Treatment of antibiotic-resistant bacteria colonizing diabetic foot ulcers by OLED induced antimicrobial photodynamic therapy.

We evaluate the efficacy of antimicrobial Photodynamic Therapy (APDT) for inactivating a variety of antibiotic-resistant clinical strains from diabetic foot ulcers. Here we are focused on APDT based on organic light-emitting diodes (OLED). The wound swabs from ten patients diagnosed with diabetic foot ulcers were collected and 32 clinical strains comprising 22 bacterial species were obtained. The isolated strains were identified with the use of mass spectrometry coupled with a protein profile database and tested for antibiotic susceptibility. 74% of isolated bacterial strains exhibited adaptive antibiotic resistance to at least one antibiotic. All strains were subjected to the APDT procedure using an OLED as a light source and 16 µM methylene blue as a photosensitizer. APDT using the OLED led to a large reduction in all cases. For pathogenic bacteria, the reduction ranged from 1.1-log to > 8 log (Klebsiella aerogenes, Enterobacter cloaca, Staphylococcus hominis) even for high antibiotic resistance (MRSA 5-log reduction). Opportunistic bacteria showed a range from 0.4-log reduction for Citrobacter koseri to > 8 log reduction for Kocuria rhizophila. These results show that OLED-driven APDT is effective against pathogens and opportunistic bacteria regardless of drug resistance.

Phage Interactions with the Nervous System in Health and Disease.

The central nervous system manages all of our activities (e.g., direct thinking and decision-making processes). It receives information from the environment and responds to environmental stimuli. Bacterial viruses (bacteriophages, phages) are the most numerous structures occurring in the biosphere and are also found in the human organism. Therefore, understanding how phages may influence this system is of great importance and is the purpose of this review. We have focused on the effect of natural bacteriophages in the central nervous system, linking them to those present in the gut microbiota, creating the gut-brain axis network, as well as their interdependence. Importantly, based on the current knowledge in the field of phage application (e.g., intranasal) in the treatment of bacterial diseases associated with the brain and nervous system, bacteriophages may have significant therapeutic potential. Moreover, it was indicated that bacteriophages may influence cognitive processing. In addition, phages (via phage display technology) appear promising as a targeted therapeutic tool in the treatment of, among other things, brain cancers. The information collected and reviewed in this work indicates that phages and their impact on the nervous system is a fascinating and, so far, underexplored field. Therefore, the aim of this review is not only to summarize currently available information on the association of phages with the nervous system, but also to stimulate future studies that could pave the way for novel therapeutic approaches potentially useful in treating bacterial and non-bacterial neural diseases.

Ceramide is implicated in humoral peripheral and intrathecal autoimmune response in MS patients.

BACKGROUND: The disturbed metabolism of ceramide (Cer) is supposed to evoke the autoimmune response, contributing to MS pathology. OBJECTIVES: To determine levels of anti-Cer immunoglobulins G (IgGs) in the CSF and serum of subjects with various phenotypes of MS, and to investigate relationships between levels of anti-Cer antibodies and MS-related variables. METHODS: IgGs isolated from serum and the CSF of 68 MS patients and appropriate controls were examined for their reactivity to Cer subspecies. Their levels were compared between the studied groups and compartments, and analyzed with regard to clinical variables. RESULTS: Increased levels of anti-C16:0-, C18:0-, C18:1-, C24:0- and C24:1-Cer IgGs were detected in the CSF and serum of MS patients in comparison with controls. For IgGs against particular Cer subspecies, correlations were found between their CSF and serum level, as well as with the Link index. Serum and the CSF anti-Cer IgGs differed between patients with clinically isolated syndrome (CIS) and relapsing-remitting MS from those with progressive MS. No correlations were found between anti-Cer IgGs and other MS-related clinical variables. CONCLUSION: Patients with MS have shown altered panels of anti-Cer IgGs in the CSF and serum, which might suggest a relevant, though limited role of Cer as a target for autoimmune humoral response. Utility of antibodies against Cer subspecies as potential markers for MS activity and progression deserves further investigations.

Correction: Gebczak et al. Evaluation of PC12 Cells' Proliferation, Adhesion and Migration with the Use of an Extracellular Matrix (CorMatrix) for Application in Neural Tissue Engineering. Materials 2021, 14, 3858.

In the original publication [...].

Three-dimensional Collagen Scaffolds in Cultures of Olfactory Ensheathing Cells Used for Severed Spinal Cord Regeneration.

BACKGROUND/AIM: The regeneration of a completely damaged spinal cord is still a challenge in modern medicine. A promising treatment method is autologous transplantation of olfactory ensheathing cells (OECs). This study aimed primarily to test methods of culturing OECs with the use of materials and reagents that are certified for pharmaceutical use in the production of an advanced cell therapy product intended for humans. MATERIALS AND METHODS: The culture of OECs was performed using various modifications of the surface of the culture vessels (with fibronectin and poly-D-lysine). The number of cells was assessed after immunofluorescence staining using anti-fibronectin and anti-p75 NGF receptor antibodies. The study compared, in terms of surgical manipulations, scaffolds with OECs prepared based on 3 types of collagen: Acid Solubilized Telo Collagen and Pepsin Solubilized Atelocollagen, and the popular Corning collagen. RESULTS: We have shown that when suspending OECs in collagen gel, it is much better to use acid-solubilized collagen (ASC) than pepsin-solubilized collagen (PSC) because the 3D collagen scaffold from ASC provides much easier handling of the product during a surgical procedure. We also found that the OEC cultures should be grown on the surface modified with fibronectin. Furthermore, we have also shown that the optimal concentration of fetal bovine serum (FBS) for culturing these cells should be around 10%. CONCLUSION: The culture of OECs based on reagents intended for human use can be successfully carried out, obtaining sufficient OECs content in the heterogeneous cell culture to produce a functional advanced therapy medicinal product.

A Thorough Synthesis of Phage Therapy Unit Activity in Poland-Its History, Milestones and International Recognition.

The year 2020 marked 15 years of the Phage Therapy Unit in Poland, the inception of which took place just one year after Poland's accession to the European Union (2004). At first sight, it is hard to find any connection between these two events, but in fact joining the European Union entailed the need to adapt the regulatory provisions concerning experimental treatment in humans to those that were in force in the European Union. These changes were a solid foundation for the first phage therapy center in the European Union to start its activity. As the number of centers conducting phage therapy in Europe and in the world constantly and rapidly grows, we want to grasp the opportunity to take a closer look at the over 15-year operation of our site by analyzing its origins, legal aspects at the local and international levels and the impressive number and diversity of cases that have been investigated and treated during this time. This article is a continuation of our work published in 2020 summarizing a 100-year history of the development of phage research in Poland.

Results of a long-term uniform system of neurorehabilitation in patients with incomplete thoracic spinal cord injury.

INTRODUCTION AND OBJECTIVE: The results of kinesiotherapy treatment in patients after incomplete spinal cord injury (iSCI) are inconclusive, mostly due to different, subjective evaluation methods. The study aims to evaluate the range of functional regeneration in long-term 13 months follow-up using comparative neurophysiological tests after uniform kinesiotherapy in patients with thoracic iSCI. MATERIAL AND METHODS: Comparative tests were performed of sensory perception in dermatomes Th1-S1, electromyography (at rest-rEMG and during maximal contraction-mcEMG) in the muscles of the trunk and lower extremities, electroneurography (ENG) of the motor fibres of the lower extremities, and motor-evoked potential induced transcranially (MEP) before and after treatment in 25 iSCI patients. All subjects were treated with the same kinesiotherapeutic procedures. RESULTS: A moderate increase was found in amplitudes in rEMG and mcEMG recordings fromthe rectus abdominis and rectus femoris muscles, MEPs amplitudes, and amplitudes after peroneal nerve stimulations in ENG studies. There was no improvement in sensory perception. CONCLUSIONS: Following the proposed kinesiotherapy algorithm, patients after thoracic iSCI presented a moderate more motor than sensory functions improvement. Applied neurorehabilitation evoked normalization of muscle tension, moderate improvement of rectus abdominis and rectus femoris muscles motor units activity, and motor central and peripheral neural impulses transmission. The comparative neurophysiological assessment provide a more precise and objective insight into the functional status of afferent and efferent systems than the classical clinical approach in iSCI patients.

Awake craniotomy with dexmedetomidine during resection of brain tumours located in eloquent regions.

INTRODUCTION: An awake craniotomy (AC) is the gold standard for the resection of supra-tentorial brain tumours in eloquent areas. Intraoperative monitoring "on-demand" of essential eloquent brain functions and the increasing need to preserve higher intellectual functions during surgery requires a unique anaesthetic approach during AC. Dexmedetomidine is considered the first-choice pharmacological agent for sedation during AC. MATERIAL AND METHODS: Twenty-six patients with a single brain tumour located in areas of eloquent brain function were enrolled in this prospective study. The patients underwent AC under conscious sedation. Motor-evoked potentials and brainstem-evoked auditory potentials were measured using neurophysiological tests during surgery to assess brain potentials. Intraoperative brain relaxation was reached using a modified Bristow scale. Neuromonitoring and psychological tests were maintained until meningeal closure. RESULTS: All operations were carried out successfully, and no reoperations were needed. No significant impact on circulatory and respiratory parameters was observed during conscious sedation based on dexmedetomidine. Neither instrumental airway support nor conversion to general anaesthesia was necessary. Brain relaxation was good in 84% of cases. Intraoperative epileptic episodes were observed in 15% of the patients. Neuro-logical and psychological monitoring was satisfactory. Unaltered muscle force was observed postoperatively in 88% of the patients. CONCLUSIONS: AC performed under conscious sedation, and dexmedetomidine infusion without instrumental airway support, was safe and well-tolerated by patients with comfortable physiological sleep for most of the procedure. This approach to AC was associated with minimal risk of perioperative adverse events and may be particularly beneficial in patients with severe comorbidities.

The Long-Term Effect of Treatment Using the Transcranial Magnetic Stimulation rTMS in Patients after Incomplete Cervical or Thoracic Spinal Cord Injury.

Repetitive transcranial magnetic stimulation (rTMS) may support motor function recovery in patients with incomplete spinal cord injury (iSCI). Its effectiveness mainly depends on the applied algorithm. This clinical and neurophysiological study aimed to assess the effectiveness of high-frequency rTMS in iSCI patients at the C2-Th12 levels. rTMS sessions (lasting 3-5 per month, from 2 to 11 months, 5 months on average) were applied to 26 iSCI subjects. The motor cortex was bilaterally stimulated with a frequency at 20-25 Hz and a stimulus strength that was 70-80% of the resting motor threshold (15.4-45.5% maximal output) during one therapeutic session. Surface electromyography (sEMG) recordings at rest and during maximal contractions and motor evoked potential (MEP) recordings were performed from the abductor pollicis brevis (APB) and the tibialis anterior (TA) muscles. The same neurophysiological studies were also performed in patients treated with kinesiotherapy only (K group, n = 25) and compared with patients treated with both kinesiotherapy and rTMS (K + rTMS). A decrease in sEMG amplitudes recorded at rest from the APB muscles (p = 0.001) and an increase in sEMG amplitudes during the maximal contraction of the APB (p = 0.001) and TA (p = 0.009) muscles were found in the K + rTMS group. A comparison of data from MEP studies recorded from both APB and TA muscles showed significant changes in the mean amplitudes but not in latencies, suggesting a slight improvement in the transmission of spinal efferent pathways from the motor cortex to the lower spinal centers. The application of rTMS at 20-25 Hz reduced spasticity in the upper extremity muscles, improved the recruitment of motor units in the upper and lower extremity muscles, and slightly improved the transmission of efferent neural impulses within the spinal pathways in patients with C2-Th12 iSCI. Neurophysiological recordings produced significantly better parameters in the K + rTMS group of patients after therapy. These results may support the hypothesis about the importance of rTMS therapy and possible involvement of the residual efferent pathways including propriospinal neurons in the recovery of the motor control of iSCI patients.

Evaluation of PC12 Cells' Proliferation, Adhesion and Migration with the Use of an Extracellular Matrix (CorMatrix) for Application in Neural Tissue Engineering.

The use of extracellular matrix (ECM) biomaterials for soft tissue repair has proved extremely successful in animal models and in some clinical settings. The aim of the study was to investigate the effect of the commercially obtained CorMatrix bioscaffold on the viability, proliferation and migration of rat pheochromocytoma cell line PC12. PC12 cells were plated directly onto a CorMatrix flake or the well surface of a 12-well plate and cultured in RPMI-1640 medium and a medium supplemented with the nerve growth factor (NGF). The surface of the culture plates was modified with collagen type I (Col I). The number of PC12 cells was counted at four time points and then analysed for apoptosis using a staining kit containing annexin V conjugate with fluorescein and propidium iodide (PI). The effect of CorMatrix bioscaffold on the proliferation and migration of PC12 cells was tested by staining the cells with Hoechst 33258 solution for analysis using fluorescence microscopy. The research showed that the percentage of apoptotic and necrotic cells was low (less than 7%). CorMatrix stimulates the proliferation and possibly migration of PC12 cells that populate all levels of the three-dimensional architecture of the biomaterial. Further research on the mechanical and biochemical capabilities of CorMatrix offers prospects for the use of this material in neuro-regenerative applications.

Isolation and Characterization of Phages Active against Paenibacillus larvae Causing American Foulbrood in Honeybees in Poland.

The aim of this study was the isolation and characterization, including the phage effect on honeybees in laboratory conditions, of phages active against Paenibacillus larvae, the causative agent of American Foulbrood-a highly infective and easily spreading disease occurring in honeybee larva, and subsequently the development of a preparation to prevent and treat this dangerous disease. From the tested material (over 2500 samples) 35 Paenibacillus spp. strains were obtained and used to search for phages. Five phages specific to Paenibacillus were isolated and characterized (ultrastructure, morphology, biological properties, storage stability, and genome sequence). The characteristics were performed to obtain knowledge of their lytic potential and compose the final phage cocktail with high antibacterial potential and intended use of future field application. Preliminary safety studies have also been carried out on healthy bees, which suggest that the phage preparation administered is harmless.

Distinctive sphingolipid patterns in chronic multiple sclerosis lesions.

Multiple sclerosis (MS) is a CNS disease characterized by immune-mediated demyelination and progressive axonal loss. MS-related CNS damage and its clinical course have two main phases: active and inactive/progressive. Reliable biomarkers are being sought to allow identification of MS pathomechanisms and prediction of its course. The purpose of this study was to identify sphingolipid (SL) species as candidate biomarkers of inflammatory and neurodegenerative processes underlying MS pathology. We performed sphingolipidomic analysis by HPLC-tandem mass spectrometry to determine the lipid profiles in post mortem specimens from the normal-appearing white matter (NAWM) of the normal CNS (nCNS) from subjects with chronic MS (active and inactive lesions) as well as from patients with other neurological diseases. Distinctive SL modification patterns occurred in specimens from MS patients with chronic inactive plaques with respect to NAWM from the nCNS and active MS (Ac-MS) lesions. Chronic inactive MS (In-MS) lesions were characterized by decreased levels of dihydroceramide (dhCer), ceramide (Cer), and SM subspecies, whereas levels of hexosylceramide and Cer 1-phosphate (C1P) subspecies were significantly increased in comparison to NAWM of the nCNS as well as Ac-MS plaques. In contrast, Ac-MS lesions were characterized by a significant increase of major dhCer subspecies in comparison to NAWM of the nCNS. These results suggest the existence of different SL metabolic pathways in the active versus inactive phase within progressive stages of MS. Moreover, they suggest that C1P could be a new biomarker of the In-MS progressive phase, and its detection may help to develop future prognostic and therapeutic strategies for the disease.

Treatment of patients with cervical and upper thoracic incomplete spinal cord injury using repetitive transcranial magnetic stimulation.

PURPOSE: To evaluate the short- and long-term effectiveness of repetitive transcranial magnetic stimulation with parameters based on results of comparative neurophysiological studies in patients with incomplete spinal cord injury. Results may help to understand mechanisms responsible for regeneration of the incomplete spinal cord after injury. METHODS: Repetitive transcranial magnetic stimulation sessions (three to five sessions per month for not less than 5 months) to 15 patients with C4-Th2 incomplete spinal cord injury were applied with individually designed parameters. One session consisted of bilateral stimulation of the primary motor cortex (for 10 min each with 800 stimuli in 2-s lasting trains and the inter-train intervals of 28 s) with frequency at 20-22 Hz and stimulus strength that was 70%-80% of the resting motor threshold (0.84-0.96 T). Recordings of surface electromyography at rest and during the attempt of maximal muscle contractions and motor evoked potentials were performed from abductor pollicis brevis and tibialis anterior muscles bilaterally. Amplitude parameters of surface electromyography and motor evoked potentials were used as outcomes. All neurophysiological tests were comparatively applied before and after treatment. RESULTS: Decrease in surface electromyography amplitudes recorded at rest from abductor pollicis brevis (p = 0.009), increase in surface electromyography amplitudes during maximal contraction of abductor pollicis brevis (p = 0.03) and increase in motor evoked potential parameters recorded from abductor pollicis brevis (p = 0.04) were found. CONCLUSION: Proposed repetitive transcranial magnetic stimulation algorithm reduced the increased muscle tension in upper extremity muscles, improved the function of upper extremity muscle motor units and slightly improved the transmission of efferent neural impulses within spinal pathways. Besides functional recovery in descending spinal pathways, repetitive transcranial magnetic stimulation may also inhibit inevitable pathological changes in nerves.

Partial Recovery of Proprioception in Rats with Dorsal Root Injury after Human Olfactory Bulb Cell Transplantation.

Transplanted human olfactory ensheathing cells (hOECs) were mixed with collagen into a unilateral transection of four dorsal roots (C6-T1) in a rat model. By mixing with collagen, the limited numbers of hOEC were maximized from an olfactory bulb biopsy and optimize cavity filling. Cyclosporine was administered daily to prevent immune rejection. Forelimb proprioception was assessed weekly in a vertical climb task. Half of the rats receiving hOEC transplants showed some functional improvement ("responders") over six weeks of the study while the other half did not ("nonresponders") and performed similarly to "injured only" rats. Transplanted cells were seen at both one week and six weeks after the surgical procedure; many were concentrated within the lesion cavity, but others were found with elongated processes in the overlying connective tissue. There were some fibers in the injury area associated with transplanted cells that were immunostained for neurofilament and TUJ1. Responder and nonresponder rats were compared with regard to microglial activation within the deep dorsal horn of cervical levels C7, C8 and also axon loss within the cuneate fasciculus at cervical level C3. Little difference was seen in microglial activation or axonal loss that could account for the improved proprioception in the responders group. This preliminary study is the first to transplant human olfactory bulb cells into a rat model of dorsal root injury; by refining each component part of the procedure, the repair potential of OECs can be maximized in a clinical setting.

Antiphage activity of sera during phage therapy in relation to its outcome.

AIM: The aim was to study the association between the phage neutralization of patients' sera and the clinical outcome of phage therapy (PT). PATIENTS: About 62 patients with various bacterial infections receiving PT as well as 30 healthy volunteers were studied. MATERIALS & METHODS: Antiphage activity of sera (AAS) was examined using the phage neutralization test of different types of phages before and during PT in relation to the route of phage administration and correlated with the results of PT. RESULTS & CONCLUSION: The analysis of the association between AAS level and clinical results indicated that the level of AAS is not correlated with the outcome of PT.

Antibody Production in Response to Staphylococcal MS-1 Phage Cocktail in Patients Undergoing Phage Therapy.

In this study, we investigated the humoral immune response (through the release of IgG, IgA, and IgM antiphage antibodies) to a staphylococcal phage cocktail in patients undergoing experimental phage therapy at the Phage Therapy Unit, Medical Center of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy in Wroclaw, Poland. We also evaluated whether occurring antiphage antibodies had neutralizing properties toward applied phages (K rate). Among 20 examined patients receiving the MS-1 phage cocktail orally and/or locally, the majority did not show a noticeably higher level of antiphage antibodies in their sera during phage administration. Even in those individual cases with an increased immune response, mostly by induction of IgG and IgM, the presence of antiphage antibodies did not translate into unsatisfactory clinical results of phage therapy. On the other hand, a negative outcome of the treatment occurred in some patients who showed relatively weak production of antiphage antibodies before and during treatment. This may imply that possible induction of antiphage antibodies is not an obstacle to the implementation of phage therapy and support our assumption that the outcome of the phage treatment does not primarily depend on the appearance of antiphage antibodies in sera of patients during therapy. These conclusions are in line with our previous findings. The confirmation of this thesis is of great interest as regards the efficacy of phage therapy in humans.

Phage Therapy: Combating Infections with Potential for Evolving from Merely a Treatment for Complications to Targeting Diseases.

Antimicrobial resistance is considered to be one of the greatest challenges of medicine and our civilization. Lack of progress in developing new anti-bacterial agents has greatly revived interest in using phage therapy to combat antibiotic-resistant infections. Although a number of clinical trials are underway and more are planned, the realistic perspective of registration of phage preparations and their entering the health market and significantly contributing to the current antimicrobial crisis is rather remote. Therefore, in addition to planning further clinical trials, our present approach of phage treatment carried out as experimental therapy (compassionate use) should be expanded to address the growing and urgent needs of increasing cohorts of patients for whom no alternative treatment is currently available. During the past 11 years of our phage therapy center's operation, we have obtained relevant clinical and laboratory data which not only confirm the safety of the therapy but also provide important information shedding more light on many aspects of the therapy, contributing to its optimization and allowing for construction of the most appropriate clinical trials. New data on phage biology and interactions with the immune system suggest that in the future phage therapy may evolve from dealing with complications to targeting diseases. However, further studies are necessary to confirm this promising trend.

The Effect of Bacteriophage Preparations on Intracellular Killing of Bacteria by Phagocytes.

Intracellular killing of bacteria is one of the fundamental mechanisms against invading pathogens. Impaired intracellular killing of bacteria by phagocytes may be the reason of chronic infections and may be caused by antibiotics or substances that can be produced by some bacteria. Therefore, it was of great practical importance to examine whether phage preparations may influence the process of phagocyte intracellular killing of bacteria. It may be important especially in the case of patients qualified for experimental phage therapy (approximately half of the patients with chronic bacterial infections have their immunity impaired). Our analysis included 51 patients with chronic Gram-negative and Gram-positive bacterial infections treated with phage preparations at the Phage Therapy Unit in Wroclaw. The aim of the study was to investigate the effect of experimental phage therapy on intracellular killing of bacteria by patients' peripheral blood monocytes and polymorphonuclear neutrophils. We observed that phage therapy does not reduce patients' phagocytes' ability to kill bacteria, and it does not affect the activity of phagocytes in patients with initially reduced ability to kill bacteria intracellularly. Our results suggest that experimental phage therapy has no significant adverse effects on the bactericidal properties of phagocytes, which confirms the safety of the therapy.

Functional regeneration of supraspinal connections in a patient with transected spinal cord following transplantation of bulbar olfactory ensheathing cells with peripheral nerve bridging.

Treatment of patients sustaining a complete spinal cord injury remains an unsolved clinical problem because of the lack of spontaneous regeneration of injured central axons. A 38-year-old man sustained traumatic transection of the thoracic spinal cord at upper vertebral level Th9. At 21 months after injury, the patient presented symptoms of a clinically complete spinal cord injury (American Spinal Injury Association class A-ASIA A). One of the patient's olfactory bulbs was removed and used to derive a culture containing olfactory ensheathing cells and olfactory nerve fibroblasts. Following resection of the glial scar, the cultured cells were transplanted into the spinal cord stumps above and below the injury and the 8-mm gap bridged by four strips of autologous sural nerve. The patient underwent an intense pre- and postoperative neurorehabilitation program. No adverse effects were seen at 19 months postoperatively, and unexpectedly, the removal of the olfactory bulb did not lead to persistent unilateral anosmia. The patient improved from ASIA A to ASIA C. There was improved trunk stability, partial recovery of the voluntary movements of the lower extremities, and an increase of the muscle mass in the left thigh, as well as partial recovery of superficial and deep sensation. There was also some indication of improved visceral sensation and improved vascular autoregulation in the left lower limb. The pattern of recovery suggests functional regeneration of both efferent and afferent long-distance fibers. Imaging confirmed that the grafts had bridged the left side of the spinal cord, where the majority of the nerve grafts were implanted, and neurophysiological examinations confirmed the restitution of the integrity of the corticospinal tracts and the voluntary character of recorded muscle contractions. To our knowledge, this is the first clinical indication of the beneficial effects of transplanted autologous bulbar cells.