selenoprotein P, which transports selenium to peripheral tissues, and urinary selenium metabolites, which regulate whole-body selenium. At intakes of selenium near the nutritional requirement, Sepp1(-/-) mice had whole-body selenium concentrations 72 to 75% of Sepp1(+/+) mice. Genotype did not affect dietary intake of selenium. Sepp1(-/-) mice excreted in their urine approximately 1.5 times more selenium in relation to their whole-body selenium than did Sepp1(+/+) mice. In addition, Sepp1(-/-) mice gavaged with (75)SeO(2-)(3) excreted 1.7 to 2.4 times as much of the (75)Se in the urine as did Sepp1(+/+) mice. These findings demonstrate that deletion of selenoprotein P raises urinary excretion of selenium. When urinary small-molecule (75)Se was injected intravenously into mice, over 90% of the (75)Se appeared in the urine within 24 h, regardless of selenium status. This shows that urinary selenium is dedicated to excretion and not to utilization by tissues. Our results indicate that deletion of selenoprotein P leads to increased urinary selenium excretion. We propose that the absence of selenoprotein P synthesis in the liver makes more selenium available for urinary metabolite synthesis, increasing loss of selenium from the organism and causing the decrease in whole-body selenium and some of the decreases observed in tissues of Sepp1(-/-) mice.     

Photodynamic antimicrobial activity of new porphyrin derivatives against methicillin resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Methicillin resistant Staphylococcus aureus (MRSA) with multiple drug resistance patterns is frequently isolated from skin and soft tissue infections that are involved in chronic wounds. Today, difficulties in the treatment of MRSA associated infections have led to the development of alternative approaches such as antimicrobial photodynamic therapy. This study aimed to investigate photoinactivation with cationic porphyrin derivative compounds against MRSA in in-vitro conditions. In the study, MRSA clinical isolates with different antibiotic resistance profiles were used. The newly synthesized cationic porphyrin derivatives (P_M, P_E, P_PN, and P_PL) were used as photosensitizer, and 655 nm diode laser was used as light source. Photoinactivation experiments were performed by optimizing energy doses and photosensitizer concentrations. In photoinactivation experiments with different energy densities and photosensitizer concentrations, more than 99% reduction was achieved in bacterial cell viability. No decrease in bacterial survival was observed in control groups. It was determined that there was an increase in photoinactivation efficiency by increasing the energy dose. At the energy dose of 150 J/cm² a survival reduction of over 6.33 log₁₀ was observed in each photosensitizer type. While 200 μM P_M concentration was required for this photoinactivation, 12.50 μM was sufficient for P_E, P_PN, and P_PL. In our study, antimicrobial photodynamic therapy performed with cationic porphyrin derivatives was found to have potent antimicrobial efficacy against multidrug resistant S. aureus which is frequently isolated from wound infections.     

Age-dependent resistance to Cryptosporidium muris (strain MCR) infection in golden hamsters and mice

An age-dependent aspect of resistance to Cryptosporidium muris (strain MCR) infection was monitored in Syrian golden hamsters, Mesocricetus auratus, at 1-, 5- and 10-week of age and in ICR mice. Mus musculus, at 3-, 12-, and 15-week of age orally inoculated with a single dose of 2 x 10(6) oocysts, respectively. The prepatent periods for both animals were similar, independent of age, but the patency was significantly longer in younger hamsters (P < 0.001) and a long tendency in younger mice. Hamsters infected at 1-week of age excreted about 10 times higher oocysts than those at 5- and 10-week of age. However, the total oocyst output was similar among mice of different ages. There was a good correlation between the length of the patency and the total oocyst output in hamsters (R = 0.9646), but not in mice (R = 0.4561). The immunogenicity of the parasite to homologous challenge infections was very strong in hamsters and relatively strong in mice. These results indicate that acquired resistance to C. muris infection is age-related and the innate resistance is independent of age of hamsters, and that both innate and acquired resistance, on the contrary, are irrespective of age of mice.