Biofilm production and antibiotic susceptibility of Staphylococcus epidermidis strains from Hidradenitis Suppurativa lesions.

BACKGROUND: An aberrant interaction between commensal skin bacteria and the host skin immune system is considered important in the pathogenesis of hidradenitis suppurativa (HS). OBJECTIVE: In this study, we investigated the antibiotic susceptibility and biofilm-forming capabilities of S. epidermidis strains isolated from HS patients. METHODS: Skin biopsies were taken from active HS lesions such as inflammatory nodules and/or sinuses and non-involved skin from 26 patients and cultured under optimal microbiological conditions for 24 h. Planktonic growth, biofilm production, antibiotic susceptibility and biofilm eradication by clindamycin, doxycycline, rifampicin and tetracycline were tested including a laboratory control strain of S. epidermidis for reference. RESULTS: Staphylococcus epidermidis was cultured in 16 of 26 HS patients (62%). In total 27 different S. epidermidis isolates were identified; 16 (59%) from non-involved skin and 11 (41%) from HS lesions. All bacterial strains showed planktonic growth. Twenty-four of 27 (89%) isolates were strong biofilm producers in vitro. The biofilm-forming capability varied amongst the strains from non-involved skin and lesional skin. Twenty-four strains had an intermediate to resistant antibiotic susceptibility to clindamycin (89%). Rifampicin was the most effective antibiotic at inhibiting planktonic growth and at eradication of biofilm (P < 0.05). CONCLUSION: We observed a slight increase in S. epidermidis virulence, characterized by resistance to commonly used antibiotics, increased biofilm production and resistance to biofilm eradication. In particular, the reduced sensitivity to tetracycline and clindamycin, two standard antibiotics in the treatment of HS, is alarming. Rifampicin, also important in HS treatment, showed the greatest efficacy at eradicating the biofilm at low MIC concentrations.

Results of a Direct Search Using Synchrotron Radiation for the Low-Energy (229)Th Nuclear Isomeric Transition.

We report the results of a direct search for the (229)Th (I(π)=3/2(+)←5/2(+)) nuclear isomeric transition, performed by exposing (229)Th-doped LiSrAlF(6) crystals to tunable vacuum-ultraviolet synchrotron radiation and observing any resulting fluorescence. We also use existing nuclear physics data to establish a range of possible transition strengths for the isomeric transition. We find no evidence for the thorium nuclear transition between 7.3 eV and 8.8 eV with transition lifetime (1-2) s≲τ≲(2000-5600) s. This measurement excludes roughly half of the favored transition search area and can be used to direct future searches.

Selection of resistance by antimicrobial coatings in the healthcare setting.

Antimicrobial touch surfaces have been introduced in healthcare settings with the aim of supporting existing hygiene procedures, and to help combat the increasing threat of antimicrobial resistance. However, concerns have been raised over the potential selection pressure exerted by such surfaces, which may drive the evolution and spread of antimicrobial resistance. This review highlights studies that indicate risks associated with resistance on antimicrobial surfaces by different processes, including evolution by de-novo mutation and horizontal gene transfer, and species sorting of inherently resistant bacteria dispersed on to antimicrobial surfaces. The review focuses on antimicrobial surfaces made of copper, silver and antimicrobial peptides because of the practical application of copper and silver, and the promising characteristics of antimicrobial peptides. The available data point to a potential for resistance selection and a subsequent increase in resistant strains via cross-resistance and co-resistance conferred by metal and antibiotic resistance traits. However, translational studies describing the development of resistance to antimicrobial touch surfaces in healthcare-related environments are rare, and will be needed to assess whether and how antimicrobial surfaces lead to resistance selection in these settings. Such studies will need to consider numerous variables, including the antimicrobial concentrations present in coatings, the occurrence of biofilms on surfaces, and the humidity relevant to dry-surface environments. On-site tests on the efficacy of antimicrobial coatings should routinely evaluate the risk of selection associated with their use.

A near full-length open reading frame next generation sequencing assay for genotyping and identification of resistance-associated variants in hepatitis C virus.

BACKGROUND: The current treatment options for hepatitis C virus (HCV), based on direct acting antivirals (DAA), are dependent on virus genotype and previous treatment experience. Treatment failures have been associated with detection of resistance-associated substitutions (RASs) in the DAA targets of HCV, the NS3, NS5A and NS5 B proteins. OBJECTIVE: To develop a next generation sequencing based method that provides genotype and detection of HCV NS3, NS5A, and NS5 B RASs without prior knowledge of sample genotype. STUDY DESIGN: In total, 101 residual plasma samples from patients with HCV covering 10 different viral subtypes across 4 genotypes with viral loads of 3.84-7.61 Log IU/mL were included. All samples were de-identified and consequently prior treatment status for patients was unknown. Almost full open reading frame amplicons (∼ 9 kb) were generated using RT-PCR with a single primer set. The resulting amplicons were sequenced with high throughput sequencing and analysed using an in-house developed script for detecting RASs. RESULTS: The method successfully amplified and sequenced 94% (95/101) of samples with an average coverage of 14,035; four of six failed samples were genotype 4a. Samples analysed twice yielded reproducible nucleotide frequencies across all sites. RASs were detected in 21/95 (22%) samples at a 15% threshold. The method identified one patient infected with two genotype 2b variants, and the presence of subgenomic deletion variants in 8 (8.4%) of 95 successfully sequenced samples. CONCLUSIONS: The presented method may provide identification of HCV genotype, RASs detection, and detect multiple HCV infection without prior knowledge of sample genotype.

The binding of IgG1 containing immune complexes to the FcR of allogenically activated T cells induces changes in the membrane potential and the cell surface charge.

The effect on membrane potential and cell surface charge of binding immune complexes containing IgG1 and IgG2a monoclonal antibodies to Fc receptors was studied in resting and allogenically activated murine T cells. IgG1 complexed by antigen or heat aggregation induced electrophysiological changes on activated T cells. A biphasic alteration of membrane potential was detected by measurement of the intra- and extracellular distribution of the fluorescent dye, DiOC6. A short-lived hyperpolarization, detectable for 4-6 min after adding the respective ligand, was followed by a longer lasting depolarization. The cell surface charge, measured by cell electrophoresis, was also changed. This alteration was detected 2-4 hr after addition of immune complexes and disappeared by the 8th hr of incubation. Monoclonal antibody 2.4.G2, reactive with mouse FcR, induced a similar membrane potential response on activated T cells, but did not affect the cell surface charge. Monomeric IgGs and complexes of IgG2a did not modify these parameters. FcR ligands had no effect on the studied characteristics of resting T cells.

A comparison of the kinetics of the macrophage electrophoretic mobility (MEM) and the tanned sheep erythrocyte electrophoretic mobility (TEEM) tests.

In this study the macrophage electrophoretic mobility (MEM) test was modified by using tanned sheep erythrocytes in place of guinea pig peritoneal macrophages as the indicator cells of lymphocyte sensitization to antigens. This modification is named the tanned sheep erythrocyte electrophoretic mobility (TEEM) test, and a comparison of the kinetics of the two systems allowed the following conclusions to be made: 1) Treatment of freshly drawn sheep red blood cells with a concentration of 1/40,000 tannic acid produced optimum results in the TEEM test. 2) Lymphocyte-antigen and lymphocyte-number response curves show similarity in the two test systems. 3) A plateau response with slowing factor is achieved at a lower dilution in the TEEM test than in the MEM test. 4) Whilst similarity in the first stage reaction was found in the two systems, in the second stage of the test (at 37 degrees C) tanned sheep red cells gave a plateau response after 45 min instead of the 60 min found in the MEM test. 5) The two slowing factors showed similar gel filtration patterns with molecular weights between 13,000 and 15,000 daltons, and had equivalent activity in both test systems. 6) The disadvantages of the guinea pig macrophage as an indicator cell are discussed. 7) The TEEM test seems simpler to perform than the MEM test and may be widely applicable in clinical immunology for the estimation of lymphocyte sensitization.

[The macrophage electrophoretic mobility LAD test - a diagnostic method for multiple sclerosis.]. / Der Makrophagen-Elektrophorese-Mobilitäts-LAD-Test als diagnostisches Verfahren für die multiple Sklerose.

With the MEM test (Field) one can establish a cellular immune reaction because the sensitized lymphocytes release the macrophage slowing factor (MSF) upon interaction with the appropriate antigen. A macrophage migration inhibition was detected in some neurological diseases with destruction of the parenchyma. The modification MEM-LAD (linoleic acid depression) test made further differentiation possible in the 146 neurological patients and normals. The reduction of macrophage mobility inhibition was 94.7+/-4.7% in multiple sclerosis (MS) cases as compared with that of normals of 55.1+/-3.7% and of other neurological diseases of 47.8+/-7.1%. There were no significant differences due to the course and duration of the disease or to immunosuppressive therapy. The pathogenically important results in relatives of MS patients with values between the MS and normal group (78.5+/-0.7%) in mothers suggested a familial (genetic) disposition. The same value was found in a monozygotic twin of an MS patient. The results in the children studied showed that besides the endogenic metabolic component the aetiopathogenically important exogenic factors can operate early in life. In correlation with the principle of the MEM-LAD test the suppressive action of linoleic acid can result in a further therapeutic concept.

[Demonstration of a factor in cerebrospinal fluid with inhibitory activity for electrophoretic cell mobility in multiple sclerosis (author's transl)]. / Nachweis eines Faktors mit elektrophoretischer Zellmobilitätshemmung im Liquor cerebrospinalis bei Multipler Sklerose

Inhibition of electrophoretic cell migration using cerebrospinal fluid (CSF) directly was investigated by the modified MEM (macrophage electrophoretic mobility) and TEEM (tanned sheep erythrocyte electrophoretic mobility) tests, respectively. An inhibitory activity of macrophage slowing factor (MSF)--one of in vivo lymphokines--in CSF was established in cases of multiple sclerosis (17.5 +/- 3.8%), and neurolues. The value of this MSF assay turned out to be significantly different from the remaining inflammatory ailments of the nervous system (10.1 +/- 6.8%). Results of other neurological diseases were found to be very much lower (5.1 +/- 4.2%). It seems important, for immunopathogenesis and the diagnosis of neuroimmunological diseases with enhanced cellular immunoreaction, to evaluate MSF activity in CSF. To characterize the active factor in CSF (and serum) these fluids were fractionated by gel filtration chromatography as well as supernatants from lymphocyte-antigen incubation in MS patients. The main activity for inhibition of electrophoretic cell mobility was eluated in the same fraction in these fluids. It could be shown that units have a molecular weight of about 15000 Daltons; this value for MSF lies below those for other inhibitory lymphokines.

Anomalous lymphocyte-antigen reaction in relatives of multiple sclerosis patients. A study of a possible genetic factor in the disease.

A combined familial study of multiple sclerosis (MS) in England and in the Rostock area of the GDR using the macrophage electrophoretic mobility (MEM)-LAD test embracing 132 relatives has revealed a closely similar pattern of distribution of "anomalous" LAD (Linoleic Acid Depression) values in relatives (77% type of reaction) to that originally reported in the British study. The anomaly in predominantly associated with females--all mothers of MS patients being affected, whilst daughters and sisters are also represented. In addition unusual full MS type of reaction (90% reduction) has been found in some children related to patients. There is clearly a genetic element in the development of MS probably mainfested in the inborn mishandling of unsaturated fatty acids suggested by Thompson; no recognizable pattern of inheritance is noticeable even within the combined material. There is evidence that the metabolic anomaly alone does not inevitably lead to MS, and the full abnormality may be present at an early age. A survey about the examinations and a selection of characteristic family trees of MS are given, illustrating the manner in which the 77% type anomaly is distributed with occasional omission of a generation.

Aspects of cellular immunity in multiple sclerosis. Antigen-reactivity of lymphocytes and lymphokine activity.

Some new results of cell-mediated immunity in multiple slcerosis (MS) are presented, based on the determination of charge-changing lymphokines as products of antigen sensitive lymphocytes (C PAL), obtained by several forms of the electrophoretic mobility (EM) method. Lymphocytes from MS react to myelin basic protein (BP), the reactivity in other neurological diseases depending on the degree of destruction of nervous parenchyma. Applying a membrane-associated antigen from normal brain (NTA), positive reactivity of MS lymphocytes was obtained. Besides the usual determination of lymphokines in vitro the sensitive EM test allows the demonstration of lymphokine activities in vivo; i.e. in body fluids such as cerebrospinal fluid (CSF) and serum. In CSF of MS a high lymphokine activity was found. The differentiation of lymphokines and comparison between in vitro and in vivo activity were carried out. Moreover, a characteristic lymphokine pattern for MS with high activities in all regions of molecular weight, especially in the CSF, could be detected. On the basis of these findings, important also from the pathogenetic point of view, a diagnostic scheme for MS is suggested, consisting of a program of determination of the immuno-reactive CSF syndrome and some special procedures, including examination of lymphocyte reactivity.

The use of protein beads as immunoadsorbent for the column fractionation of lymphocytes.

Beads of calf serum proteins (CSB) with antigen-antibody complexes or antigen by activation with glutaraldehyde were used as immunoadsorbents for the fractionation of mouse lymphocytes. T-lymphocytes could be separated by this method with high purity. The precursors of antibody-forming cells were completely eliminated from spleen and bone marrow cell suspensions. The unspecific binding of lymphocytes to CBS or Sepharose was similar and not selective for T or B lymphocytes.

Determination of cell mediated immunity against tumour associated antigens in patients of ENT.

In an unconventional assay system (MEM Test) Caspary & Field claimed in 1971 to have detected lymphocyte sensitization to a common tumour antigen in all patients with cancer. There was no evidence of histogenetic specificity to the reaction and their conclusions are in direct contradiction to those of all workers who have studied the cytotoxic activity of lymphocytes on tumour cells. To improve the specificity of the test system, another type of tumour antigen was used in MEM Test incubation. Tumour-associated antigens were prepared according to the hypertonic salt extraction method introduced by Reisfeld, Leonard, Meltzer et al. As shown by the results, information could be gained concerning the existence of a malignant tumour and its location.

Design and implementation of a neuromuscular training program following anterior cruciate ligament reconstruction.

Neuromuscular training programs are increasingly integrated into clinical practice for lower extremity rehabilitation. A few rehabilitation programs have been evaluated for patients with anterior cruciate ligament (ACL) deficiency and for injury prevention, but there is limited scientific evidence of the effect of neuromuscular training following ACL reconstruction. Therefore, a neuromuscular training program was developed for patients after ACL reconstruction. The objective of the neuromuscular training was to improve the ability to generate a fast and optimal muscle firing pattern, to increase dynamic joint stability, and to relearn movement patterns and skills necessary during activities of daily living and sports activities. The main areas considered when designing the postoperative rehabilitation program after ACL reconstruction were: ACL graft healing and ACL strain values during exercises, proprioception and neuromuscular control, and clinical studies on the effect of neuromuscular training programs. The rehabilitation program consists of balance exercises, dynamic joint stability exercises, jump training/plyometric exercises, agility drills, and sport-specific exercise. The patients exercise 3 times a week for 6 months. The scientific and clinical evidence for the rehabilitation program are described and the main exercises in the program are outlined.

A contribution to immunological tumour diagnostics in urology.

Fifty-four patients with malignant and other diseases of the urinary bladder and the prostate were examined for lymphocyte sensitization by means of electrophoretic mobility test (EM test). In this antigens from brain tissue (EF) and from malignomas of kidney, urinary bladder and prostate were used. Positive HEP values were obtained in 40 patients out of 42 with malignant tumours and in 5 patients out of 12 with other kidney diseases. With the corresponding tumour-associated antigens (TAA) in the EM-test, the histological tumour diagnosis could be confirmed in every case. Positive findings after using EF as well as the corresponding TAA are with great probability an indication of a malignant disease.

[Method of electrophoretic mobility of macrophages and its use in clinical immunology]. / Metod élektroforeticheskoi podvizhnosti makrofagov i ego primenenie v klinicheskoi immunologii.

A test of measurement of the electrophoretic mobility of macrophages (EMM) is used for detection of the product secreted by the sensitized lymphocytes after the contact with the antigen. Thus, by reduction of the macrophage mobility it is possible to assess the sensitization level of the lymphocyte population under study. This offers a possibility of using this test for the diagnosis of some infectious diseases, for provisional diagnosis of malignant growth, and also of destructive affections of the nervous system. The EMM test finds wide application in the determination of compatibility of donor's and the recipient's tissues before the transplantation, and also to assess the efficacy of immunodepressive therapy.

Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli.

Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites, but there are substantial roadblocks to their therapeutic application. High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity of these peptides and their susceptibility to proteolytic degradation, while subsequent purification of recombinant peptides often requires multiple steps and has not been cost-effective. Here we have developed methodologies appropriate for large-scale industrial production of HDPs; in particular, we describe (i) a method, using fusions to SUMO, for producing high yields of intact recombinant HDPs in bacteria without significant toxicity and (ii) a simplified 2-step purification method appropriate for industrial use. We have used this method to produce seven HDPs to date (IDR1, MX226, LL37, CRAMP, HHC-10, E5 and E6). Using this technology, pilot-scale fermentation (10L) was performed to produce large quantities of biologically active cationic peptides. Together, these data indicate that this new method represents a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans.

Bovine lactoferrin and lactoferricin interfere with intracellular trafficking of Herpes simplex virus-1.

Although both lactoferrin (Lf), a component of the innate immune system of living organisms, and its N-terminal pepsin cleavage product lactoferricin (Lfcin) have anti-herpes activity, the precise mechanisms by which Lf and Lfcin bring about inhibition of herpes infections are not fully understood. In the present study, experiments were carried out to characterize the activity of bovine Lf and Lfcin (BLf and BLfcin) against the Herpes simplex virus-1 (HSV-1). HSV-1 cellular uptake and intracellular trafficking were studied by immunofluorescence microscopy. In comparison to the untreated infected control cells, both the BLf- and BLfcin-treated cells showed a significant reduction in HSV-1 cellular uptake. The few virus particles that were internalized appeared to have a delayed intracellular trafficking. Thus, in addition to their interference with the uptake of the virus into host cells, Lf and Lfcin also exert their antiviral effect intracellularly.

The novel adjuvant combination of CpG ODN, indolicidin and polyphosphazene induces potent antibody- and cell-mediated immune responses in mice.

The need to enhance the immunogenicity of purified subunit antigens and modulate resulting immune responses has prompted the development of new adjuvants. Here, the ability of CpG oligodeoxynucleotides (ODN), a bovine host defence peptide indolicidin, and polyphosphazene to synergistically combine and enhance innate and adaptive immune responses was examined in mice. In vitro, the adjuvant combination of CpG ODN, indolicidin and polyphosphazene (CpG/indol/PP) enhanced the secretion of TNF-alpha, IL-12p40, and IL-6 by bone marrow-derived DCs (BMDCs) when compared to the individual components. When co-formulated with ovalbumin (OVA), CpG/indol/PP formed antigen-adjuvant complexes, and enhanced antibody and cell-mediated responses in mice, via both MHC I and II pathways, promoting a more balanced antibody-mediated and type 1-biased cell-mediated immune response. Furthermore, substitution of the proline residues of indolicidin with arginine increased the synergistic adjuvant effect of the peptide, and induced significantly higher IgG1 and IgG2a titers and IFN-gamma secretion, as well as increased uptake by antigen presenting cells. These results clearly demonstrate that the use of a combination of CpG ODN, indolicidin, and polyphosphazene as adjuvant can significantly enhance an antigen-specific immune response.