[Transfusion in autologous and allogenic hematopoietic stem cell transplant: Guidelines from the Francophone Society of Bone Marrow Transplantation and Cellular Therapy (SFGM-TC)]. / Transfusion dans l'autogreffe et l'allogreffe de cellules souches hématopoïétiques chez l'adulte et l'enfant : recommandations de la Société francophone de greffe de moelle et de thérapie cellulaire (SFGM-TC).

The recommendations of the French Health and Drug Safety Authorities (HAS/ANSM-Haute Autorité de santé/Agence nationale de sécurité du médicament) are known, but there are always new developments underway. With regards to CMV suppression, there is the introduction of platelet glycoprotein Ia and the Intercept (Amotosalem+UVA) inactivation method which addresses bacterial risk. The irradiation of platelets is included in the recommendations to ensure HEV-negative plasma post allograft. In terms of blood transfusion safety, these measures as well as the broader spectrum of Ia, particularly for arboviruses, are a real breakthrough. The survey conducted in clinical services and the services providing blood products for transfusion along with a literature review have shown that several improvements need to be made. The first is a reduction of transfusions of concentrated red blood cells with introduction at a threshold of 7g/dL during hospitalization of patients without a fragile clinical status. The second improvement would address transfusion of refractory thrombocytopenia, encouraging an increase in discussion between clinicians and those conducting the transfusion in order to consider different etiologies and to identify appropriate care protocols. Third would be the need for the transmission of information between the transplantation doctors and blood transfusion specialists in order to define an approach to transfusion care adapted to the patient's situation. It is important to inform and educate patients about transfusion protocols post allotransplant or autotransplant. It must be clearly communicated to patients that they should always have on their person their blood group documentation. This is especially true when receiving care for a hemopathy or an autologous transplant. If undergoing an allogeneic transplant, patients should also carry transfusion guidelines post autotransplant or post allotransplant along with the phone numbers for the stem cell transplantation department and the blood transfusion center responsible for their care.

Evaluation of liposomal carnosine in adjuvant arthritis.

Liposomal carnosine could overcome the problems associated with direct application of this peptide. Anti-inflammatory and antioxidant effects of liposomal and non-liposomal carnosine in adjuvant arthritis were compared. The experiments were done on healthy animals, untreated arthritic animals, arthritic animals with oral administration of carnosine, and with subcutaneous administration of liposomal carnosine, both administered in the same daily dose of 150 mg/kg b.w. during 28 days. Carnosine reduced hind paw volume on day 28. Both forms markedly decreased interleukin-1ß, matrix metalloproteinase-9 and monocyte chemoattractant protein-1 (MCP-1) in plasma on day 14. Only liposomal carnosine reduced significantly MCP-1. Malondialdehyde, 4-hydroxynonenal, resistance to Fe2+-induced oxidation and protein carbonyls were significantly corrected after administration of any form of carnosine. Liposomal carnosine corrected more effectively the oxidative stress in plasma than did carnosine. In brain tissue, our results showed protective ability of both forms of carnosine against oxidation of proteins and lipids. They also corrected the resistance to Fe2+-induced oxidation in arthritic animals. We found that only liposomal carnosine decreased the mRNA expression of inducible nitric oxide synthase in cartilage tissue. It can be concluded that the liposomal drug-delivery system is improving the pharmacological properties of carnosine administered in arthritis.

[Assessment and management of post-transplant iron overload: Guidelines of the Francophone Society of Marrow Transplantation and Cellular Therapy (SFGM-TC)]. / Évaluation et prise en charge de la surcharge en fer post-greffe : recommandations de la Société francophone de greffe de moelle et de thérapie cellulaire (SFGM-TC).

To harmonize clinical practice in hematopoietic stem cell transplantation, the Francophone Society of Bone Marrow Transplantation and Cell Therapy (SFGM-TC) set up the sixth annual series of workshops which brought together practitioners from all member centers and took place in September 2015 in Lille. The main aim of this session was to describe the impact, evaluation and treatment of post-transplant iron overload.

Influence of amine-modified poly(vinyl alcohol)s on vibrating-membrane nebulizer performance and lung toxicity.

A suitable aerosol droplet size and formulation output rate is essential for the therapy of lung diseases under application of nebulizers. The current study investigated the potential of amine-modified poly(vinyl alcohol)s as excipients for inhalation delivery. A change of conductivity (effective at <0.1mg/ml) and viscosity (effective at >0.1mg/ml) of samples that were supplemented with charge-modified polymers had a significant influence on the generated droplet size (shift from ~8 to ~4 µm) and formulation throughput rate (shift from ~0.2 to ~1.0 g/min), where polymers with a higher amine density (and molecular weight) showed an elevated activity. Biocompatibility assessment of polymers in A549 cells and an isolated lung model resulted in cell lysis and lung edema formation dependent on the type (degree of amine substitution) and dose of polymer applied. Suitable compositions and concentrations of amine-modified poly(vinyl alcohol)s were identified with respect to an optimized nebulizer performance and acceptable biocompatibility. Charge-modified polymers represent novel excipients with potential to improve inhalation therapy.

A chiral, low-cytotoxic [Ni15]-wheel complex.

A new chiral [Ni15] complex with a Schiff-base ligand derived from o-vanillin and L-glutamic acid is presented, emphasizing the properties relevant for biology and materials science. The formation of the complex molecules in solution is confirmed by AFM and dynamic light scattering studies. The compound is weakly antiferromagnetic with considerable admixture of excited states, comprising negligibly interacting [Ni3] units. Studies of the interactions with two cell lines indicate low cytotoxicity.

Functionalized polyglycerol amine nanogels as nanocarriers for DNA.

Polyglycerol based nanogels (nPG) can function as cellular delivery systems. These nPGs are synthesized with different amine densities (nPG amines) by acid-catalyzed epoxide-opening polymerization using a mini-emulsion approach and surface modification. All the synthesized nanogels are characterized by NMR, dynamic light scattering, and ζ-potential, showing slightly positive surface charge and a homogeneous size of ≈100 nm. The use of these systems for delivery applications is demonstrated with regard to polyplex formation, cytotoxicity, and cellular uptake studies. It is depicted that the CE50 value of the high loaded nPG amines is eight times higher than the low loaded ones. The influence of the amine loading percentage on the nanogel and the effects of polyvalency in these architecture is discussed.

Cell attachment on poly(3-hydroxybutyrate)-poly(ethylene glycol) copolymer produced by Azotobacter chroococcum 7B.

BACKGROUND: The improvement of biomedical properties, e.g. biocompatibility, of poly(3-hydroxyalkanoates) (PHAs) by copolymerization is a promising trend in bioengineering. We used strain Azotobacter chroococcum 7B, an effective producer of PHAs, for biosynthesis of not only poly(3-hydroxybutyrate) (PHB) and its main copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), but also alternative copolymer, poly(3-hydroxybutyrate)-poly(ethylene glycol) (PHB-PEG). RESULTS: In biosynthesis we used sucrose as the primary carbon source and valeric acid or poly(ethylene glycol) 300 (PEG 300) as additional carbon sources. The chemical structure of PHB-PEG and PHB-HV was confirmed by 1H nuclear-magnetic resonance (1H NMR) analysis. The physico-chemical properties (molecular weight, crystallinity, hydrophilicity, surface energy) and surface morphology of films from PHB copolymers were studied. To study copolymers biocompatibility in vitro the protein adsorption and COS-1 fibroblasts growth on biopolymer films by XTT assay were analyzed. Both copolymers had changed physico-chemical properties compared to PHB homopolymer: PHB-HV and PHB-PEG had less crystallinity than PHB; PHB-HV was more hydrophobic than PHB in contrast to PHB-PEG appeared to have greater hydrophilicity than PHB; whereas the morphology of polymer films did not differ significantly. The protein adsorption to PHB-PEG was greater and more uniform than to PHB and PHB-PEG copolymer promoted better growth of COS-1 fibroblasts compared with PHB homopolymer. CONCLUSIONS: Thus, despite low EG-monomers content in bacterial origin PHB-PEG copolymer, this polymer demonstrated significant improvement in biocompatibility in contrast to PHB and PHB-HV copolymers, which may be coupled with increased protein adsorption and hydrophilicity of PEG-containing copolymer.

The terpolymer produced by Azotobacter chroococcum 7B: effect of surface properties on cell attachment.

The copolymerization of poly(3-hydroxybutyrate) (PHB) is a promising trend in bioengineering to improve biomedical properties, e.g. biocompatibility, of this biodegradable polymer. We used strain Azotobacter chroococcum 7B, an effective producer of PHB, for biosynthesis of not only homopolymer and its main copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), but also novel terpolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-poly(ethylene glycol) (PHB-HV-PEG), using sucrose as the primary carbon source and valeric acid and poly(ethylene glycol) 300 (PEG 300) as additional carbon sources. The chemical structure of PHB-HV-PEG was confirmed by (1)H nuclear-magnetic resonance analysis. The physico-chemical properties (molecular weight, crystallinity, hydrophilicity, surface energy) of produced biopolymer, the protein adsorption to the terpolymer, and cell growth on biopolymer films were studied. Despite of low EG-monomers content in bacterial-origin PHB-HV-PEG polymer, the terpolymer demonstrated significant improvement in biocompatibility in vitro in contrast to PHB and PHB-HV polymers, which may be coupled with increased protein adsorption, hydrophilicity and surface roughness of PEG-containing copolymer.

The use of isothermal titration calorimetry and molecular dynamics to show variability in DNA transfection performance.

The mechanism causing variability in DNA transfection efficacy for low-molecular-weight pDMAEMA (poly(2-(dimethylamino)ethyl methacrylate) and pDMAEMA-b-pHEMA (poly(2-(dimethyl amino)ethylmethacrylate)-block-poly(2-hydroxyl methacrylate)) has so far remained unclear, apart from the evidence of beneficial effects of the pHEMA grafting. This study has explicitly characterized the electrostatically driven self-assembly process of linear polymethacrylate polymers with DNA-generating nanocarriers for efficient gene transfection. Isothermal titration calorimetry (ITC) showed clear differences in binding-heat profiles of homo-polycationic and pHEMA grafted polymers with DNA. Polyethylene imine, a branched polycationic polymer of 25kDa with high transfection potential that has previously been successfully used in transfection experiments, demonstrated a heat flow profile that was partly identical to pDMAEMA-b-pHEMA. Computational molecular dynamics (MD) simulated the folding process of polymer in water from a linear to a coiled state: homo-pDMAEMA and pHEMA grafts reduced their overall positive charge accessibility upon folding, down to 45% and 63%, respectively. The homo-pDMAEMA formed the globular conformation more preferably than pHEMA grafts, thus impeding electrostatic interaction with DNA. These findings substantiate the known disadvantage of low-molecular-weight linear polymers compared to higher-molecular-weight polymers in transfection performance; here we have disclosed the ability of a non-cationic chain elongation to be beneficial for the self-assembly process. The combination of MD and ITC has proved to be a suitable approach for carrier-payload interaction studies and may be used to predict the efficacy of a polymer as a nanocarrier from the flexibility of its structure.

Optimising the self-assembly of siRNA loaded PEG-PCL-lPEI nano-carriers employing different preparation techniques.

Amphiphilic cationic block copolymers consisting of poly(ethylene glycol), poly(ε-caprolactone) and poly(ethylene imine) spontaneously assemble to nano-sized particulate carriers, which can be utilised for complexation of nucleic acids (small-interfering RNA), representing a multifunctional vector system, designed for drug and gene delivery. Apart from polymer design and charge ratio, a more homogeneous complexation could lead to a more uniform charge distribution, subsequently increasing colloidal stability, RNA protection and consequently transfection efficiency. Microfluidic mixing techniques, bringing cationic polymer and nucleic acid together at a constant ratio during the entire mixing process, have the potential for a gentler complexation. In the present study carriers were prepared by a solvent displacement technique. In a first step complex size for addition of RNA during (addition to the aqueous or the organic phase) or after (classical pipetting or microfluidic mixing) carrier assembly was determined by dynamic light scattering. Suitable N/P ratios have previously been selected by measuring size and ζ-potential as a function of N/P. Subsequently, for the most promising techniques (loading after assembly), colloidal stability, the ability to protect RNA as well as transfection efficiency in vitro were compared. Finally, parameters for the superior microfluidic mixing process were optimised with the help of a central composite design. Generally, gentler loading leads to more homogeneous complexes. Hence, possibly due to a more consistent surface coating, loading after carrier assembly resulted in less aggregation. In comparison to bulk mixing, microfluidic assembly exhibited smaller diameters (179±11 vs. 230±97nm), less heterogeneity (PDI=0.205±0.028 vs. 0.353±0.161), enhanced RNA protection (RNA recovery=30.6±1.0 vs. 15.4±1.4%) as well as increased transfection performance (34.8±1.5 vs. 24.5±2.2% knockdown). Therefore, microfluidic complexation represents a reproducible alternative for formulating gene delivery carriers with superior colloidal stability, RNA protection and transfection efficiency.

Amphiphilic and biodegradable hy-PEI-g-PCL-b-PEG copolymers efficiently mediate transgene expression depending on their graft density.

Novel biodegradable amphiphilic copolymers hy-PEI-g-PCL-b-PEG were prepared by grafting PCL-b-PEG chains onto hyper-branched poly(ethylene imine) as non-viral gene delivery vectors. Our investigations focused on the influence of graft densities of PCL-b-PEG chains on physico-chemical properties, DNA complexation and transfection efficiency. We found that the transfection efficiencies of these polymers increased at first towards an optimal graft density (n=3) and then decreased. The buffer-capacity-test showed almost exactly the same tendency as transfection efficiency. Cytotoxicity (MTT-assay) depended on the cooperation of PEG molecular weight and graft density of PCL-b-PEG chains. With increasing the graft density, cytotoxicity, zeta-potential, affinity with DNA, stability of the polyplexes and CMC-values were reduced strongly and regularly. Increasing the excess of polymer over DNA was shown to result in a decrease of the observed particle size to 100-200 nm.

The effect of environmental pH on polymeric transfection efficiency.

Although polymers, polyplexes, and cells are exposed to various extracellular and intracellular pH environments during polyplex preparation and polymeric transfection, the impact of environmental pH on polymeric transfection has not yet been investigated. This study aims to understand the influence of environmental pH on polymeric transfection by modulating the pH of the transfection medium or the culture medium. Changes in the extracellular pH affected polymeric transfection by way of complex factors such as pH-induced changes in polymer characteristics (e.g., proton buffering capacity and ionization), polyplex characteristics (e.g., size, surface charge, and decomplexation), and cellular characteristics (e.g., cellular uptake, cell cycle phases, and intracellular pH environment). Notably, acidic medium delayed endocytosis, endosomal acidification, cytosolic release, and decomplexation of polyplexes, thereby negatively affecting gene expression. However, acidic medium inhibited mitosis and reduced dilution of gene expression, resulting in increased transfection efficiency. Compared to pH 7.4 medium, acidic transfection medium reduced gene expression 1.6-7.7-fold whereas acidic culture medium enhanced transfection efficiency 2.1-2.6-fold. Polymeric transfection was affected more by the culture medium than by the transfection medium. Understanding the effects of extracellular pH during polymeric transfection may stimulate new strategies for determining effective and safe polymeric gene carriers.

Novel lynx spider toxin shares common molecular architecture with defense peptides from frog skin.

A unique 30-residue cationic peptide oxyopinin 4a (Oxt 4a) was identified in the venom of the lynx spider Oxyopes takobius (Oxyopidae). Oxt 4a contains a single N-terminally located disulfide bond, Cys4-Cys10, and is structurally different from any spider toxin studied so far. According to NMR findings, the peptide is disordered in water, but assumes a peculiar torpedo-like structure in detergent micelles. It features a C-terminal amphipathic α-helical segment (body; residues 12-25) and an N-terminal disulfide-stabilized loop (head; residues 1-11), and has an unusually high density of positive charge in the head region. Synthetic Oxt 4a was produced and shown to possess strong and broad-spectrum cytolytic and antimicrobial activity. cDNA cloning showed that the peptide is synthesized in the form of a conventional prepropeptide with an acidic prosequence. Unlike other arachnid toxins, Oxt 4a exhibits striking similarity with defense peptides from the skin of ranid frogs that contain the so-called Rana-box motif (a C-terminal disulfide-enclosed loop). Parallelism or convergence is apparent on several levels: the structure, function and biosynthesis of a lynx spider toxin are mirrored by those of Rana-box peptides from frogs.

Self-assembled biodegradable amphiphilic PEG-PCL-lPEI triblock copolymers at the borderline between micelles and nanoparticles designed for drug and gene delivery.

Amphiphilic PEG-PCL-PEI triblock copolymers self-assemble into nano-scaled, positively charged, multifunctional carriers, suitable for drug and gene delivery. A set of block copolymers with varying hydrophilic/hydrophobic ratio (systematically altered at the borderline of micelle and particle forming polymers) was synthesized, characterized and assembled into carriers. A detailed structural characterization in the liquid state of these assemblies was carried out: carrier size was determined using dynamic light scattering, cryogenic scanning electron microscopy and atomic force microscopy. Nuclear magnetic resonance analyses elucidated carrier's core-shell structure. ζ-potential and thickness of the hydrophilic outer polymer shell were determined by laser Doppler anemometry. Subsequently the impact of carrier's structure on its features (stability and toxicity) was investigated. Polymers hydrophilic in nature formed small (<40 nm) micelle-like carriers, whilst hydrophobic polymers aggregated to larger particle-like assemblies (>100 nm). Monitoring carrier size as a function of initial polymer concentration clarified different assembly mechanisms. Shell thickness, colloidal stability and toxicity were found to depend on the length of the hydrophilic polymer block. Due to controllable size, charge, stability and toxicity, this class of novel carriers is a promising candidate for prospective co-delivery of drugs and nucleic acids.

Biophysical characterization of hyper-branched polyethylenimine-graft-polycaprolactone-block-mono-methoxyl-poly(ethylene glycol) copolymers (hy-PEI-PCL-mPEG) for siRNA delivery.

A library of mono-methoxyl-poly(ethylene glycol)-block-poly(ε-caprolactone) (mPEG-PCL) modified hyperbranched PEI copolymers (hy-PEI-PCL-mPEG) was synthesized to establish structure function relationships for siRNA delivery. These amphiphilic block-copolymers were thought to provide improved colloidal stability and endosomal escape of polyplexes containing siRNA. The influence of the mPEG chain length, PCL segment length, hy-PEI molecular weight and the graft density on their biophysical properties was investigated. In particular, buffer capacity, complex formation constants, gene condensation, polyplex stability, polyplex size and zeta-potential were measured. It was found that longer mPEG chains, longer PCL segments and higher graft density beneficially affected the stability and formation of polyplexes and reduced the zeta-potential of siRNA polyplexes. Significant siRNA mediated knockdown was observed for hy-PEI25k-(PCL900-mPEG2k)(1) at N/P 20 and 30, implying that the PCL hydrophobic segment played a very important role in siRNA transfection. These gene delivery systems merit further investigation under in vivo conditions.

Disulfide-stabilized helical hairpin structure and activity of a novel antifungal peptide EcAMP1 from seeds of barnyard grass (Echinochloa crus-galli).

This study presents purification, activity characterization, and (1)H NMR study of the novel antifungal peptide EcAMP1 from kernels of barnyard grass Echinochloa crus-galli. The peptide adopts a disulfide-stabilized α-helical hairpin structure in aqueous solution and thus represents a novel fold among naturally occurring antimicrobial peptides. Micromolar concentrations of EcAMP1 were shown to inhibit growth of several fungal phytopathogens. Confocal microscopy revealed intensive EcAMP1 binding to the surface of fungal conidia followed by internalization and accumulation in the cytoplasm without disturbance of membrane integrity. Close spatial structure similarity between EcAMP1, the trypsin inhibitor VhTI from seeds of Veronica hederifolia, and some scorpion and cone snail toxins suggests natural elaboration of different functions on a common fold.

Novel class of spider toxin: active principle from the yellow sac spider Cheiracanthium punctorium venom is a unique two-domain polypeptide.

Venom of the yellow sac spider Cheiracanthium punctorium (Miturgidae) was found unique in terms of molecular composition. Its principal toxic component CpTx 1 (15.1 kDa) was purified, and its full amino acid sequence (134 residues) was established by protein chemistry and mass spectrometry techniques. CpTx 1 represents a novel class of spider toxin with modular architecture. It consists of two different yet homologous domains (modules) each containing a putative inhibitor cystine knot motif, characteristic of the widespread single domain spider neurotoxins. Venom gland cDNA sequencing provided precursor protein (prepropeptide) structures of three CpTx 1 isoforms (a-c) that differ by single residue substitutions. The toxin possesses potent insecticidal (paralytic and lethal), cytotoxic, and membrane-damaging activities. In both fly and frog neuromuscular preparations, it causes stable and irreversible depolarization of muscle fibers leading to contracture. This effect appears to be receptor-independent and is inhibited by high concentrations of divalent cations. CpTx 1 lyses cell membranes, as visualized by confocal microscopy, and destabilizes artificial membranes in a manner reminiscent of other membrane-active peptides by causing numerous defects of variable conductance and leading to bilayer rupture. The newly discovered class of modular polypeptides enhances our knowledge of the toxin universe.

Triazine dendrimers as nonviral vectors for in vitro and in vivo RNAi: the effects of peripheral groups and core structure on biological activity.

A family of triazine dendrimers, differing in their core flexibility, generation number, and surface functionality, was prepared and evaluated for its ability to accomplish RNAi. The dendriplexes were analyzed with respect to their physicochemical and biological properties, including condensation of siRNA, complex size, surface charge, cellular uptake and subcellular distribution, their potential for reporter gene knockdown in HeLa/Luc cells, and ultimately their stability, biodistribution, pharmacokinetics and intracellular uptake in mice after intravenous (iv) administration. The structure of the backbone was found to significantly influence siRNA transfection efficiency, with rigid, second generation dendrimers displaying higher gene knockdown than the flexible analogues while maintaining less off-target effects than Lipofectamine. Additionally, among the rigid, second generation dendrimers, those with either arginine-like exteriors or peripheries containing hydrophobic functionalities mediated the most effective gene knockdown, thus showing that dendrimer surface groups also affect transfection efficiency. Moreover, these two most effective dendriplexes were stable in circulation upon intravenous administration and showed passive targeting to the lung. Both dendriplex formulations were taken up into the alveolar epithelium, making them promising candidates for RNAi in the lung. The ability to correlate the effects of triazine dendrimer core scaffolds, generation number, and surface functionality with siRNA transfection efficiency yields valuable information for further modifying this nonviral delivery system and stresses the importance of only loosely correlating effective gene delivery vectors with siRNA transfection agents.

Trafficking microenvironmental pHs of polycationic gene vectors in drug-sensitive and multidrug-resistant MCF7 breast cancer cells.

While multidrug resistance (MDR) has been a significant issue in cancer chemotherapy, delivery resistance to various anti-cancer biotherapeutics, including genes, has not been widely recognized as a property of MDR. This study aims to provide a better understanding of the transfection characteristics of drug-sensitive and drug-resistant cells by tracing microenvironmental pHs of two representative polymer vectors: poly(L-lysine) and polyethyleneimine. Drug-sensitive breast MCF7 cells had four- to seven-times higher polymeric transfection efficiencies than their counterpart drug-resistant MCF7/ADR-RES cells. Polyplexes in MCF7/ADR-RES cells after endocytosis were exposed to a more acidic microenvironment than those in MCF7 cells; the MDR cells show faster acidification rates in endosomes/lysosomes than the drug-sensitive cells after endocytosis (in the case of PLL/pDNA complexes, approximately pH 5.1 for MCF7/ADR-RES cells vs. approximately pH 6.8 for MCF7 cells at 0.5 h post-transfection). More polyplexes were identified trapped in acidic subcellular compartments of MCF7/ADR-RES cells than in MCF7 cells, suggesting that they lack endosomal escaping activity. These findings demonstrate that the design of polymer-based gene delivery therapeutics should take into account the pH of subcellular compartments.

N-terminal amphipathic helix as a trigger of hemolytic activity in antimicrobial peptides: a case study in latarcins.

In silico structural analyses of sets of alpha-helical antimicrobial peptides (AMPs) are performed. Differences between hemolytic and non-hemolytic AMPs are revealed in organization of their N-terminal region. A parameter related to hydrophobicity of the N-terminal part is proposed as a measure of the peptide propensity to exhibit hemolytic and other unwanted cytotoxic activities. Based on the information acquired, a rational approach for selective removal of these properties in AMPs is suggested. A proof of concept is gained through engineering specific mutations that resulted in elimination of the hemolytic activity of AMPs (latarcins) while leaving the beneficial antimicrobial effect intact.