Background: Cervical dystonia (CD) patients have impaired working memory, processing speed and visual-motor integration ability. We used magnetoencephalography (MEG) to investigate changes in cerebral oscillations in CD patients during an executive function test, before and after administration of botulinum toxin. Methods: MEG data were collected from five CD patients while they performed a visual continuous performance task (CPT), before and after they received a botulinum toxin injection. MEG data was also collected on five controls matched for age and gender. Coherence source imaging was performed to quantify network connectivity of subjects. Results: Controls demonstrated two errors with visual CPT; CD patients demonstrated six and three errors pre- and post-botulinum toxin respectively. After botulinum toxin, mean time from cue to correct response was 0.337 s in controls, 0.390 s in patients before botulinum toxin injection, and 0.366 s after the injection. Differences in coherence between controls and patients were found in the following brain regions: Fronto-frontal, fronto-parietal, fronto-striatal, fronto-occipital, parieto-parietal and temporo-parietal. Intrahemispheric and interhemispheric networks were affected. Post injection, there was minimal change in coherence in the above-mentioned networks. Discussion: Neuropsychological testing suggests difference in coherence in frontal circuits between CD cases and controls during the visual CPT, which may reflect subjects’ increased difficulty with the task. Botulinum toxin is associated with minimal improvement with executive function in CD. Dystonia is defined as a variable pattern of sustained involuntary movement characterized by over-active muscle contractions, which are often associated with voluntary movement . While iVarious aspects of altered cognition have been described in dystonia. For example, Romano et al. showed impairment in working memory, processing speed, visual motor ability and short-term memory in patients with cranio-cervical dystonia . TestingIn addition to neuropsychological testing, functional connectivity has been a topic of interest in dystonia. Abnormal fronto-striatal connectivity and cerebellar involvement has been reported in hereditary dystonia using 18F-fluoro-deoxyglucose positron emission tomography (FDG-PET) and diffusion tensor imaging (DTI) in the resting state . Using rMagnetoencephalography coherence source imaging (MEG-CSI) has also been used to investigate CD ,10. Our N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) protein family, which are responsible for mediation of the fusion of synaptic vesicles with the neuronal presynaptic plasma membrane [Botulinum toxins function by cleaving members of the soluble membrane . Proteolmembrane .While studies with fMRI and other functional imaging techniques provide insight into CD in a resting state, not much is known about task-specific changes in functional connectivity in CD patients. In this current project, our aim was to use MEG CSI to investigate changes in CD patients’ cerebral oscillations at the network level during performance of a task assessing executive function.MEG data were collected from five patients with CD , before To be included in the study, patients needed to have gained significant benefit from the previous injections. MEG studies were conducted between 2 and 3 weeks post injection. For comparison, MEG data was also collected from five age- and gender-matched controls lacking any neurological disorder. The controls were not injected as part of this study.MEG recordings were conducted while patients and controls were performing a visual continuous performance test (CPT). In this task, random letters were shown briefly for 150 ms with a 1.8 s inter-stimulus interval . Participants were instructed to respond as quickly as possible by pressing a button when the “X” target stimulus followed the “A” cue stimulus . This CPOscillations of spontaneous neural activity can be detected by MEG and seen in the in MEG waveform recordings. These neuronal oscillations can be quantified by applying a short-time Fast Fourier Transform (stFFT). After transformation to a time-frequency representation, the strength of network interactions can be determined by calculating the coherence, which is a measure of synchrony between signals from different regions for each FFT frequency. Using MEG-Coherence Source Imaging (MEG-CSI), Elisevich et al. found regions of high coherence primarily located in the cerebral cortex and localized in the epileptogenic regions, and a seizure-free outcome was associated with removal of these highly coherent networks ,16,17.A detailed description of the MEG based methodology used in this study has been previously published by our lab in a resting state MEG study of CD patients .This study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Permission was received from Henry Ford Hospital Institutional Review Board (IRB # 9460) in April 2015 and last renewed in April 2018. All persons gave their written informed consent prior to their inclusion in the study. No patient-identifiable information was included in the text.During the visual CPT, control subjects made two errors in total. CD patients made six errors during the pre-botulinum toxin treatment recording and three errors post-botulinum toxin. The average time from visual cue to correct response was 0.337 seconds in controls, 0.390 seconds in CD patients pre-botulinum toxin and 0.366 seconds in CD patients post-botulinum toxin .p < 0.05). Pathways with a statistically significant difference in coherence between patients and controls were the following: Fronto-frontal, fronto-parietal, fronto-temporal, fronto-striatal, fronto-occipital, parieto-parietal, parieto-striatal and temporo-parietal  were statistically significantly different between the two groups  .After treatment, out of a total of 1431 networks, 193 interhemispheric networks and 105 intrahemisheric networks (24 right and 81 left) were statistically significantly different between the two groups. Coherence between the same pathways remained statistically significant .On comparing patients post- and pre-botulinum toxin, out of a total of 1431 networks only five networks were statistically significantly different: fronto-parietal, fronto-cingulate, fronto-occipital, parieto-insular and parieto-temporal.p < 0.05).Coherence was lower in the alpha band pre-treatment compared to post-treatment in three of the five CD patients. Beta and Gamma band coherence was lower in three of the five patients, post-treatment (This is the first study using MEG-CSI to assess task specific network connectivity in patients with CD and to assess the impact of botulinum toxin treatment giving symptomatic benefit for dystonic features. Our study reiterates the importance of frontal networks in the cerebral cortex in tasks that assess attention and other components of executive function. It further indicates the importance of the parietal lobe in selective attention.As previously stated, the spectrum of executive function in dystonia has been of great interest, even though studies to date have reported inconsistencies ,5. TestiOur previous study assessing changes in coherence using resting state had shown that compared to controls, CD patients showed a state of lower amplitude and frequency of synchronicity, and a lower frequency of synchronicity of oscillating neurons, or coherence . With taOur study is also the first to assess the effect of botulinum toxin treatment on network connectivity while performing a task assessing executive function using MEG-CSI. Previously, we showed that efficacious botulinum toxin use is associated with a change in coherence in specific pathways in the resting state, suggesting a role of sensorimotor integration in clinical benefit . Our curPallidal oscillatory activity in theta-alpha range correlates with involuntary muscle activity in dystonia . In theiIn our study, our sample size precluded us from commenting on spectral signals in controls compared to CD patients and with botulinum toxin injection.A significant contribution of our study is its use of MEG-CSI as a means of imaging brain function. This technique offers high temporal and spatial resolution and provides a direct measure of neuronal activity, unlike PET and fMRI, which measure changes in metabolic responses and blood flow and offer less temporal resolution . The magOur study has several limitations. We did not employ rating scales to assess changes in motor improvement in CD at the time of MEG-CSI testing. The control subjects did not receive saline injections as a part of the protocol to act as a control for the post-injection CD patients. While an independent component analysis was carried out to filter out heart sounds and other noise, it is possible that some movement artifacts might have partly confounded the results. While we looked at 1431 network connections per patient, our sample size of five subjects in this pilot study limits the generalizability of our results. A larger sample size is needed to carry out a more thorough comparison of neuropsychological testing and MEG-CSI findings. A larger number of participants would further allow us to characterize potential difference in coherence spectra between controls and patients, and with and without botulinum toxin.Our pilot study, based on MEG data on five CD patients and age and sex matched controls predominantly shows a difference in coherence in the frontal circuits, those associated with executive functioning, during the visual CPT. This may reflect increased difficulty with the task in CD patients, which is also suggested by neuropsychological testing. Using a larger sample size, the difference in MEG coherence and spectral signals, between CD patients and controls, at resting state and with specific cognitive-tasks needs to be explored further.


Open Access Atlas of Otolaryngology, Head and Neck Operative Surgery and Open Access Guide to Audiology and Hearing Aids for Otolaryngologists, as well as the African Head and Neck Society (AfHNS) Clinical Practice Guidelines for Head and Neck Cancer in Developing Countries and Limited Resource Settings. The author outlines the simplicity, advantages, and popularity of this form of publication and why societies and individuals should embrace open access publishing to benefit especially those studying and practicing in developing countries. He discusses some of the challenges related to open access publishing and calls for medical societies to become involved in evaluating the quality of open access texts and videos for their members.Accessing educational and scientific material is key to improving otolaryngology care in developing countries. Yet current financial models of publishers restrict access to academic information. This article describes the author’s experience with self-publishing 2 open access textbooks,  Open Access Atlas of Otolaryngology, Head and Neck Operative Surgery (OAA) and Open Access Guide to Audiology and Hearing Aids for Otolaryngologists. More than 100 authors from >20 countries have contributed, and volunteers have translated chapters into Portuguese, Spanish, and French. Recently, the African Head and Neck Society (www.afhns.org) launched another open access resource, the African Head and Neck Society (AfHNS) Clinical Practice Guidelines for Head and Neck Cancer in Developing Countries and Limited Resource Settings. This article describes the author’s experience with these open access resources.Accessing educational and scientific material is key to improving otolaryngology (ENT) care in developing countries. Yet many trainees, practitioners, and researchers cannot afford textbooks and pay-to-view journals. The author self-publishes 2 open access textbooks, OAA (http://www.entdev.uct.ac.za/guides/open-access-atlas-of-otolaryngology-head-neck-operative-surgery) provides detailed descriptions of surgical procedures. The authors are mostly international leaders who volunteered to contribute. It includes surgical procedures no longer performed in developed countries such as hammer and gouge mastoidectomy, which would not be included in modern textbooks. Being in electronic format, chapters are very detailed with numerous photographs and videoclips. Chapters are still being added, and existing chapters are edited from time to time. This illustrates an important advantage of electronic textbooks: they do not have to be completed before being published, as chapters can be added over time. OAA: as additional chapters are added, it becomes better known and translations are added. The OAA received the Open Education Consortium 2017 Award for Open Education Excellence, a tribute to its contributors (http://www.oeconsortium.org/projects/open-education-awards-for-excellence/2017-oe-award-winners-oer-categories/).The 2 This textbook, Open Access Guide to Audiology and Hearing Aids for Otolaryngologists (http://www.entdev.uct.ac.za/guides/open-access-guide-to-audiology-and-hearing-aids-for-otolaryngologists), aims to improve audiologic care by ENTs. Only 4 countries in sub-Saharan Africa train audiologists. Hence, audiology and hearing aid fittings are generally done by ENTs, as occurs in some central European countries.https://creativecommons.org). Readers may use material provided it is referenced. Hyperlinks to individual chapters are maintained on the IFOS Developing World ENT website (www.entdev.uct.ac.za.guides/).Chapters are submitted in Microsoft Word format, and the author (J.J.F.) edits, formats, and uploads a PDF version to the University of Cape Town webserver. The textbooks are registered with the Creative Commons, a nonprofit corporation that issues public copyright licenses . Although intended for developing countries, 8 of the 10 top users are developed countries Table 1.Editors have little idea what readers wish to read when planning a textbook. However, online textbooks, of which chapters are individually downloaded, provide such data Table 2.Even though it is tempting to monetize open access textbooks to raise funds for worthy causes, this was not done, as the authors contributed with the understanding that it is a philanthropic project.www.afhns.org) launched this novel open access resource in 2019. African surgeons and oncologists formulated the guidelines (https://developingworldheadandneckcancerguidelines.com/) in consultation with American and European colleagues to ensure resource-appropriate best care for patients in developing countries. Thyroid, parotid, and submandibular gland guidelines have been released and are precisely tailored to availability of diagnostic investigations and therapeutic interventions. Guidelines for other head and neck sites will be released in the future. This ability to self-publish web-based guidelines permits countries in the “Global South” to assume leadership and take responsibility for their own regions without having to work through established institutions or publishers. That there were 1630 visitors and 7860 page views of the guidelines in the first 3 months demonstrates the power of web-based publishing.International guidelines have limited value in limited resource settings because of lack of cytology, imaging, (chemo)radiation, complex surgery, and even thyroid and calcium replacement after thyroidectomy. Clinicians in the “Global South” should therefore develop resource-appropriate guidelines for developing countries, rather than be guided by institutions in the “Global North,” as they know best what the challenges, limitations, and possibilities are to deliver care. The AfHNS (Apart from the benefits previously described, reasons why societies and individual authors should adopt an open access policy for educational material include the following:Our open access texts have not cost a cent to produceAuthors retain copyrightPublishers cannot discontinue publicationsChapters can be edited/updated, even on a mobile phoneReaches remote areas including conflict zones where textbooks are inaccessibleNiche topics can be includedCan be read on a mobile phoneElectronic texts will become translatable into many languagesThe author’s experience demonstrates how open access publishing is a great leveler across developed and developing worlds by making educational material freely available and empowering one to bypass established publishers and institutions.However, open access publishing presents challenges such as the pay-to-publish model, a barrier for authors from developing countries, and concerns about the quality of predatory journals. Just as governments are regulating social media, medical societies should identify reputable YouTube videos, textbooks, and journals for their membership.Johannes J. Fagan, conceived, designed, and authored the article.Competing interests: None.Sponsorships: None.Funding source: None.


Unfortunately, the diffusible nature of these ions renders them difficult to study with traditional microscopy in situ within ex vivo brain tissue sections. This mini-review examines the recent progress in the field, using direct elemental mapping techniques to study ion homeostasis during normal brain physiology and pathophysiology, through measurement of ion distribution and concentration in ex vivo brain tissue sections. The mini-review examines the advantages and limitations of specific techniques: proton induced X-ray emission (PIXE), X-ray fluorescence microscopy (XFM), secondary ion mass spectrometry (SIMS), laser-ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), and the sample preparation requirements to study diffusible ions with these methods.Diffusible ions (Na Cell membrane potential for example, is maintained by ion pumps that carefully control intra-cellular and extra-cellular concentrations of Na+, K+, Mg2+, Ca2+, and Cl–  enables communication along nerve cells in the form of electrical currents . The brain has evolved to use a number of neurotransmitter systems , upon which neurotransmitter-to-receptor binding opens ion channels in cell membranes. The opening of ion channels results in rapid diffusion of ions across the intra-cellular/extra-cellular concentration gradient, thus generating an action potential. Elegant approaches have been developed to study electrical impulses generated by ion influxes during neuron and glial signaling , or to directly image changes in specific ionic gradients in vitro . Direct measurement of ionic homeostasis within tissues, in animal models of brain injury or pathology has been more difficult to achieve.Diffusible ions also perform specialized functions in brain nerve cells, e.g., neurotransmission. The diffusible nature of the aforementioned ions  following perturbation of brain physiology, often achieved with agonist or antagonists that activate or deactivate ion channels or receptors. Ion homeostasis can be directly monitored and 23Na , but spaand 23Na , have enn injury , ischemin injury , hemorrhn injury , schizopn injury , and epin injury , to namen injury . This miex vivo imaging, including all the analytical methods described in this article. Practically, it is impossible for ex vivo analysis to completely reproduce the in vivo condition. The overarching aim of sample preparation for ex vivo analyses should therefore, be to best preserve the in vivo chemical composition and distribution. In some cases, where substantive changes in chemical composition or distribution may occur, analyses may still be valid if the results from ex vivo analysis are at least proportional to the in vivo condition. These considerations are especially important for ex vivo analyses of concentration and distribution of diffusible ions, which are easily re-distributed or leached from the sample by aqueous reagents used during perfusion or chemical fixation   induced X-ray emission (PIXE) to elemental mapping are contained within numerous reviews. Although detailed explanation of the technique is beyond the scope this mini-review, in brief: PIXE arises from ionization of an atom through ejection of a core electron  to yield a core-hole. The core-hole is subsequently filled via relaxation of an outer shell electron, which releases an X-ray with an energy characteristic of the relaxation process involved. The energy of the emitted X-ray photon, therefore, is proportional to the stabilization energy gained when an outer shell electron fills the inner core-hole. This process fundamentally differs from X-ray fluorescence microscopy  as the core hole is generated by an ion beam as opposed to an incident photon (X-ray) absorption. This underlying difference gives rise to an inherent relative increase in sensitivity of PIXE measurements relative to XFM, for detection of lighter elements, such as diffusible ions (Naand Cl–) . Unfortuand Cl–) . The spaand Cl–) . Therefoand Cl–) , PIXE isand Cl–) . PIXE haischemia , and epiischemia . Indeed,epilepsy . Interesoccurred .In contrast to PIXE, XFM is dependent on the creation of a core hole through absorption of an X-ray photon, which promotes a core electron to an unoccupied orbital (resonant absorption) or the continuum (ionization). Detailed reviews on the theory and instrumentation of XFM for elemental mapping are found within the references below. The diffraction-limited spatial resolution that can be achieved with the short wavelength of X-rays used for XFM is on the nano scale, but this is rarely achieved (see following review articles for further details) . Typical+, K+, Mg2+, Ca2+, and Cl–) occurs over the energy range 1000–3000 eV and photons in this relatively low-energy range impose technical limitations for both PIXE and XFM measurements. The Be window on energy dispersive X-ray detectors, such as Si drift detectors absorb much of the emitted X-ray fluorescence across this energy range. Further, Ar(g), which composes ∼0.93% of air, as well as the variable amount of water vapor (humidity) and sample-to-detector pathlength will all attenuate much of the emitted X-ray fluorescence signal at these low energies ((g)-filled sample environments for PIXE and XFM experiments may be required  that occur in experimental rodent models of ischemic stroke , the ischemic penumbra , and healthy tissue, following stroke  following epileptic seizures in rodent models  is a key driver of cell damage during seizures or after ischemic stroke. Cat models , b, and t models .– levels in brain parenchyma surrounding the lateral ventricles . The most widely used application of mass spectrometry for elemental mapping is laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) (Mass spectrometry offers an inherent advantage to detect lighter ions such as Na-ICP-MS) . The spa-ICP-MS) , which i-ICP-MS) . Thus, w-ICP-MS) . However-ICP-MS) .Although suitable to the study of diffusible ions, LA-ICP-MS has mainly found widespread use in the study of transition metals in brain tissues . This is+, Mg2+, K+, Ca2+, and Cl–) (–1), and can detect both the lighter elements as well as most of the heavier elements (+ or O–) to scan the sample surface to generate secondary ions for detection, and it routinely achieves elemental mapping to spatial resolution of 50–100 nm (2). In this respect, nano-SIMS complements LA-ICP-MS, with the latter being used for survey scans of larger areas, while the former can be used to probe sub-cellular details. The preservation of distributions of diffusible ions also represents a challenge for nano-SIMS analysis due to its requirement to preserve distributions of diffusible ions at nanoscale. As analysis must be conducted in an ultra-high vacuum, high-pressure freezing is often utilized to rapidly freeze a small volume of tissue, followed by freeze substitution and resin infiltration at low temperatures  to enable nano-SIMS analysis of these diffusible ions  offers the analytical advantages associated with mass spectrometry – excellent detection limits and sensitivity to light elements  . Compareelements . Nano-SI0–100 nm . Unfortuble ions . The senischemia , traumatischemia , and metischemia . Nano-SIischemia , associaischemia .+, Mg2+, Cl–, K+, Ca2+), in situ, in ex vivo tissue sections. Such capability is much needed, due to the important roles such ions play in healthy brain physiology and pathophysiology. The unique advantages and limitations of the various elemental mapping techniques  can be leveraged during experimental design, to develop strategies most suited to target lighter ions  or heavier ions , and for analyses at sub-cellular, or cellular spatial resolution. It is anticipated that increased awareness of these capabilities to study diffusible ions will see further integration of these methods into the “routine-toolbox” available for the modern neuroscientists.Although direct elemental mapping techniques have been most readily applied to study transition and heavy metal ions within the CNS, with careful selection of sample preparation protocols they are also well suited to study diffusible ions , ischemic stroke (MP and MK), intra-cerebral hemorrhage (FC), and traumatic brain injury . HJ, EM, MP, and MH were specifically interested in the development of analytical methodology to study diffusible ions. All authors were involved in the writing and editing of the manuscript, specific author contributions (writing and editing) were: DH: primary Ph.D. student conducting literature review, writing manuscript, and preparing figures. WA: honors student assisted DH in literature review and figure preparation. NS and HJ: expertise input for nano-SIMS. EM: expertise input for PIXE and LA-ICP-MS. RR and MF: expertise input for traumatic brain injury. FC: expertise input for intra-cerebral hemorrhage. RT and VL: expertise input for blood-brain barrier and glial cells. MP and MK: expertise input for ischemic stroke. MH: overall conceptual design for review, supervisor of DH and WA – advising in writing and figure design.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Nivolumab is an immune checkpoint inhibitor (ICI) that has shown efficacy for treating non-small cell lung cancer and has become a standard therapy for previously treated non-small cell lung cancer. Moreover, immune-related adverse events of ICI therapy are well-known. Malignant pericardial effusions occasionally arise in patients with lung cancer. There have been a few reports of pericardial effusion in non-small cell lung cancer after nivolumab administration. However, the cause of this condition is controversial; the possibilities include serositis as an immune-related adverse event or pseudo-progression.Case Presentation: This report presents two cases of pericardial effusion with tamponade in lung cancer during treatment with nivolumab. Both patients experienced temporal increases in pericardial effusions followed by effusion regression. In one case, nivolumab administration was continued after performance of pericardiocentesis, without an increase in pericardial effusion. In the other case, temporal simultaneous increases in both the pericardial effusion and the primary tumor were detected, followed by simultaneous regression in both the effusion and the tumor. These findings support the fact that the pericardial effusions were caused by pseudo-progression.Conclusions: Pericardial effusion with tamponade can occur in lung cancer patients being treated with nivolumab; moreover, some of these effusions might be caused by pseudo-progression. In the case of putative pseudo-progression, continuation of nivolumab administration might be allowable with strict follow up. Nivolumab, an anti-programmed death 1 antibody, is an immune checkpoint inhibitor (ICI) that has shown efficacy for treating non-small cell lung cancer (NSCLC) (Immune checkpoint inhibitor (ICI) therapy is well-known for affecting the phenomenon of pseudo-progression in solid tumors . Pseudo-Malignant pericardial effusion occasionally arises in patients with malignant tumors, most commonly cancerous lung tumors . MoreoveHerein, we report two cases of pericardial effusion with tamponade in lung cancer patients during treatment with nivolumab. The pericardial effusions in the two cases were both malignant. The increases in the effusions were temporary and followed by decreases; therefore, these findings suggest pseudo-progression.A 65-year-old man with a 68 pack-year smoking history consulted his primary care physician with the chief complaint of a productive cough. Subsequently, a large mass lesion of his right lung was detected on chest X-ray, and he was referred to our hospital. He was further examined through contrast-enhanced computed tomography (CT), which revealed a mass lesion with a 92-mm diameter, extending from the middle lobe of his right lung to the upper mediastinum, lymphadenopathy of the mediastinum and bilateral neck, swelling of bilateral adrenal grands, intraperitoneal dissemination, and slight pericardial effusion. After further examination, he was diagnosed with adenocarcinoma of the lung, cT4N3M1c, stage IVB (8th edition of the TNM classification for lung cancer). Neither epidermal growth factor receptor (EGFR) mutations nor an anaplastic lymphoma kinase (ALK) gene rearrangement were detected. The patient was treated with four cycles of carboplatin and pemetrexed. Nearly all lesions diminished in size; however, intraperitoneal dissemination worsened.nab-paclitaxel monotherapy; however, the efficacy of these treatment regimens was limited. Eighteen months after pericardiocentesis, the patient died of lung cancer progression; however, pericardial effusion had not increased.Nivolumab therapy was then initiated for the patient (3 mg/kg every 2 weeks) as a second-line therapy. His serum carcinoembryonic antigen (CEA) level before initiation of nivolumab therapy was 143.7 ng/ml; his chest X-ray and CT are presented as Figures nab-paclitaxel, and the treatment was effective for all previously detected lesions; however, multiple brain metastases arose. He then received whole brain irradiation, and these new lesions showed reduction. Subsequently, he was treated with three cycles of pemetrexed as a second-line chemotherapy; however, the primary lesion showed regrowth.A 71-year-old man with a 25 pack-year smoking history visited our hospital with the chief complaints of productive cough and dyspnea. Subsequently, a massive left pleural effusion was detected on chest X-ray. He was then examined through contrast-enhanced CT, which revealed a massive left pleural effusion, a mass lesion with a 36-mm diameter, in the lower lobe of his left lung, and slight pericardial effusion. After further examination, the patient was diagnosed with adenocarcinoma of the lung, cT4N3M1a, stage IVA. Neither EGFR mutations nor an ALK gene rearrangement were detected. He was treated with four cycles of carboplatin and Nivolumab therapy was then initiated for the patient (3 mg/kg every 2 weeks) as a third-line therapy. The chest X-ray and CT before initiation of nivolumab therapy are presented as Figures In this study, we made two important clinical observations. First, we showed that pericardial effusion with tamponade can occur in lung cancer patients during nivolumab treatment, findings similar to those in five previous reports –8. In twSecond, some pericardial effusions with tamponade in lung cancer patients during nivolumab treatment might be caused by pseudo-progression. The present two cases showed temporal increasing of pericardial effusions, followed by effusion regression; in addition, the temporal increasing of pericardial effusions were accompanied by tumor marker decreases. In Case 1, nivolumab administration was continued after pericardiocentesis, and pericardial effusion did not increase again. In Case 2, temporal simultaneous increases of the pericardial effusion and the primary tumor were detected, followed by simultaneous regression in both the effusion and the tumor. These findings support the fact that pericardial effusions can be caused by pseudo-progression; however, in Case 2, we could not completely rule out the cause of pericardial effusion as being an irAE, because nivolumab therapy was discontinued after pericardiocentesis. In one previous report, two cases showed increasing malignant pericardial effusions after nivolumab administration, followed by effusion regression; therefore, the pleural effusions were thought to have resulted from pseudo-progression .The cause of pericardial effusion after nivolumab administration is thought to be either nivolumab-induced serositis or pseudo-progression; however, making a distinction between the two causes is difficult. In the present report, Case 1 showed an increased lymphocyte count in the pericardial effusion. One previous report considered a low lymphocyte count in pericardial effusion to indicate pseudo-progression , and theThe mechanism behind pseudo-progression's causing pericardial effusion is unclear. An increase in malignant pericardial effusion has been reported to have a close association with vascular endothelial growth factor (VEGF) , and VEGIn conclusion, pericardial effusion with tamponade can occur in lung cancer patients during treatment with nivolumab, and some of these effusions might be caused by pseudo-progression. In the case of putative pseudo-progression, continuation of nivolumab administration might be allowable with strict observation and follow up of the patient. We, therefore, suggest that in similar cases, making a determination of pericardial effusion caused by pseudo-progression may improve the outcome of NSCLC patients by allowing continuation of nivolumab therapy.Informed consent was obtained from the patients for publication of the findings of this case report.MY conceived of the study and drafted the manuscript. WD, NS, KF, and TO collected the data. KK, YM, NM, MT, and NO participated in writing and editing as well as collection of data. NH edited the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The synthesis and application of porphyrins has seen a huge shift towards research in porphyrin bio-molecular based systems in the past decade. The preferential localization of porphyrins in tumors, as well as their ability to generate reactive singlet oxygen and low dark toxicities has resulted in their use in therapeutic applications such as photodynamic therapy. However, their inherent lack of bio-distribution due to water insolubility has shifted research into porphyrin-nanomaterial conjugated systems to address this challenge. This has broadened their bio-applications, viz. bio-sensors, fluorescence tracking, in vivo magnetic resonance imaging (MRI), and positron emission tomography (PET)/CT imaging to photo-immuno-therapy just to highlight a few. This paper reviews the unique theranostic role of porphyrins in disease diagnosis and therapy. The review highlights porphyrin conjugated systems and their applications. The review ends by bringing current challenges and future perspectives of porphyrin based conjugated systems and their respective applications into light. The early detection of diseases plays a vital role in their successful treatment and recovery. However, currently available detection methods such as biopsy procedures  , blood tAn ideal drug should be able to deliver the appropriate drug quantities at a controlled rate and time to the desired site. However, a number of challenges in this respect have been reported with current drug and/or delivery systems suffering from altered bio-distribution, clearance issues from the body, and uncontrolled release with non-matching pharmacodynamics . Researc1) and reacts with molecular oxygen in the surrounding environment to form singlet state oxygen (1O2). In photodynamic therapy, porphyrins based photosensitizers are the most widely used as photosensitizing drugs. PDT porphyrin based photosensitizers (PBP) are categorized into three generations. First-generation PBP have seen limited use due to long half-life, chemical impurity, low attenuation coefficients, and excessive accumulation onto the skin. The second-generation PBP seemingly addressed the short-comings of the first generation; however, they lacked the desired water solubility required for biological application and the ability of the required light to penetrate into tissue for the treatment of deep-seated tumors. This has resulted in the development of third-generation PBP with increased cancer cell-specific accumulation. Third-generation PBP are centered on the use of conjugated systems of the porphyrins linked to moieties such as targeting bio-molecules, nanomaterials , and other moieties  to improve the specificity of the porphyrins . Wi. Wi28]. 4) (2 relaxivity of 140.57 s−1 FemM−1. An in vitro experiment of the DDNP-SPIONs binding to β-amyloid aggregates showed that the combination (DDNP-SPIONs) induced the fluorescence enhancement of the DDNP-SPIONs. The research by Venter et al., 2018 -labeled cationic porphyrin, viz. 5,10,15,20-tetra(4-methylpyridyl) porphyrin (TMP), has found use in PET radiotracer for tumor imaging. Preliminary bio-evaluation studies on [68Ga]-labeled TMP showed potential as radio-labeled porphyrin derivative for tumor imaging .Helicpbat pylori (Hp) biofilms using protoporphyrin IX (PP IX) and co-porphyrin I (PI) for fluorescence labeling of bacteria  loaded liposomes. The group then applied positron emission tomography (PET) imaging to estimate the location of 4T1 mammary tumors. To complement PET, fluorescence imaging using a NIR fluorescence fiber optic probe was used to pinpoint the exact tumor site. The use of 64Cu-Dox-PoP liposomes in PET and fluorescence imaging to identify and quantify liposome accumulation further shows the capabilities of porphyrin conjugates within dual functionalities.Diagnostic imaging has long been used as a tool to recognize, define, and identify tumors. However, the concept of dual/multi-imaging was only introduced in the early 2000s. Prior to that, clinicians often used trial-and-error with regards to the use of imaging as means of diagnosis. Medical imaging has since advanced with the introduction of nuclear medicine (where radioactive substances are applied for the diagnosis and treatment of disease). Lou et al., 2017 , chelateThere are three distinctive pathways for the synthesis of meso substituted ABCD-type porphyrins: (a) Total synthesis, (b) mixed condensation, and (c) functionalization of preformed systems . Althoug4 is also still not fully reached by researchers and therefore still remains a widely investigated research area. The misuse and over-prescription of antibiotics has resulted in the increase in antibiotic resistance in gram-positive and gram-negative bacteria. This has resulted in the need for alternative methods such as antimicrobial photodynamic therapy (aPDT) for disease treatment. The photo-physical properties of porphyrins allow for their use in aPDT with meso-tetraarylporphyrins as the most commonly used porphyrins in aPDT. Porphyrins such as 5,10,15-tris (1-methylpyridinium-4-yl)-20-(pentafluorophenyl) porphyrin tri-iodide [2TriMAPP) [Self-aggregation of porphyrins still remains as one of the major challenges reported by many researchers. This is particularly experienced with anionic porphyrins such as tetrakis (4-sulfonatophenyl)-porphyrin and its derivatives which results in suppressed fluorescence and reduction in the singlet oxygen generation. A number of strategies have been reported to resolve the aggregation challenges commonly reported. However, to date, all reported studies are application based . Consensi-iodide , tetrakii-iodide , and 5,1TriMAPP)  and theiTriMAPP) ,63,64.Herein, we report on the use of porphyrins and their related compounds in a variety of diagnostic tools  and treatment modalities . Although significant research has been done in presenting the potential of these highly aromatic molecules as the “next generation” drugs, a lot of research still needs to be exploited as limited research has been explored in relation to their bio-distribution, mode of action/mechanism of action in-vivo, and most importantly, in their production up-scaling. The conjugation of porphyrins with various moieties ranging from small sized nanomaterials to high molecular weight polymers has also been exploited. However, limited literature is available on their pre-clinical work in different in-vivo cancer models. Despite porphyrins having proved to be highly efficient in PDT application with positive clinical trials, their application as first line cancer treatment (even in skin cancers) is still not well exploited compared to other cancer treatment modalities. Moreover, there has been no reported attempt to proceed towards clinical trials of porphyrin conjugated systems which would draw closer towards drug development of these sophisticated systems, even though the use of porphyrins in dual modality has provided a fascinating and state-of-the-art approach in disease diagnosis and treatment. Numerous porphyrin derivatives or analogues have been synthesized, such as the chlorines, phthalocyanines, and naphthalocyanines, for diagnostic and theranostic application. Furthermore, these porphyrin derivatives are known for their improved stability compared to bare porphyrins, higher coefficient extinctions (~105/M/cm) and longer wavelengths in the near-infrared region. They suffer from a number of synthetic and functionalization challenges. Moreover, there is limited literature on their toxicity profiles and improved synthetic methods aimed to improve their biocompatibility. It is therefore necessary that the porphyrin analogues are further exploited as their NIR capabilities would be highly advantageous in optical imaging techniques. To conclude this review, it is also worth mentioning pharmacological properties of porphyrins  as an untouched focus area both by scientific researchers and industrial practitioners. This has further hampered the progression of porphyrin use in a clinical set-up. Moreover, the lack of reported tuning of synthetic methods to enable synthesizable porphyrin agents with dual or tri-modality functionalities has further contributed to the lack of porphyrin conjugated systems being credited as bio-compatible systems. Therefore, there is a need for exploration beyond in vitro and in vivo studies.


Mycobacterium tuberculosis resistant to isoniazid and rifampin) each year. Little is known about the optimal treatment for these children.An estimated 32,000 children develop multidrug-resistant tuberculosis . Overall, 764 of 975 (78%) had a successful treatment outcome at the conclusion of therapy: 548/731 (75%) of confirmed and 216/244 (89%) of clinically diagnosed children . Treatment was successful in only 56% of children with bacteriologically confirmed TB who were infected with HIV who did not receive any antiretroviral treatment (ART) during MDR-TB therapy, compared to 82% in children infected with HIV who received ART during MDR-TB therapy . In children with confirmed MDR-TB, the use of second-line injectable agents and high-dose isoniazid (15–20 mg/kg/day) were associated with treatment success . These findings for high-dose isoniazid may have been affected by site effect, as the majority of patients came from Cape Town. Limitations of this study include the difficulty of estimating the treatment effects of individual drugs within multidrug regimens, only observational cohort studies were available for inclusion, and treatment decisions were based on the clinician’s perception of illness, with resulting potential for bias.To inform the pediatric aspects of the revised World Health Organization (WHO) MDR-TB treatment guidelines, we performed a systematic review and individual patient data (IPD) meta-analysis, describing treatment outcomes in children treated for MDR-TB. To identify eligible reports we searched PubMed, LILACS, Embase, The Cochrane Library, PsychINFO, and BioMedCentral databases through 1 October 2014. To identify unpublished data, we reviewed conference abstracts, contacted experts in the field, and requested data through other routes, including at national and international conferences and through organizations working in pediatric MDR-TB. A cohort was eligible for inclusion if it included a minimum of three children (aged <15 years) who were treated for bacteriologically confirmed or clinically diagnosed MDR-TB, and if treatment outcomes were reported. The search yielded 2,772 reports; after review, 33 studies were eligible for inclusion, with IPD provided for 28 of these. All data were from published or unpublished observational cohorts. We analyzed demographic, clinical, and treatment factors as predictors of treatment outcome. In order to obtain adjusted estimates, we used a random-effects multivariable logistic regression  adjusted for the following covariates: age, sex, HIV infection, malnutrition, severe extrapulmonary disease, or the presence of severe disease on chest radiograph. We analyzed data from 975 children from 18 countries; 731 (75%) had bacteriologically confirmed and 244 (25%) had clinically diagnosed MDR-TB. The median age was 7.1 years. Of 910 (93%) children with documented HIV status, 359 (39%) were infected with HIV. When compared to clinically diagnosed patients, children with confirmed MDR-TB were more likely to be older, to be infected with HIV, to be malnourished, and to have severe tuberculosis (TB) on chest radiograph  affects 32,000 children per year, requires longer treatment with much more toxic medications than drug-susceptible tuberculosis. Unfortunately, little is know about the optimal treatment for children with MDR TB.This study reviewed treatment and outcome data from children around the world in order to better understand the management of MDR-TB in children.This study also sought to understand the risk factors for poor treatment outcomes in children with MDR-TB.This study informed the World Health Organization guidelines on treatment of MDR-TB in children.We performed a systematic review and individual patient data meta-analysis on clinical characteristics and treatment outcomes on 975 children from across 18 countries.Children were analyzed in two separate groups, those with bacteriologically confirmed MDR-TB and those who were clinically diagnosed with MDR-TB.We found that, in general, children do well when treated with the second-line MDR-TB medications , despite the fact that there was a high burden of severe disease.Malnutrition and not being treated for HIV (if the child was HIV-positive) during TB treatment significantly increased the risk of poor outcomes.Second-line injectable agents and high-dose isoniazid were associated with treatment success. However, a high proportion of children with non-severe disease who received no second-line injectable agents still did well; therefore, children with non-severe disease may be able to be spared from these toxic medications.Consideration should be given to using high-dose isoniazid in treatment regimens, and if children have non-severe disease, the possibility of excluding second-line injectable agents from the treatment regimen should be considered.HIV treatment should be started as soon as is possible, and malnutrition should be aggressively treated. Mycobacterium tuberculosis with resistance to at least isoniazid and rifampin) in 2015 . A,8. A7,8]In order to identify eligible reports, we searched PubMed, LILACS , Embase, The Cochrane Library, PsychINFO, and BioMedCentral databases through 1 October 2014, with a search strategy using a combination of the search terms “tuberculosis,” “multidrug resistance,” “MDR-TB,” “multidrug-resistant,” and “children,” both as exploded MeSH  headings and free-text terms, and without language restriction. The specific search strategies for Pubmed and Embase are presented in To identify additional published and unpublished data, we contacted experts in the field of pediatric MDR-TB. We also requested data through other routes, including at national and international conferences and training events, and through international and in-country organizations working in pediatric MDR-TB, including the Sentinel Project on Pediatric Drug-Resistant Tuberculosis, the WHO Childhood TB Sub-Group, Médecins Sans Frontières (MSF), the United States Centers for Disease Control and Prevention (CDC) and European Centre for Disease Prevention and Control (ECDC), The Union, and the National Institutes of Health (NIH).All abstracts were screened by EPH and a researcher with Cochrane South Africa to select full text reports to review. All full text reports were reviewed independently by two reviewers  to assess for eligibility, except reports in Russian, French, Dutch, and Spanish, which were reviewed by a single reviewer . Disagreements about study selection were resolved by a third reviewer. If eligibility was unclear because of missing information, two attempts were made to contact authors of the primary report. After two unsuccessful attempts, reports were excluded.The quality of individual studies was described using a modified version of the Newcastle-Ottawa approach for cohort studies adapted for use in pediatric MDR-TB . An examThe authors of all eligible studies and cohorts were contacted to request IPD. De-identified IPD were included following written agreement from the original authors, or the lead clinical physician in the case of unpublished data.Data were requested on factors that could influence treatment decision and outcome, including:Participants: age, sex, nutritional status, HIV status and antiretroviral treatment (ART), adult MDR-TB source case information, bacteriologically confirmed versus clinical diagnosis , diseaseIntervention: use of individual drugs and the duration of drug use in the treatment regimen.Outcomes: acid-fast bacilli (AFB) smear microscopy (smear) and culture conversion, adverse events, and WHO-defined treatment outcomes, including cure, treatment completion, treatment failure, loss to follow-up, not evaluated, and death  as either severe or non-severe, based on the reported chest radiographic findings using adapted Wiseman criteria ; disagreAlthough we collected information on adverse effects, we were unable to complete formal analyses given the limitations in the data.Data from children with confirmed pre-extensively drug-resistant (pre-XDR)-TB  were combined with those from children with MDR-TB. Children with confirmed extensively drug-resistant TB  were excluded from analysis and will be reported on separately. This was done because patients with XDR-TB generally have very different treatment requirements, such that their outcomes would not be representative of the MDR-TB and pre-XDR-TB cohorts. When analyzing the association between individual drug use and treatment success, treatment outcomes were dichotomized as either successful or unsuccessful and stratified by confirmed versus clinically diagnosed MDR-TB. Successful treatment outcome was defined as cure or treatment completion . Unsuccen = 44 children) and one site that did not report on the severity of extrapulmonary disease (n = 2 children). For the patients at sites that had no data from which to estimate a mean value, their missing values were replaced in the analysis by the mean value of the variable in the entire sample. At sites where most patients had these values reported, for individual patients who had missing data on HIV status (n = 65), sex (n = 3), severe extrapulmonary disease (n = 68), and malnutrition (n = 43), missing values were replaced by their respective site's mean value of the variable. Children from countries with very low HIV prevalence who did not have an HIV test were assumed to be HIV uninfected following consultation with site investigators. A p-value of 0.05 was taken as the limit of statistical significance. All statistical analyses were performed using SAS software .With respect to the analysis of the estimates of the association between individual antituberculosis medications (used as part of a multidrug regimen) and treatment success, in order to obtain adjusted estimates, we used a random-effects multivariable logistic regression  with adaptive quadrature approximation. Our models for predictors of drug effect on TB treatment outcome only included those children with bacteriologically confirmed MDR-TB. Patients were considered to be clustered within cohorts such that intercepts and slopes of the main exposure variables were allowed to vary across cohorts. This was done to account for unmeasured differences in patient populations and other site-specific characteristics. Estimates were adjusted for the following covariates: age (as a continuous variable), sex, HIV infection, malnutrition, severe extrapulmonary disease, or the presence of severe disease on chest radiograph. There were nine sites that did not report any information on patients' nutritional status  had confirmed MDR-TB by DST and 244 (25%) had clinically diagnosed MDR-TB. Most data predated the rollout of GeneXpert MTB/RIF; the vast majority of confirmed patients were diagnosed using culture. Of the 731 children with a confirmed diagnosis, 68 (9.3%) had pre-XDR-TB . It should be noted that 68% of confirmed patients had no additional information regarding resistance to fluoroquinolones and/or second-line injectable agents. Of the 244 children with a clinical diagnosis of MDR-TB, 164 (67%) had clinical TB with a known close source case with DST-confirmed MDR-TB, 14 (6%) had failure of first-line therapy  despiteKey demographic and clinical characteristics, stratified by confirmed versus clinically diagnosed MDR-TB, are shown in TB treatment outcomes are summarized in In multivariable analysis, among children with confirmed MDR-TB, HIV infection  and malnutrition  were associated with reduced odds of successful treatment outcome versus treatment failure or death . MalnutrWe also conducted analyses to assess estimates of the association between individual antituberculosis medications, used as part of a multidrug regimen, and treatment success  to inforAmong the clinically diagnosed children , 60 (25%) received no injectable agent or less than 1 month of injectable agent. Fifty (83%) of these children had successful treatment outcomes.Later generation fluoroquinolones were defined as moxifloxacin, gatifloxacin, sparfloxacin, or high-dose levofloxacin. Only 63/885 (7%) children in total were treated with moxifloxacin . One child with confirmed disease received both gatifloxacin and sparfloxacin. Information on the dose of levofloxacin was available in only 93/885 (11%) of children. This infrequent use precluded useful analysis of the clinical impact of fluoroquinolones on treatment outcome.This systematic review and IPD meta-analysis of pediatric MDR-TB represents a global collaborative effort of the pediatric TB community. It has for the first time, to our knowledge, generated a data set of children treated for MDR-TB in multiple countries, which has informed the revised 2016 WHO MDR-TB treatment guidelines .A striking finding is the high proportion (78%) of successful treatment outcomes amongst all children, with 75% success in children with confirmed MDR-TB and 89% in those with clinically diagnosed disease. These good outcomes are despite the high prevalence of comorbidities , severe pulmonary (49%) and extrapulmonary (13%) TB, and the abscence of the newer, safer, and more effective drugs. A previous systematic review, without IPD, reported a similar proportion of children with MDR-TB successfully treated (82%) . These oThe association of HIV infection and malnutrition with reduced odds of successful TB treatment is consistent with the published literature ,40. ChilIn this data set, children infected with HIV were more likely to have confirmed MDR-TB, despite reports of higher rates of paucibacillary TB disease in adults infected with HIV. This may be because the majority (95%) of children infected with HIV were from South Africa, where diagnostic tools and approaches may differ. There may also be hesitancy to start MDR-TB treatment in addition to ART; thus, treatment delays may lead to children having more severe TB disease, resulting in more bacteriologically confirmed disease. Children infected with HIV also tended to be older, as prevention of mother to child transmission (PMTCT) initiatives were rolling out during the study period in South Africa; this may have contributed to more children infected with HIV having a confirmed diagnosis.katG mutation and lower level resistance by the inhA mutations) [nhA mutation’s low-level isoniazid resistance but is unlikely to overcome the high-level isoniazid resistance due to a katG gene mutation [inhA mutations (61% of the isoniazid resistant mutations among pediatric patients [A prominent finding was the benefit of high-dose (15–20 mg/kg/day) isoniazid. Twenty-one percent of children with confirmed MDR-TB received high-dose isoniazid; however, the majority of these children were from Cape Town. Despite attempts to statistically control for site-specific effect, it is possible that other unmeasured site-related effects, including the quality of clinical care, could have contributed to this finding. However, the fact that the point estimate for the benefit of high-dose isoniazid remained unchanged when South African data were examined demonstrates that this benefit remains, despite possible site-specific effect. When high-dose isoniazid is considered as part of MDR-TB treatment but the mutation conferring isoniazid resistance is unknown, the local epidemiology of isoniazid resistance mutations should be considered (high-level isoniazid resistance is conferred by the tations) . High-domutation . The Wespatients ), which patients –50.Among children with confirmed MDR-TB, the use of second-line injectable agents was associated with successful treatment versus failure/death. Although we found improved outcomes in children with confirmed disease when treated with injectable agents, we saw high levels of treatment success in children with clinically diagnosed MDR-TB who received no second-line injectable agents or who had received these parenteral antibiotics for less than 1 month. Eighty-three percent (50/60) of the children with clinically diagnosed disease  who received no or less than 1 month’s treatment with a second-line injectable agent had successful treatment outcomes. These data need to be interpreted with caution and the use of second-line injectable agents in children needs more study. We did not collect data on the reason for using an injectable-free regimen, but possibilities include less severe disease, the presence of resistance to an injectable agent , and perceived and actual injectable-associated adverse events.Nonetheless, our findings lend support to not using second-line injectable agents in children with non-severe MDR-TB ,18,51. AOur results were used to inform the revision of the 2016 WHO MDR-TB treatment guidelines, which state that the harms associated with second-line injectable agents may outweigh potential benefits, and therefore injectable agents may be excluded from the treatment regimens of children with non-severe forms of MDR-TB disease .A strength of our study was the inclusion of children with both bacteriologically confirmed and clinically diagnosed TB. Because of the paucibacillary nature of pediatric TB and the challenges of obtaining clinical samples, approximately 60%–70% of children treated globally for TB will be clinically diagnosed ,35. The A criticism of studies that include clinically diagnosed children is that some of these children may not have MDR-TB and so treatment outcomes may not reflect true MDR-TB disease. In this study, among children with a clinical diagnosis, 67% had clinical TB with a known MDR-TB source case, and another 6% had failure of first-line therapy despite good adherence. Given that studies have shown that children are most likely to share the TB strain of their household contact, it is likely that the majority of children in this cohort did in fact have MDR-TB –39.The most significant limitation of this study is that the estimated treatment effects of individual drugs should be interpreted with caution because the analysis could not isolate the benefit of each drug. We could only compare regimens with and without each drug. If particular drugs were used or not used together—because of disease severity or resistance profiles or toxicity—there is potential for confounding by indication that would produce biased effect estimates for each drug. Also, treatment decisions were based on the clinician’s perception of illness, with resulting potential for bias.Furthermore, we were not able to compare specific regimens given the large variation in drugs used and treatment duration as part of individualized treatment. All studies included were retrospective or prospective observational cohort studies; there were no clinical trials. However, given that the study was an IPD, some adjustments could be made for covariates at the individual level, which is not possible in study-level meta-analyses. This worked to mitigate the risk of confounding and other potential biases. The sample size was modest compared to adult IPD data sets, and estimates were frequently imprecise, while some associations were not estimated because of limited data. There were no data available yet on the novel drugs, delamanid and bedaquiline, and data on linezolid were insufficient to include in this analysis. Data on late-generation fluoroquinolones and clofazimine were sparse. Children who were lost to follow-up and not evaluated  were not included in this analysis, because their treatment outcome was unknown. Relapse was also not evaluated, as data on outcomes after completing antituberculosis treatment were rarely reported. This could have resulted in overestimation of favorable treatment outcome.A further limitation is that, apart from DST for isoniazid and rifampicin, data about a strain’s susceptibility to other drugs  were frequently not available for analysis; therefore, we could not assess the association between combinations of likely effective drugs (based on DST results) and outcomes. This may have also led to an underestimate of the benefit of drugs to which the strain was susceptible. Additionally, data on adverse events were incompletely reported and could not be included in the analysis. This scarcity of the reporting of adverse events also contributed to us being unable to distinguish treatment failure due to lack of efficacy from treatment failure due to drug intolerance.Data were limited regarding the duration of posttreatment follow-up or posttreatment outcomes; therefore, we could not evaluate recurrence, relapse, or the effect of total duration of treatment on MDR-TB outcomes. Data on history of previous TB treatment were sparse and could not be analyzed.All studies used consecutive sampling of children enrolled in MDR-TB treatment, which should have minimized selection bias. However, it is possible that the diagnosis of MDR-TB could have been missed in some children due to passive case finding at most study sites and general difficulties in diagnosing TB and MDR-TB in children. Children in whom the diagnosis may have been missed, often children with clinically diagnosed TB, who would usually have less severe, more paucibacillary disease, might have had different clinical characteristics and different outcomes than those included in the selected studies and therefore may have better treatment outcomes if they had in fact been enrolled. Thus, the likelihood of selection bias cannot be ruled out.An additional limitation of this study was that the literature published after October 2014 was not included. In order to determine what effect this may have on our findings, we did a search of the pediatric MDR-TB literature from October 2014 to the present. The studies were reviewed, either full studies or abstracts, and details of studies that had the potential to be included in our study are included in Children treated for MDR-TB have good treatment outcomes—even those with comorbidities and severe disease. That children tend to have much better treatment outcomes than adults raises the possibility that children may be specifically suited to do well with shorter, less intense treatment regimens. This deserves further study. Our study provides evidence that a substantial proportion of children with clinically diagnosed MDR-TB treated without second-line injectable agents have successful outcomes and that high-dose isoniazid may improve treatment success in some settings. Given the limitations of the current study, these findings should be interpreted cautiously but deserve further evaluation in subsequent studies. The high mortality and low treatment success rates among children infected with HIV who were not on ART during TB treatment demonstrates the importance of testing children with TB for HIV and initiating ART as soon as possible.Data regarding the treatment outcomes and pharmacokinetic and safety profiles of bedaquiline, delamanid, later generation fluoroquinolones, linezolid, and clofazimine in children are urgently needed in children with MDR-TB, including in children infected with HIV. Future research is also needed on pediatric outcomes involving the shorter MDR-TB regimen ,50. ConsS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 Text(DOC)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.


Clostridium difficile associated colitis. In the IC group, the period from the transplantation up to diagnosis was significantly shorter than acute GI-GVHD group . In the acute GI-GVHD group, tortoiseshell-like mucosal patterns were significantly more common than the IC group . Furthermore, there were some cases presenting normal mucosal appearance despite the diagnosis with acute GI-GVHD by pathological findings. Clinically, we should consider IC when abdominal symptoms appeared in the early period after HSCT. Endoscopically, tortoiseshell-like mucosal pattern was a characteristic feature of acute GI-GVHD. In addition, it is essential to perform mucosal biopsy for diagnose of acute GI-GVHD even in patients showing the normal mucosal appearance.The aim of this study is to elucidate the differences of the clinicopathological characteristics between acute gastrointestinal (GI)-graft-versus-host disease (GVHD) and infectious colitis (IC) after hematopoietic stem cell transplantation (HSCT). Of the 282 patients who underwent HSCT at our institution between January 1991 and December 2015, we could investigate 182 patients in detail. Of the 182 patients, we selected those who underwent colonoscopy and were diagnosed with acute GI-GVHD or IC after HSCT. Patients’ backgrounds, colonoscopic findings, and pathological findings were retrospectively analyzed. There were 30 patients who had colonoscopy performed and diagnosed with acute GI-GVHD or IC after HSCT. Of the 30 patients, 20 had acute GI-GVHD and 10 had IC. All the cases of acute GI-GVHD were diagnosed by endoscopic biopsy and 4 of the IC patients had  Hematopoietic stem cell transplantation (HSCT), also known as bone marrow transplantation (BMT), peripheral blood stem cell transplantation (PBSCT), and cord blood transplantation (CBT), involves the intravenous infusion of hematopoietic stem cells collected from the bone marrow, peripheral blood, and umbilical cord blood, respectively. HSCT has been performed with increasing frequency for the common treatment of numerous malignant and nonmalignant diseases, such as leukemia, malignant lymphoma (ML), multiple myeloma (MM), myelodysplastic syndromes (MDS), aplastic anemia (AA), and other disorders of the hematopoietic system. The development of acute and chronic graft-versus-host disease (GVHD), which can lead to life-threatening processes , is one Acute GVHD is defined as GVHD that occurs within the first 100 days of allogeneic -HSCT. Acute GVHD is a major cause of morbidity and mortality in patients who undergo HSCT . AlthougClostridium difficile [CD], Pseudomonas, and Klebsiella species), fungi (Aspergillus and Candida species), viruses , and parasites. In particular, CD and CMV infections are the most commonly occurring GI infections after HSCT. The treatment of acute GI-GVHD is quite different from IC. Therefore, it is important to distinguish acute GI-GVHD from IC in patients with HSCT for initiating optimal treatments.Patients with acute GI-GVHD usually present with frequent watery diarrhea, abdominal pain, and hematochezia; similar complaints are also seen in patients with HSCT accompanied with infectious colitis (IC). In the pre- and early post-HSCT phases, patients are at a high risk for developing infections, which are caused by bacteria  and were diagnosed with acute GI-GVHD or IC after HSCT. The patients who did not undergo CS, those who had a history of severe GI disease before the transplantation, and those who coexisted with GI-GVHD and IC were excluded.CD toxin testing, CMV antigenemia, and endoscopic biopsy were performed. Endoscopic biopsy was performed to diagnose CMV enteritis  from acute GI-GVHD and IC is required. TMA was excluded based on the laboratory findings and pathological findings, which pointed to the presence of hyaline thrombi in the capillaries, or fibrinoid necroses of the terminal arterioles of the intestine.CS was performed to observe the entire colon and terminal ileum. However, in patients with poor condition or cases involving difficult colonoscopy (difficulty in intubation), the endoscopic observation was limited to the site where the scope could reach. The biopsy samples were collected from the normal mucosa and the intestinal mucosa, which demonstrated endoscopic features, such as redness, edema, erosion, and ulceration. CS was performed using an Olympus colonoscope  with indigo carmine dye contrast, and narrow band imaging, with or without magnifying observation, by experienced endoscopists. Sedatives were administered according to the patients’ conditions.Patients’ characteristics , endoscopic and histopathological findings, and clinical courses were reviewed from medical charts and films, retrospectively.The significant differences in the variable characteristics were investigated using Fisher’s exact test for categorical variables and a Student’s t-test for continuous variables. EZR , versionThe backgrounds of the 182 patients are shown in CD toxin testing and fecal cultures were performed in all the patients. In addition, CS was performed in 40 of these patients. Of the 40 patients, acute GI-GVHD was observed in 20 patients, and IC was observed in 10 patients . Remaining 10 patients were performed not only colonoscopy but also esophagogastroduodenoscopy (EGD). Then, they were not diagnosed with GI-GVHD or IC by the result of endoscopic histologic examination and stool culture. It was considered that the abdominal symptoms of these 10 patients were associated with pretreatments prior to HSCT. No patients with TMA were observed.Subject analysis is shown in p <0.001). In acute GI-GVHD group, tortoiseshell-like mucosal pattern was found in 10, 4, and 2 of 14 patients (including duplicate) in rectosigmoid, left-side, and right-side colon, respectively. No significant differences in erosion/ulceration  abdominal symptoms appeared in HSCT patients with IC significantly earlier than those with acute GI-GVHD; (2) a tortoise shell-like mucosal pattern was a characteristic endoscopic feature of acute GI-GVHD; and (3) sufficient amount of biopsy specimens should be taken from the GI tract because some patients with acute GI-GVHD had endoscopically normal mucosal appearance.CD infection (CDI) from auto-HSCT was 6.5 days. According to Willems et al. [No reports were found comparing the difference of the mean time to diagnosis between acute GI-GVHD and IC. Wild et al.  showed ts et al. , the medIn the present study, a tortoiseshell-like mucosal pattern was a characteristic endoscopic finding. Ten percent of the patients diagnosed with acute GI-GVHD showed endoscopically normal mucosal appearance. In some cases, skin biopsy can be used for GVHD diagnosis; however, biopsy from GI can also be used because its diagnostic rate is higher (58%) than that of the skin biopsy (46%) . A positThe limitations of this study included the fact that (1) the proportion of patients with acute GI-GVHD was lower than those observed in other studies –6, (2) tThis study was conducted to evaluate the differences of the clinicopathological characteristics between patients with acute GI-GVHD and with IC. Clinically, the presence of IC should be considered when abdominal symptoms appeared in the early period after HSCT. Endoscopically, tortoiseshell-like mucosal pattern is a characteristic feature of acute GI-GVHD. Biopsy should be performed even in patients showing normal mucosal appearance to diagnose acute GI-GVHD. Future studies utilizing prospective and larger sample sizes are therefore necessary to validate the results of the present study and to identify useful markers to distinguish between acute GI-GVHD and IC.The authors declare no conflict of interest. This work was supported in part by Health and Labour Sciences Research Grants for research on intractable diseases from the Ministry of Health, Labour and Welfare of Japan (Investigation and Research for intractable Inflammatory Bowel Disease) and Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number JP17J02428 (to TI). The funders of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report.


Extracellular vesicles (EVs) are small vesicles involved in intercellular communication. Data is emerging that EVs and their cargo have potential as diagnostic biomarkers and treatments for brain diseases, including traumatic brain injury and epilepsy. Here, we summarize the current knowledge regarding changes in EV numbers and cargo in status epilepticus (SE) and traumatic brain injury (TBI), which are clinically significant etiologies for acquired epileptogenesis in animals and humans. We also review encouraging data, which suggests that EVs secreted by stem cells may serve as recovery-enhancing treatments for SE and TBI. Using Gene Set Enrichment Analysis, we show that brain EV-related transcripts are positively enriched in rodent models of epileptogenesis and epilepsy, and altered in response to anti-seizure drugs. These data suggest that EVs show promise as biomarkers, treatments and drug targets for epilepsy. In parallel to gathering conceptual knowledge, analytics platforms for the isolation and analysis of EV contents need to be further developed. Each year, 2.2 million patients are newly diagnosed with epilepsy. Thus, a new epilepsy diagnosis occurs every 13 sec. Despite the fact that more than 20 anti-seizure drugs have been approved for clinical use, seizures for about 30% of epilepsy patients remain uncontrolled [According to the World Health organization, more than 50 million people worldwide have epilepsy  exosomes, that have an endosomal origin (30–150 nm); (2) microvesicles , that bud from the cellular outer membrane (100–1000 nm); and (3) apoptotic bodies that are released during apoptosis (50–5000 nm) ,23. The In this review, we address the current methodological limitations that should be considered when performing EV-related studies and interpreting their results. We summarize the current knowledge of the role of extracellular vesicles in epilepsy and epileptogenesis with a focus on post-traumatic epileptogenesis (PTEgenesis). Finally, we report our in silico analysis of changes to EV-related transcripts after TBI and SE in brain tissue in relation to anti-seizure drug treatment.http://evtrack.org/, accessed on: 15 January 2019) was introduced in 2017. EV-TRACK is a database for EV-related studies and its aim is to centralize experimental parameters of the rapidly expanding EV-related research [The interest for EVs has grown rapidly and the methods used in EV research are constantly evolving. The mission of the International Society for Extracellular Vesicles (ISEV) is to advance extracellular vesicle research globally. In 2014, ISEV determined and published guidelines for the minimal requirements for the analysis of EVs (MISEV) . The guiresearch . Sequential ultracentrifugation, density gradient centrifugation, size-exclusion chromatography, and various precipitation-based methodologies applying different reagents have all been used for the isolation of EVs . Each of7 to 109 EVs but 1016 lipoprotein particles [When isolating EVs from plasma or serum, lipoproteins are one of the major sources of contamination. The number of EVs is only a tiny fraction of the number of lipoproteins in plasma. Simonsen (2017) estimated that 1 mL of plasma contains 10articles . Unfortuarticles ,30. Lipoarticles ,32. In aarticles ,34,35. RRecently, sucrose-density gradient, sucrose-cushion, and iodixanol floatation density gradient methods have been used to isolate EVs from brain tissue ,37,38. WmicroRNAs and other circulating small RNAs are common targets for biomarker discovery from liquid biopsy for different indications ,40. MostWhen examining the miRNA cargo of EVs in plasma, it is important to remove other potential carriers of miRNA during EV-purification. Recently, we evaluated precipitation-based purification methods and showed that vesicle-free miRNAs are found in the purified EV-pellet . Van DeuIn December 2017, ISEV organized a workshop on “extracellular vesicles as disease biomarkers”. The summary published in the Journal of Extracellular Vesicles highlighted gaps in current knowledge related to pre-analytical issues affecting EV purification and the need to use standardized isolation and analysis methods including the use of the EV-TRACK and MISEV .One cell type can secrete several populations of vesicles carrying RNA. Lässer et al. studied one cell line and found extracellular RNA in two density fractions with very distinct RNA and protein profiles . For exa® reagent to precipitate EVs and reported that L1-CAM specific  vesicles comprised only 5–10% of the total number of plasma EVs [Currently, there are no comprehensive studies that define the origins of plasma EVs. This is particularly true for brain-derived EVs. To obtain a general idea of the origin of plasma vesicles, Arraud et al. used cryo-transmission electron microscopy combined with gold-immunolabelling and cell-type-specific antibodies. These authors observed spherical, tubular, and large membrane particles in the EV fraction and estimated that more than 50% of plasma EVs originate from platelets (CD41-positive EVs) or erythrocytes (CD235a-positive EVs) . In anotasma EVs . www.proteinatlas.org, accessed on: 15 January 2019), L1-CAM is expressed also in the kidney, but its presence in kidney-derived plasma EVs is not clear.The search of brain-cell specific EV markers is ongoing. L1-CAM and GluR2 are found in the EVs released by cultured cortical neurons, and consequently, they have been used as neuron-specific EV-markers ,56,57,58The secretion of EVs into body fluids is altered in several diseases, including neurodegenerative diseases . ConBased on recent definitions, epileptogenesis refers to the development and extension of tissue capable of generating spontaneous seizures, resulting in (a) development of an epileptic condition and/or (b) progression of the epilepsy after it is established . ImportaTo our knowledge, four articles have analyzed EVs in epilepsy. Huttner et al. quantified the amount of somatic cell marker CD133 on membrane particles in the CSF of partial epilepsy patients. They found increased levels of CD133 on membrane particles and suggested that it may indicate increased membrane shedding . HoweverCurrently, there are no prognostic biomarkers to identify subjects who have a high risk of epileptogenesis after TBI . We willEV numbers increase in the CCI-induced mouse models of TBI ,67,68. ABohman et al. measured the EV number in the serum of fluid-percussion injury (FPI)-induced piglets . They foIn contrast to the above studies, Midura et al. reported that the total numbers of EVs in mouse plasma decreased at 3–24 h after weight-drop-induced TBI, but the number returned to the control levels by 3 d post-TBI . Several articles have addressed the changes in EV numbers within plasma and CSF of patients with TBI. Morel et al. observed an increase in the number of EVs in human plasma at the acute phase (day 0) of severe TBI. During the following days, EV numbers decreased progressively and reached the control level by day 10 . NekludoEV numbers have also been reported to increase in human CSF after TBI. Morel et al. observed that the number of EVs in CSF was elevated at day 0 after TBI. The EV number returned to control levels by day 10 after TBI. Based on antibody-mediated capture, EVs were mainly derived from platelets (GPIb+) and endothelial cells (CD31+), suggesting they represented blood-brain barrier (BBB) damage or injured blood vessels . Patz etAlthough several studies showed changes in EV numbers after both experimental and clinical TBI, large variation in methodology used for the isolation and analysis of EVs should be noted . Most ofTaken together, these studies show that the EV numbers in plasma and CSF change after experimental and clinical TBI. All but one study showed an increase in the number of EVs. Based on the studies, EV numbers in plasma and CSF appear to be highest during the first 24 h after the injury both in animal models and human patients. After 24 h, the EV numbers gradually return to normal levels. Interestingly, Midura et al. reported that the number of EVs was lowest during the first 24 h after the injury, which contradicts other studies. In addition to changes in EV numbers, some studies have reported changes in EV size. However, due to huge variation in the EV isolation and quantification methods, the studies mentioned in this review are not conclusive regarding the changes in EV number and size in CSF and plasma after TBI.Several studies have reported altered miRNA profiles in brain tissue and plasma after status epilepticus (SE) and TBI, and these findings have been comprehensively reviewed earlier ,40,79. EHarrison et al. isolated EVs from the brains of CCI-injured mice at 7 d post-TBI and pinpointed five differentially expressed miRNAs, of which miR-21 had the highest increase compared to controls . CirculaIn 2013, Patz et al. performed a miRNA array analysis of EVs isolated from the CSF of severe TBI patients. They found 81 mature miRNAs of which miR-9 and miR-451 were the only ones differentially expressed compared to controls . An inteYan et al. studied patients with mesial temporal lobe epilepsy with hippocampal sclerosis (mTLE-HS) and found 50 differentially expressed miRNAs in plasma EVs. Two miRNAs were upregulated and 48 miRNAs were downregulated . Raoof et al. performed miRNA array analysis of CSF samples from patients with TLE or SE. They isolated EV-related and argonaute-2–related miRNAs from pooled CSF samples and measured the top hits, using microarray and quantitative PCR. They found unique distribution patterns for different miRNAs between EVs and Argonaute2. For example, miR-19b-3p was found in the EV fraction in patients with multiple sclerosis that served as controls to epilepsy patients. In TLE and SE patients, miR-19b-3p was bound to argonaute-2 . The samhttp://microvesicles.org/, accessed on: 1 December 2018) [To investigate changes in EV-related gene expression in brain tissue, we analysed available transcriptomics data from rodent models of epileptogenesis and epilepsy. For the analysis of epileptogenesis  models, we used microarray data from the hippocampus and cortex sampled at 32 h after lateral FPI  or 4 moner 2018) . EnrichmEV-related gene expression was positively enriched in the ipsilateral cortex and hippocampus sampled at 32 h after lateral FPI C,D. ThisPositive enrichment of EV-related genes was also observed in the hippocampus 3 months after amygdala stimulation-induced epilepsy A. No enrThese data indicate that EV-related gene expression in the brain is increased acutely in the FPI model of TBI as well as chronically in the amygdala stimulation model of epilepsy and the CCI model of TBI. These changes could be involved in epileptogenesis and these preliminary observations encourage further exploration of the role of EVs in epilepsy.Studies applying mesenchymal stem cell (MSC) as a therapy for neurological diseases (reviewed in ) includiThe beneficial role of EV therapy after TBI has been reported in several studies . Zhang eMore detailed analysis of the mechanism of EV therapy in TBI has been described recently. Gao et al. showed that endothelial colony-forming cell (ECFC)-derived EVs improved the neurological functional recovery after CCI in mice . In partThe knowledge on EV isolation from cell culture media and the commonly used analysis methods are constantly developing. Dominkus et al. reported that when using ultracentrifugation purification, commonly used fluorescent PKH26 dye forms nanoparticles that resemble EVs. Particles are also capable of internalizing at least to primary astrocytes in a similar manner as EVs. Therefore, they can cause false positive results unless proper negative controls are included in the experiments . The addTo our knowledge, only Long et al. have described the beneficial effects of EV therapy in epilepsy. They used MSC-EVs to treat pilocarpine-induced SE in mice. Intranasally-administered EVs reached the hippocampus within 6 h following administration. Long et al. found a decrease in proinflammatory signals and an increase in anti-inflammatory cytokines within the hippocampus at 24 h post-SE, reduced neuron loss 4d post-SE, promoted normal neurogenesis in the hippocampus, and preserved cognitive functions in the chronic phase . These rTreatment of epilepsy with anti-seizure drugs can last for tens of years, and one question is: do anti-seizure drugs affect EVs? So far, there is no literature available. To investigate whether anti-seizure drugs modify EV gene expression, we performed an in silico analysis using LINCS database, containing drug-induced transcriptomics profiles of cell lines to assess the possible effect of anti-seizure drugs on the expression of EV-related genes . We founCarbamazepine showed a negative enrichment in all cell lines , indicating decreased expression of EV-related genes A. PrimidNext, we investigated whether antiepileptic drugs modify EV-related gene expression also in vivo. We used phenytoin and levetiracetam-induced transcriptomic datasets of the rat cerebral cortex, brainstem, and hippocampus (GEO: GSE2880) . By usinOur in silico analyses suggests that carbamazepine, primidone, and gabapentin can modify the EV-related gene expression in vitro, and phenytoin in vivo. In most cases, the gene expression was decreased. Interestingly, our literature analysis indicated an increase in the expression of EV-related genes in epileptogenesis and epilepsy in several rodent models. Further studies are needed to explore whether the modification of epileptogenesis-related EVs contributes to the mechanisms of actions of anti-seizure or antiepileptogenic drugs.Extracellular vesicles show promise as diagnostic and prognostic biomarkers for epileptogenesis and epilepsy as several studies have reported acute changes in EV numbers and their cargo after epileptogenic brain insults such as SE or TBI. Our in silico analysis suggested that post-injury modulation of EV-related gene expression could be long-lasting. Importantly, EVs are also emerging as therapeutic agents to improve recovery such as SE and TBI. However, understanding the mechanistic and therapeutic potential of EVs in epileptogenesis and epilepsy and the effect of anti-seizure drugs on EV metabolism and cargo are in their infancy. Further studies are urgently needed to explore the potential of EVs as antiepileptogenic and co-morbidity-modifying agents as well as targets for treatment to improve the prognosis of epilepsy and those who are at risk of developing epilepsy after brain insults.