PEGylated liposomes with NGR ligand and heat-activable cell-penetrating peptide–doxorubicin conjugate for tumor-specific therapy

Cell-penetrating peptides (CPPs) mediated tumor-oriented nanocarriers have been widely studied by researchers recently. However, applications of CPPs in vivo were usually hampered by their loss in untargeted tissues and enzymatic degradation. These shortfalls required strategies to camouflage CPPs before their arrival at the targeted site. In this work, we constructed a thermosensitive liposome (TSL) containing Asparagines–Glycine–Arginine (NGR) peptide as the targeting moiety and heat-activable cell-penetrating peptide–doxorubicin conjugate for enhancing specific cancer therapy. Different to the masking strategies of CPPs reported, CPPs existing in conjugation form of CPPs and doxorubicin (CPP-Dox) were hidden in TSL to cloak and protect CPPs. Meanwhile, NGR moiety and local tumor hyperthermia were utilized to achieve specific targeting of CPPs to the tumor. The nanocarrier (CPP-Dox/NGR-TSL) prepared in this work possessed suitable physiochemical properties such as small particle size of about 90 nm, high drug encapsulation efficiency of approximately 95%, good stability in the medium containing 10% fetal bovine serum (FBS) and so on. In vitro experiments on Human fibrosarcoma cells (HT-1080) and human breast adenocarcinoma cells (MCF-7) verified the specific targeting ability and enhanced intracellular drug delivery of the liposomes to HT-1080 cells. Furthermore, comparing with NGR-targeted TSL containing Dox (Dox/NGR-TSL), the results of intravenous administration showed CPP-Dox/NGR-TSL significantly inhibited tumor growth in nude mice xenografted HT-1080 tumors and excellent body safety. In conclusion, the nanocarrier constructed in this study would be a safe and efficiently drug delivery system for specific cancer treatment.     

Utilization of a sol–gel method for encapsulation of doxorubicin

The sol-gel pre-doping method was used to encapsulate doxorubicin in silica gels and optimum conditions of preparation of drug-loaded gel were established, ensuring both reproducible and effective results of quantitative encapsulation of doxorubicin and its gradual but complete release. Doxorubicin was encapsulated in polysiloxane polymers using the method based on sol-gel encapsulation without a catalyst, with an acid catalyst (HCl) and a base catalyst (NH₃). The time of gelation of the gel loaded with doxorubicin, encapsulation efficiency of the drug and the degree of release of the drug from the gel are all affected by the kind of catalyst (acidic or basic) or its absence at the gel preparation stage, and the temperature of the gelation process. The time of sol gelation when using the NH₃ or HCl catalyst was 9 days at 21°C, 2 days at 30°C and 1.5 days at 37°C, while for the gel prepared without a catalyst it was 90 days at 21°C, 75 days at 30°C and 70 days at 37°C. The efficiency of doxorubicin encapsulation was 99.5 ± 0.5% (w/w) for acid-catalyzed gel, 98.9 ± 1.01% (w/w) for base-catalyzed gel and 86.4 ± 11.6% (w/w) for non-catalyzed gel. A 100% (w/w) release of doxorubicin by diffusion through pores was found only in the case of base-catalyzed gel after a 140-h incubation time. For acid-catalyzed gel and non-catalyzed gel, the total amounts of released doxorubicin after 140 h of incubation were 3-5% (w/w) and 9-11% (w/w), respectively. The stability of doxorubicin encapsulated in the three kinds of gel matrices was found to be improved compared to the stability of a free form of the drug in solution.     

Poly(amidoamine) dendrimer–drug conjugates with disulfide linkages for intracellular drug delivery

Understanding and improving drug release kinetics from dendrimer–drug conjugates are key steps to improve their in vivo efficacy. N-Acetyl cysteine (NAC) is an anti-inflammatory agent with significant potential for clinical use in the treatment of neuroinflammation, stroke and cerebral palsy. There is a need for delivery of NAC which can enhance its efficacy, reduce dosage and prevent it from binding plasma proteins. For this purpose, a poly(amidoamine) dendrimer–NAC conjugate that contains a disulfide linkage was synthesized and evaluated for its release kinetics in the presence of glutathione (GSH), cysteine (Cys), and bovine serum albumin (BSA) at both physiological and lysosomal pH. The results indicate that the prepared conjugate can deliver ～60% of its NAC payload within 1 h at intracellular GSH concentrations at physiological pH, whereas the conjugate did not release any drug at plasma GSH levels. The stability of the conjugate in the presence of bovine serum albumin at plasma concentrations was also demonstrated. The efficacy of the dendrimer–NAC conjugate was measured in activated microglial cells (target cells in vivo) using the reactive oxygen species (ROS) assay. The conjugates showed an order of magnitude increase in antioxidant activity compared to free drug. When combined with intrinsic and ligand-based targeting with dendrimers, these types of GSH sensitive nanodevices can lead to improved drug release profiles and in vivo efficacy.     

High-performance PEGylated Mn–Zn ferrite nanocrystals as a passive-targeted agent for magnetically induced cancer theranostics

An effective magnetic nanocrystals (MNCs)-mediated theranostics strategy as a combination of simultaneous diagnostics and heating treatment of tumors by using magnetic resonance imaging (MRI) and alternating current magnetic field (ACMF) is successfully developed. In this strategy, we had firstly synthesized a well-established Mn–Zn ferrite MNCs coated with PEG-phospholipids (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol copolymers, DSPE-PEG2000). The monodisperse PEGylated MNCs with core–shell structure (15 nm) exhibited excellent performance, such as high magnetism of 98 emu g⁻¹ Fe, relaxivity coefficient (r2) of 338 mm⁻¹ s⁻¹, and specific absorption rate (SAR) value of 324 W g⁻¹ Fe. It was proved that the obtained MNCs with an average diameter of 48.6 nm can drastically minimize the recognition and phagocytosis of macrophages, simultaneously improve their biocompatibility in vitro. These advantages endowed them with efficient passive targeting ability in vivo for prominent tumor MRI and magnetically induced heating when exposed to ACMF, based on enhanced permeability and retention (EPR) effects. To ensure sufficient accumulation of MNCs within tumors for targeted hyperthermia, we described the use of MNCs with a well-tolerated intravenous single dose of 18 mg Fe/kg mouse body weight, achieving repeatedly injection and hyperthermia within a subcutaneous breast cell carcinoma mouse model. With an ACMF of 12 A at 390 kHz, the tumor surface sites could be heated to approximately 43 °C in 30 min based on MNCs-mediated intravenous injections. The long-lasting hyperthermia could effectively induce the apoptosis of tumor cells, inhibit the angiogenesis of tumor vessels, and finally suppress the tumor growth within a certain period of time.     

Cell-selective intracellular drug delivery using doxorubicin and α-helical peptides conjugated to gold nanoparticles

Cell penetrating peptides (CPPs), which can enter a cell through the cell membrane, have potential research applications in the fields of drug delivery, gene therapy, and cancer therapy. However, CPPs are associated with problems such as low cell selectivity, low cell penetrating activity, and cell toxicity. To overcome the disadvantages of CPPs, we constructed a drug delivery system by developing 25 nm gold nanospheres (GNSs) conjugated to four α-helical CPPs from our peptide library. We examined the applicability of this cell-selective drug delivery system by evaluating its cell-penetrating and cell death activities and comparing them with those activities of the TAT peptide. Using the 25 nm GNS, we obtained higher cell death induction activity by the anti-cancer drug doxorubicin compared with our previous study using a 41 nm GNS. After entering the cell, the peptide-conjugated 25 nm GNS accumulated around the cell nucleus. High cell selectivity by α-helical CPP sequences was also demonstrated. Our results indicate that these α-helical peptide and 25 nm GNS conjugates are useful elements in an efficient cell-selective drug delivery system.     

A multi-stimuli responsive gold nanocage–hyaluronic platform for targeted photothermal and chemotherapy

Noninvasive and pinpointed intracellular drug release that responds to multiple stimulus is still a formidable challenge for cancer therapy. Herein, we reported a multi-stimuli responsive platform based on drug loaded gold nanocages @ hyaluronic acid (AuNCs-HA) for pinpointed intracellular drug release. These well-prepared nanohybrids could specifically recognize cancer cells via HA-CD44 interactions and be efficiently endocytosed by receptor-mediated process. Subsequently, the coated HA molecules could be degraded in lysosomes, resulting in the release of encapsulated drug. In addition, by taking advantage of the excellent photothermal properties, the AuNCs could accelerate the release of encapsulated drug and induce a higher therapeutic efficacy upon near-infrared (NIR) irradiation. In vitro results confirmed that the encapsulated drug could only be pinpointedly released in intracellular environments, which permitted high therapeutic efficacy against cancer cells and minimized the side effects. Importantly, as compared to that of the two therapies independently, a complete inhibition of tumor growth treated with the combination of chemotherapy and photothermal therapy was observed in vivo. Taken together, our present study provides new insights into developing pinpointed, multi-stimuli responsive intracellular drug release systems for synergistic cancer therapy.     

Self-assembled pH-responsive hyaluronic acid–g-poly(l-histidine) copolymer micelles for targeted intracellular delivery of doxorubicin

Hyaluronic acid (HA) was conjugated with hydrophobic poly(l-histidine) (PHis) to prepare a pH-responsive and tumor-targeted copolymer, hyaluronic acid–g-poly(l-histidine) (HA-PHis), for use as a carrier for anti-cancer drugs. The effect of the degree of substitution (DS) on the pH-responsive behaviour of HA-PHis copolymer micelles was confirmed by studies of particles of different sizes. In vitro drug release studies demonstrated that doxorubicin (DOX) was released from HA-PHis micelles in a pH-dependent manner. In vitro cytotoxicity assays showed that all the blank micelles were nontoxic. However, MTT assay against Michigan Cancer Foundation-7 (MCF-7) cells (overexpressed CD44 receptors) showed that DOX-loaded micelles with a low PHis DS were highly cytotoxic. Cellular uptake experiments revealed that these pH-responsive HA-PHis micelles taken up in great amounts by receptor-mediated endocytosis and DOX were efficiently delivered into cytosol. Moreover, micelles with the lowest DS exhibited the highest degree of cellular uptake, which indicated that the micelles were internalized into cells via CD44 receptor-mediated endocytosis and the carboxylic groups of HA are the active binding sites for CD44 receptors. Endocytosis inhibition experiments and confocal images demonstrated that HA-PHis micelles were internalized into cells mainly via clathrin-mediated endocytosis and delivered to lysosomes, triggering release of DOX into the cytoplasm. These results confirm that the biocompatible pH-responsive HA-PHis micelles are a promising nanosystem for the intracellular targeted delivery of DOX.     

Biodegradable and amphiphilic block copolymer–doxorubicin conjugate as polymeric nanoscale drug delivery vehicle for breast cancer therapy

Polymeric nanoparticles have shown great promise as attractive vehicles for drug delivery. In this study, we designed, prepared and characterized biodegradable amphiphilic triblock HPMA copolymer–doxorubicin (copolymer–DOX) conjugate based nanoparticle as enzyme-sensitive drug delivery vehicle. The enzyme-sensitive peptide GFLGKGLFG was introduced to the main chain of the copolymer with hydrophilic and hydrophobic blocks. The triblock HPMA polymer–DOX conjugate with high molecules (Mw 90 kDa) can be degraded to product with low molecule weight (Mw 44 kDa) below the renal threshold. The copolymer–DOX conjugate can self-assemble into compact nanoparticle, which was characterized by scanning electron microscope (SEM) and atomic force microscope (AFM) studies. This polymeric nanoparticle substantially enhanced antitumor efficacy compared to the free DOX, exhibiting much higher effects on inhibiting proliferation and inducing apoptosis on the 4T1 murine breast cancer model confirmed by the evidences from mice weight shifts, tumor growth curves, tumor growth inhibition (TGI), immunohistochemical analysis and histological assessment. The in vivo toxicity evaluation demonstrated that the polymeric nanoparticle reduced DOX-induced toxicities and presented no significant side effects to normal organs of both tumor bearing and healthy mice as measured by body weight shift, blood routine test and histological analysis. Therefore, the triblock HPMA copolymer–DOX conjugate based nanoparticle is promising as a potential drug delivery vehicle for breast cancer therapy.     

Thiolated Glycol Chitosan Bearing α-Cyclodextrin for Sustained Delivery of PEGylated Human Growth Hormone

Abstract

The extensive use of human growth hormone (hGH), emerging as protein therapeutics, has been limited by its instability in biological fluids and short biological half-life. In this study, thiolated glycol chitosan bearing α-cyclodextrin (TGC-CD), in situ cross-linked by poly(ethylene glycol)-diacrylate (PEG-DA), was synthesized to develop a sustained release system for PEGylated hGH (PEG-hGH). TGC-CD could form a stable inclusion complex with PEG-hGH by the physical interaction between the inner cavity of CD and PEG. Such a complex was readily cross-linked in the presence of PEG-DA via a Michael-type addition reaction. From the in vitro release experiments of PEG-hGH, it was confirmed that PEG-hGH was completely released from the complex for 12 h in PBS (pH 7.4), whereas the release rate of PEG-hGH was significantly reduced by the chemical cross-linking of the complex. PEG-hGH, released from the cross-linked complexes, maintained its structural integrity, which was demonstrated using circular dichroism spectroscopy. Overall, TGC-CD might be useful for sustained delivery of PEG-hGH.     

The posterior pedicled inferior turbinate–nasoseptal flap: a potential combined flap for skull base reconstruction

Objectives

To develop a combined pedicled flap comprising the mucoperiosteum and mucoperichondrium of the inferior turbinate, lateral nasal wall, nasal floor, and nasal septum based on the posterior lateral nasal artery, a branch of the sphenopalatine artery, for the reconstruction of skull base defects resulting from endoscopic expanded endonasal approaches.

Methods

Eleven fresh adult cadaver heads were dissected. Arterial distribution patterns of the inferior turbinate, lateral nasal wall, nasal floor, and nasal septum were investigated. The posterior pedicled inferior turbinate–nasoseptal flap was designed, measured, and harvested, and its ability to cover ventral skull base defects was examined.

Results

The inferior turbinate artery and/or posterior lateral nasal artery had 3.19 ± 1.47 (range 2–7) branches [mean outer diameter of largest branch, 0.40 ± 0.10 (range 0.24–0.60) mm] that anastomosed with the nasoseptal artery. These anastomosing arteries allowed the posterior lateral nasal artery to supply arterial blood to the nasoseptal mucoperichondrium and mucoperiosteum. Mean flap length was 100.65 ± 5.61 (range 91.43–109.44) mm, and minimum and maximum widths were 25.21 ± 2.29 (range 22.36–30.23) and 44.53 ± 5.02 (range 36.45–54.10) mm, respectively. Mean flap area was 3090.69 ± 288.08 (range 2612.97–3880.09) mm². The flap covered defects extending from the frontal sinus to the foramen magnum in all specimens.

Conclusions

Harvesting of a posterior pedicled inferior turbinate–nasoseptal flap is feasible. It should be considered a useful option for the reconstruction of large defects involving the anterior skull base, planum sphenoidale, sella turcica, and/or clivus.

     

A PEGylated Fab’ Fragment against Tumor Necrosis Factor for the Treatment of Crohn Disease

Antibodies, having a high specificity for their particular target, are increasingly being used as therapeutic agents with functions including agonist, antagonist, and targeted drug delivery. The use of many biologic therapies, including antibody fragments, is generally limited by their rapid clearance from plasma. A commonly used approach to extend exposure to biologic therapies is the attachment of polyethylene glycol.Tumor necrosis factor (TNF)-alpha is a multifunctional cytokine involved in the regulation of immune responses. Elevated levels of TNFalpha are found in a wide range of diseases, including the chronic inflammatory conditions rheumatoid arthritis, psoriasis, and Crohn disease (CD). Anti-TNFalpha antibodies have proved highly efficacious in the treatment of these conditions. In addition, they have proved invaluable for investigating the role of TNFalpha in disease etiology.Based on evidence showing that neutralizing antibodies to TNFalpha were effective in animal models of CD, anti-TNFalpha antibody treatments were assessed in clinical trials. Interestingly, the anti-TNFalpha antibody etanercept proved ineffective at achieving remission in active CD despite potently neutralizing soluble TNFalpha. This indicated that an additional mode of action is also involved in the efficacy of the anti-TNFalpha agents adalimumab, certolizumab pegol, and infliximab in CD; one suggestion was apoptosis. However, etanercept, like adalimumab and infliximab, can induce apoptosis. Furthermore, certolizumab pegol (which has demonstrated efficacy in CD) does not cause complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, apoptosis, or necrosis of neutrophils, all measured in vitro. These functional differences observed with certolizumab pegol stem from its unique structure that does not include the crystallizable fragment (Fc) portion present in the other anti-TNFalpha agents, and the way in which it signals through membrane TNF.It is well established that bacteria are a major part of the inflammatory process in CD. The property identified that reflected the efficacies of the anti-TNFalpha agents etanercept, adalimumab, certolizumab pegol, and infliximab in CD was the ability to inhibit the cytokine production by monocytes that is induced by bacterial lipopolysaccharide. It may therefore be the case that this mode of action is important for efficacy in CD.     

Folic acid–Pluronic F127 magnetic nanoparticle clusters for combined targeting,diagnosis, and therapy applications

Superparamagnetic iron oxides possess specific magnetic properties in the presence of an external magnetic field, which make them an attractive platform as contrast agents for magnetic resonance imaging (MRI) and as carriers for drug delivery. In this study, we investigate the drug delivery and the MRI properties of folate-mediated water-soluble iron oxide incorporated into micelles. Pluronic^® F127 (PF127), which can be self-assembled into micelles upon increasing concentration or raising temperatures, is used to decorate water-soluble polyacrylic acid-bound iron oxides (PAAIO) via a chemical reaction. Next, the hydrophobic dye Nile red is encapsulated into the hydrophobic poly(propylene oxide) compartment of PF127 as a model drug and as a fluorescent agent. Upon encapsulation, PAAIO retains its superparamagnetic characteristics, and thus can be used for MR imaging. A tumor-specific targeting ligand, folic acid (FA), is conjugated onto PF127–PAAIO to produce a multifunctional superparamagnetic iron oxide, FA–PF127–PAAIO. FA–PF127–PAAIO can be simultaneously applied as a diagnostic and therapeutic agent that specifically targets cancer cells that overexpress folate receptors in their cell membranes. PF127–PAAIO is used as a reference group. Based on FTIR and UV–vis absorbance spectra, the successful synthesis of PF127–PAAIO and FA–PF127–PAAIO is realized. The magnetic nanoparticle clusters of PF127–PAAIO and FA–PF127–PAAIO are visualized by transmission electron microscope (TEM). FA–PF127–PAAIO, together with a targeting ligand, displays a higher intracellular uptake into KB cells. This result is confirmed by laser confocal scanning microscopy (CLSM), flow cytometry, and atomic absorption spectroscopy (AAS) studies. The hysteresis curves, generated by using a superconducting quantum interference device (SQUID) magnetometer analysis, demonstrate that the magnetic nanoparticles are superparamagnetic with insignificant hysteresis. The MTT assay explains the negligible cell cytotoxicity of PF127–PAAIO and FA–PF127–PAAIO. In KB cells, the in vitro MRI study indicates the better T₂-weighted images in FA–PF127–PAAIO than in PF127–PAAIO.     

Phosphatidylserine-microbubble targeting-activated microglia/macrophage in inflammation combined with ultrasound for breaking through the blood–brain barrier

Background and purpose

Inflammatory reaction plays a crucial role in cerebral ischemia reperfusion (IR) injury. It has been shown that activated microglia long-term existed in cerebral ischemia and induced second injury. Therefore, we hypothesize that prepared phosphatidylserine (PS)-modified microbubbles (PS-MBs) combined with ultrasound-targeted microbubble destruction (UTMD) can safely open the blood–brain barrier (BBB) and target activated microglia for inflammatory area in the later stage of ischemia reperfusion.

Methods

To verify our hypothesis, rat model of IR was established, then the change of activated microglia/macrophage (M/M) and permeability of BBB at 1, 7, 14, and 21?days could be clearly observed post IR. And the activated M/M still can be observed during the whole experiment.

Results

The Evans blue extravasation of BBB gradually declined from day 1 to day 21. Compared to the control group, microbubbles containing PS were taken up more by activated M/M (approximately twofold) both in vitro and in vivo.

Conclusions

PS-MBs combined with ultrasound (US) exposure could safely open BBB, and the resulting PS nanoparticles (PS-NPs) could further target activated M/M in the neuroinflammation.

     

Antibody-conjugated PEGylated cerium oxide nanoparticles for specific targeting of Aβ aggregates modulate neuronal survival pathways

Oxidative stress has been found to be associated with the progression of neurodegenerative diseases such as Alzheimer's, Parkinson's, Lou Gehrig's, etc. In the recent years, cerium oxide nanoparticles (CNPs) have been studied as potent antioxidant agents able to exert neuroprotective effects. This work reports polyethylene glycol (PEG)-coated and antibody-conjugated CNPs for the selective delivering to Aβ aggregates, and the protective effect against oxidative stress/Aβ-mediated neurodegeneration. In this study PEG-coated and anti-Aβ antibody-conjugated antioxidant nanoparticles (Aβ-CNPs-PEG) were developed, and their effects on neuronal survival and brain-derived neurotrophic factor (BDNF) signaling pathway were examined. Aβ-CNPs-PEG specifically targets the Aβ aggregates, and concomitant rescue of neuronal survival better than Aβ-CNPs, by modulating the BDNF signaling pathway. This proof of concept work may allow in the future, once validated in vivo, for the selective delivery of CNPs only to affected brain areas.     

Surface modification with an antithrombin–heparin complex for anticoagulation: Studies on a model surface with gold as substrate

Gold was used as a substrate for immobilization of an antithrombin–heparin (ATH) covalent complex to investigate ATH as a surface modifier to prevent blood coagulation. Three different surface modification methods were used to attach ATH to gold: (i) direct chemisorption; (ii) using dithiobis(succinimidyl propionate) (DSP) as a linker molecule and (iii) using polyethylene oxide (PEO) as a linker/spacer. The ATH-modified surfaces were compared to analogous heparinized surfaces. Water contact angles and X-ray photoelectron spectroscopy confirmed the modifications and provided data on surface properties and possible orientation. Ellipsometry measurements showed that surface coverage of DSP and PEO was high. ATH and heparin densities were quantified using radioiodination and quartz crystal microbalance, respectively. The surface density of ATH was greatest on the DSP surface (0.17 μg cm^?2) and lowest on the PEO (0.05 μg cm^?2). The low uptake on the PEO surface was likely due to the protein resistance of the PEO component. Using radioiodinated antithrombin (AT), it was shown that ATH-immobilized surfaces bound significantly greater amounts from both buffer and plasma than the analogous heparinized surfaces. Immunoblot analysis of proteins adsorbed from plasma demonstrated that surfaces chemisorbed with PEO, whether or not subsequently modified with ATH, inhibited non-specific adsorption. The immunoblot response for AT was stronger on the DSP–ATH than on the heparin surfaces, thus confirming the results from radiolabelling. The ATH surfaces again showed higher selectivity for AT binding than analogous heparin-modified surfaces, indicating the enhanced anticoagulant potential of ATH for biomaterial surface modification.     

Cytoreductive conditioning for severe combined immunodeficiency – help or hindrance?

Use of chemotherapy-based conditioning-facilitated engraftment in patients with severe combined immunodeficiency (SCID) is contentious. In T- and NK lymphocyte-negative, B-lymphocyte-positive (T-B+NK+) and T-B-NK+ SCID, the osteo-medullary space is occupied by recipient hematopoietic stem cells and mature B-lymphocytes. The thymic niche is empty in T-B+NK+ SCID but fully occupied by developmentally arrested T-lymphocyte precursors in T-B-NK+ SCID. The outcome of infusion of donor stem cells differs and is dependent on genetic defect and the lymphocyte developmental arrest stage. At best, donor hematopoietic stem cell osteo-medullary engraftment induces normal B-lymphocyte function and long-term thymopoiesis; at worst, peripheral expansion of donor T-lymphocytes from the stem cell source results in a restricted T-lymphocyte receptor repertoire with possible B-lymphocyte failure. Conditioning improves immunoreconstitution but causes short- and long-term toxicities, and increased mortality. Newborn screening for SCID will propel the search for safe, effective methods of achieving donor cell engraftment and full immunoreconstitution without toxic sequalae.     

Focus floating microscopy: "gold standard" for cutaneous borreliosis?

Borrelia burgdorferi is difficult to detect in routine biopsy material from patients with skin lesions of borreliosis. In this study, a new immunohistochemical method, focus floating microscopy (FFM), was developed to detect B burgdorferi in tissue sections and was compared with polymerase chain reaction (PCR). By using standard histologic equipment, tissue sections stained with a polyclonal B burgdorferi antibody were simultaneously scanned through 2 planes: horizontally in serpentines and vertically by focusing through the thickness of the section.Borrelia were detected in 47 of 71 ticks, 34 of 66 tick bites, 30 of 32 erythema chronicum migrans cases, 41 of 43 borrelial lymphocytomas, and 50 of 51 acrodermatitis chronica atrophicans cases. FFM proved to be more sensitive than PCR (96.0% vs 45.2%) and nearly equally specific (99.4% vs 100%). All 169 control cases, except 1 false-positive case of secondary syphilis, were negative with FFM. FFM is an easy, quick, and inexpensive method to reliably detect Borrelia in cutaneous tissue sections.     

Reference assays for Clostridium difficile infection: one or two gold standards?

Accurate diagnosis of Clostridium difficile infection (CDI) is essential for optimal treatment, prevention and control. There are two reference assays for CDI diagnosis: the cell cytotoxicity assay (CCTA) and toxigenic culture (TC). Importantly, these tests actually detect different targets: CCTA detects the presence of C difficile toxins (primarily toxin B, but also toxin A), whereas TC detects the presence in the stool of C difficile with the potential to produce toxin. Not surprisingly studies comparing the results of these assays show imperfect agreement. Thus, a faecal sample may be CCTA negative but TC positive, and this raises the crucial question about the clinical significance of the presence of C difficile with the capacity to produce toxin but no actual detectable free toxin. A positive TC result indicates that a patient with diarrhoea is potentially infectious. TC also has the advantage that the cultured isolate is available for typing and for susceptibility testing. In general, however, CCTA has been shown to be a better test for the laboratory confirmation of CDI, although additional culture may be needed to optimise sensitivity. Crucially, when these reference assays are used to determine the accuracy of alternative diagnostic tests, care should be taken to compare methods with their appropriate standard (ie, compare tests that target equivalent end-points). Such issues have contributed to the variable and often suboptimal performance of rapid diagnostic tests for CDI. Further research is urgently needed to improve knowledge of the utility of routine diagnostic tests in CDI and the factors that influence their performance.