Actinobacteria as Microbial Cell Factories and Biocatalysts in The Synthesis of Chiral Intermediates and Bioactive Molecules; Insights and Applications.

Actinobacteria are one of the most intriguing bacterial phyla in terms of chemical diversity and bioactivities of their reported biomolecules and natural products, including various types of chiral molecules. Actinobacterial genera such as Detzia, Mycobacterium, and Streptomyces are among the microbial sources targeted for selective reactions such as asymmetric biocatalysis catalyzed by whole cells or enzymes induced in their cell niche. Remarkably, stereoselective reactions catalyzed by actinobacterial whole cells or their enzymes include stereoselective oxidation, stereoselective reduction, kinetic resolution, asymmetric hydrolysis, and selective transamination, among others. Species of actinobacteria function with high chemo-, regio-, and enantio-selectivity under benign conditions, which could help current industrial processing. Numerous selective enzymes were either isolated from actinobacteria or expressed from actinobacteria in other microbes and hence exploited in the production of pure organic compounds difficult to obtain chemically. In addition, different species of actinobacteria, especially Streptomyces species, function as natural producers of chiral molecules of therapeutic importance. Herein, we discuss some of the most outstanding contributions of actinobacteria to asymmetric biocatalysis, which are important in the organic and/or pharmaceutical industries. In addition, we highlight the role of actinobacteria as microbial cell factories for chiral natural products with insights into their various biological potentialities.

Macrophage-Myofibroblast Transition Contributes to Myofibroblast Formation in Proliferative Vitreoretinal Disorders.

Inflammation and fibrosis are key features of proliferative vitreoretinal disorders. We aimed to define the macrophage phenotype and investigate the role of macrophage-myofibroblast transition (MMT) in the contribution to myofibroblast populations present in epiretinal membranes. Vitreous samples from proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR) and nondiabetic control patients, epiretinal fibrovascular membranes from PDR patients and fibrocellular membranes from PVR patients, human retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied by ELISA, immunohistochemistry and flow cytometry analysis. Myofibroblasts expressing α-SMA, fibroblast activation protein-α (FAP-α) and fibroblast-specific protein-1 (FSP-1) were present in all membranes. The majority of CD68+ monocytes/macrophages co-expressed the M2 macrophage marker CD206. In epiretinal membranes, cells undergoing MMT were identified by co-expression of the macrophage marker CD68 and myofibroblast markers α-SMA and FSP-1. Further analysis revealed that CD206+ M2 macrophages co-expressed α-SMA, FSP-1, FAP-α and ß-catenin. Soluble (s) CD206 and sFAP-α levels were significantly higher in vitreous samples from PDR and PVR patients than in nondiabetic control patients. The proinflammatory cytokine TNF-α and the hypoxia mimetic agent cobalt chloride induced upregulation of sFAP-α in culture media of Müller cells but not of HRMECs. The NF-Ä¸ß inhibitor BAY11-7085 significantly attenuated TNF-α-induced upregulation of sFAP-α in Müller cells. Our findings suggest that the process of MMT might contribute to myofibroblast formation in epiretinal membranes, and this transition involved macrophages with a predominant M2 phenotype. In addition, sFAP-α as a vitreous biomarker may be derived from M2 macrophages transitioned to myofibroblasts and from Müller cells.

Sputum from patients with primary ciliary dyskinesia contains high numbers of dysfunctional neutrophils and inhibits efferocytosis.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by recurrent airway infection and inflammation. There is no cure for PCD and to date there are no specific treatments available. Neutrophils are a crucial part of the immune system and are known to be dysfunctional in many inflammatory diseases. So far, the role of the neutrophils in PCD airways is largely unknown. The purpose of this study was to investigate the phenotype and function of airway neutrophils in PCD, and compare them to blood neutrophils. METHODS: Paired peripheral blood and spontaneously expectorated sputum samples from patients with PCD (n = 32) and a control group of patients with non-PCD, non-cystic fibrosis bronchiectasis (n = 5) were collected. The expression of neutrophil-specific surface receptors was determined by flow cytometry. Neutrophil function was assessed by measuring the extent of actin polymerization, production of reactive oxygen species (ROS) and release of neutrophil extracellular traps (NETs) in response to activating stimuli. RESULTS: Sputum neutrophils displayed a highly activated phenotype and were unresponsive to stimuli that would normally induce ROS production, actin polymerization and the expulsion of NETs. In addition, PCD sputum displayed high activity of neutrophil elastase, and impaired the efferocytosis by healthy donor macrophages. CONCLUSIONS: Sputum neutrophils in PCD are dysfunctional and likely contribute to ongoing inflammation in PCD airways. Further research should focus on anti-inflammatory therapies and stimulation of efferocytosis as a strategy to treat PCD.

Bright spotty lesions as an imaging marker for neuromyelitis optica spectrum disorder.

Neuromyelitis optica spectrum disorder (NMOSD) is a rare inflammatory demyelinating disorder of the central nervous system (CNS). Aquaporin-4 (AQP4) antibodies in the serum are highly specific for the diagnosis of NMOSD, but the sensitivity remains under 90% allowing for diagnosis of AQP4 IgG seronegative disease. It remains of crucial importance to identify seronegative NMOSD myelitis as early as the first attack to initiate long-term treatment that will reduce future relapses and disability and to avoid potentially harmful treatments such as those of multiple sclerosis (MS). Over the years, many spinal imaging features have been reported to favour the diagnosis of NMOSD, but only longitudinally extensive transverse myelitis (LETM) was specific enough to make the diagnostic criteria in the AQP4 IgG seronegative cases. Bright spotty lesions (BSLs), which are defined as hyperintense lesions on axial T2-weighted images and sometimes associated with T1 low signal, are now reported to have a higher specificity and sensitivity compared to LETM in predicting a diagnosis of NMOSD against other causes of myelitis. In the review, we aim to highlight the position of BSLs in diagnosing NMOSD as well as its possible role as a prognostic factor for the clinical outcome.

Female sexual dysfunction in newly diagnosed egyptian patients with neuromyelitis optica spectrum disorder.

BACKGROUND: Few research works have explored female sexual dysfunction (FSD) in patients with Neuromyelitis optica spectrum disorder (NMOSD) which remains an ignored disease symptom. This work aimed to describe the frequency, patterns, and predictors of FSD in a sample of newly diagnosed AQP4-ab seropositive NMOSD patients. METHODS: This case-control study was conducted on 28 seropositive NMOSD patients and 31 age matched healthy controls. All included patients were asked to privately fill and hand back the following questionnaires: female sexual function index questionnaire (FSFI), Beck depression inventory II (BDI) and fatigue severity scale (FSS). Also, Modified Modified Ashworth scale (MMAS) and Expanded disability status scale (EDSS) were applied to all included patients. RESULTS: NMOSD patients had significantly lower total FSFI scores and significantly higher BDI and FSS scores than controls (P < 0.001). FSS scores were negatively correlated with total scores of FSFI as well as desire, lubrication, orgasm, and satisfaction scores. BDI scores was negatively correlated with desire and orgasm scores. The uncorrected visual FS score was negatively correlated with lower total scores of FSFI as well as arousal, orgasm, and satisfaction scores. The pain score was negatively correlated with the scores of the MMAS. The only predictors of FSFI total score were fatigue and visual disability. Visual disability was also a predictor of dysfunction in arousal and satisfaction domains, whereas spasticity in the lower limbs predicted sexual related pain. CONCLUSIONS: Sexual dysfunction in patients with NMOSD is strongly related to fatigue, depression, visual disability, and lower limbs spasticity.

The turning away of serum amyloid A biological activities and receptor usage.

Serum amyloid A (SAA) is an acute-phase protein (APP) to which multiple immunological functions have been attributed. Regardless, the true biological role of SAA remains poorly understood. SAA is remarkably conserved in mammalian evolution, thereby suggesting an important biological function. Since its discovery in the 1970s, the majority of researchers have investigated SAA using recombinant forms made available through bacterial expression. Nevertheless, recent studies indicate that these recombinant forms of SAA are unreliable. Indeed, commercial SAA variants have been shown to be contaminated with bacterial products including lipopolysaccharides and lipoproteins. As such, biological activities and receptor usage (TLR2, TLR4) revealed through the use of commercial SAA variants may not reflect the inherent nature of this APP. Within this review, we discuss the biological effects of SAA that have been demonstrated through more solid experimental approaches. SAA takes part in the innate immune response via the recruitment of leucocytes and executes, through pathogen recognition, antimicrobial activity. Knockout animal models implicate SAA in a range of functions, such as regulation of T-cell-mediated responses and monopoiesis. Moreover, through its structural motifs, not only does SAA function as an extracellular matrix protein, but it also binds extracellular matrix proteins. Finally, we here also provide an overview of definite SAA receptor-mediated functions and highlight those that are yet to be validated. The role of FPR2 in SAA-mediated leucocyte recruitment has been confirmed; nevertheless, SAA has been linked to a range of other receptors including CD36, SR-BI/II, RAGE and P2RX7.

The Antimicrobial Activity of Peripheral Blood Neutrophils Is Altered in Patients with Primary Ciliary Dyskinesia.

The airways of patients with primary ciliary dyskinesia (PCD) contain persistently elevated neutrophil numbers and CXCL8 levels. Despite their abundance, neutrophils fail to clear the airways from bacterial infections. We investigated whether neutrophil functions are altered in patients with PCD. Neutrophils from patients and healthy controls (HC) were isolated from peripheral blood and exposed to various bacterial stimuli or cytokines. Neutrophils from patients with PCD were less responsive to low levels of fMLF in three different chemotaxis assays (p < 0.05), but expression of the fMLF receptors was unaltered. PCD neutrophils showed normal phagocytic function and expression of adhesion molecules. However, PCD neutrophils produced less reactive oxygen species upon stimulation with bacterial products or cytokines compared to HC neutrophils (p < 0.05). Finally, the capacity to release DNA, as observed during neutrophil extracellular trap formation, seemed to be reduced in patients with PCD compared to HC (p = 0.066). These results suggest that peripheral blood neutrophils from patients with PCD, in contrast to those of patients with cystic fibrosis or COPD, do not show features of over-activation, neither on baseline nor after stimulation. If these findings extend to lung-resident neutrophils, the reduced neutrophil activity could possibly contribute to the recurrent respiratory infections in patients with PCD.

MRI differences between MOG antibody disease and AQP4 NMOSD.

BACKGROUND: MOG antibody and AQP4 antibody seropositive diseases are immunologically distinct subtypes of neuromyelitis optica spectrum disorders (NMOSD) with similar clinical presentations. MRI findings can be instrumental in distinguishing MOG antibody disease from AQP4 antibody NMOSD. OBJECTIVES: The aim of this study is to characterize the neuroradiological differences between MOG antibody disease and AQP4 antibody NMOSD with the aim to distinguish between the two entities. METHODS: This is a retrospective study of 26 MOG and 25 AQP4 seropositive patients in which MRI features of the brain, spinal cord, and orbit were compared. RESULTS: The majority of the abnormal findings in the MOG cohort were located on orbital MRIs, while spinal cord magnetic resonance (MR) abnormalities were more common in the AQP4 cohort. Brain abnormalities showed some overlap, but cortical gray/juxtacortical white matter involvement was distinct to MOG patients, while area postrema involvement was a rare feature. CONCLUSION: Cortical gray/juxtacortical white matter lesions on brain MRI might help distinguish MOG antibody disease from AQP4-positive NMOSD. These findings could be of value in distinguishing the two entities as early as the first presentation.

Aquaporin-4 IgG seropositivity is associated with worse visual outcomes after optic neuritis than MOG-IgG seropositivity and multiple sclerosis, independent of macular ganglion cell layer thinning.

BACKGROUND: Comparative studies of characteristics of optic neuritis (ON) associated with myelin oligodendrocyte glycoprotein-IgG (MOG-ON) and aquaporin-4-IgG (AQP4-ON) seropositivity are limited. OBJECTIVE: To compare visual and optical coherence tomography (OCT) measures following AQP4-ON, MOG-ON, and multiple sclerosis associated ON (MS-ON). METHODS: In this cross-sectional study, 48 AQP4-ON, 16 MOG-ON, 40 MS-ON, and 31 healthy control participants underwent monocular letter-acuity assessment and spectral-domain OCT. Eyes with a history of ON >3 months prior to evaluation were analyzed. RESULTS: AQP4-ON eyes exhibited worse high-contrast letter acuity (HCLA) compared to MOG-ON (-22.3 ± 3.9 letters; p < 0.001) and MS-ON eyes (-21.7 ± 4.0 letters; p < 0.001). Macular ganglion cell + inner plexiform layer (GCIPL) thickness was lower, as compared to MS-ON, in AQP4-ON (-9.1 ± 2.0 µm; p < 0.001) and MOG-ON (-7.6 ± 2.2 µm; p = 0.001) eyes. Lower GCIPL thickness was associated with worse HCLA in AQP4-ON (-16.5 ± 1.5 letters per 10 µm decrease; p < 0.001) and MS-ON eyes (-8.5 ± 2.3 letters per 10 µm decrease; p < 0.001), but not in MOG-ON eyes (-5.2 ± 3.8 letters per 10 µm decrease; p = 0.17), and these relationships differed between the AQP4-ON and other ON groups (p < 0.01 for interaction). CONCLUSION: AQP4-IgG seropositivity is associated with worse visual outcomes after ON compared with MOG-ON and MS-ON, even with similar severity of macular GCIPL thinning.

Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case.

The serum amyloid A (SAA) gene family is highly conserved and encodes acute phase proteins that are upregulated in response to inflammatory triggers. Over the years, a considerable amount of literature has been published attributing a wide range of biological effects to SAAs such as leukocyte recruitment, cytokine and chemokine expression and induction of matrix metalloproteinases. Furthermore, SAAs have also been linked to protumorigenic, proatherogenic and anti-inflammatory effects. Here, we investigated the biological effects conveyed by murine SAA3 (mu rSAA3) recombinantly expressed in Escherichia coli. We observed the upregulation of a number of chemokines including CCL2, CCL3, CXCL1, CXCL2, CXCL6 or CXCL8 following stimulation of monocytic, fibroblastoid and peritoneal cells with mu rSAA3. Furthermore, this SAA variant displayed potent in vivo recruitment of neutrophils through the activation of TLR4. However, a major problem associated with proteins derived from recombinant expression in bacteria is potential contamination with various bacterial products, such as lipopolysaccharide, lipoproteins and formylated peptides. This is of particular relevance in the case of SAA as there currently exists a discrepancy in biological activity between SAA derived from recombinant expression and that of an endogenous source, i.e. inflammatory plasma. Therefore, we subjected commercial recombinant mu rSAA3 to purification to homogeneity via reversed-phase high-performance liquid chromatography (RP-HPLC) and re-assessed its biological potential. RP-HPLC-purified mu rSAA3 did not induce chemokines and lacked in vivo neutrophil chemotactic activity, but retained the capacity to synergize with CXCL8 in the activation of neutrophils. In conclusion, experimental results obtained when using proteins recombinantly expressed in bacteria should always be interpreted with care.

MOG antibody-associated encephalomyelitis/encephalitis.

Myelin oligodendrocyte glycoprotein (MOG) antibody disease is a rare autoimmune disorder with antibodies against the MOG predominantly involving the optic nerve and spinal cord leading to vision loss and paralysis. When MOG antibody disease involves the brain, the phenotype is similar to acute disseminated encephalomyelitis (ADEM). In this review, we discuss MOG-positive cases presenting with encephalitis, encephalopathy, or ADEM-like presentation based on recently published series.

Depiction of nervous system disorders in television medical drama: A content analysis of 18 seasons of Grey's Anatomy.

BACKGROUND: Television medical dramas enjoy great popularity among the general public, and can be a source of information and misinformation about medical disorders. Nervous system disorders have always received attention in popular media, yet no studies have been performed to analyze their depiction and accuracy, to our knowledge. OBJECTIVE: To investigate the representation of neurological and neurosurgical diseases in Grey's Anatomy, one of most popular and longest running primetime medical melodramas in American television. METHODS: We performed a quantitative and qualitative content analysis of depictions of nervous system disorders in all 18 seasons of Grey's Anatomy, in addition to investigating the medical accuracy and the global quality of the portrayed medical content. RESULTS: A total of 285 depictions were identified in 314 out of 400 episodes (78.5%) of Grey's Anatomy. Most of portrayed characters were males (59.2%), Caucasians (72.6%), and adults (76.4%), with a mean age of 32.6 ± 19.7 years for those mentioned. Most of the portrayals were of acute nature (65.5%), with neurosurgical diseases constituting the majority; 85.6%, while 31.6% depicted neurological diseases, and 19.3% had both. The most common clinical presentations were altered level of consciousness (16.5%) and seizures (14.4%). Traumatic brain injury was the most common portrayed diagnosis (39.3%), followed by brain tumors (13.7%), stroke (8.4%), and spinal injury (8.1%). Management was surgical for the majority of cases (79.6%), while only 17.9% received pharmacotherapy. The prognosis of portrayed cases was generally favorable, as 79.5% showed good/full recovery, while mortality rate was 18.6%. Forty cases (14.0%) were depictions of rare to very rare diseases. As regards to medical accuracy, 74.3% of depictions were deemed accurate within reason, with a mean GQS score of 4.38 ± 1.4. Four clinical trials involving the nervous system have been depicted throughout the show. CONCLUSIONS: Neurosurgical and neurological diseases were portrayed in most episodes of Grey's Anatomy, with a good quality of its scientific content within reason. This study demonstrated that Grey's Anatomy had a good amount of medical information that could be able to improve the public perception of the specialty. However, there is still a room for significant improvement to acknowledge certain inaccuracies and misrepresentations in future episodes.

Acute-serum amyloid A and A-SAA-derived peptides as formyl peptide receptor (FPR) 2 ligands.

Originally, it was thought that a single serum amyloid A (SAA) protein was involved in amyloid A amyloidosis, but in fact, SAA represents a four-membered family wherein SAA1 and SAA2 are acute phase proteins (A-SAA). SAA is highly conserved throughout evolution within a wide range of animal species suggestive of an important biological function. In fact, A-SAA has been linked to a number of divergent biological activities wherein a number of these functions are mediated via the G protein-coupled receptor (GPCR), formyl peptide receptor (FPR) 2. For instance, through the activation of FPR2, A-SAA has been described to regulate leukocyte activation, atherosclerosis, pathogen recognition, bone formation and cell survival. Moreover, A-SAA is subject to post-translational modification, primarily through proteolytic processing, generating a range of A-SAA-derived peptides. Although very little is known regarding the biological effect of A-SAA-derived peptides, they have been shown to promote neutrophil and monocyte migration through FPR2 activation via synergy with other GPCR ligands namely, the chemokines CXCL8 and CCL3, respectively. Within this review, we provide a detailed analysis of the FPR2-mediated functions of A-SAA. Moreover, we discuss the potential role of A-SAA-derived peptides as allosteric modulators of FPR2.

Association of CNS demyelination and COVID-19 infection: an updated systematic review.

BACKGROUND: Since the declaration of COVID-19 pandemic, several case reports of demyelination of both peripheral and central nervous systems have been published. The association between CNS demyelination and viral infection has long been documented, and this link was recently reported following SARS-CoV-2 infection as well. OBJECTIVES: In this systematic review, we aim to investigate the existing literature on CNS demyelination associated with SARS-CoV-2, and the proposed pathophysiological mechanisms. METHODS: We conducted a systematic review of articles in PubMed, SCOPUS, EMBASE, Cochrane, Google Scholar and Ovid databases, from 1 January 2020 until June 15, 2021. The following keywords were used: "COVID-19", "SARS-CoV-2", "demyelination", "demyelinating disease", "multiple sclerosis", "neuromyelitis optica", and "transverse myelitis". RESULTS: A total of 60 articles were included in the final analysis of this systematic review and included 102 patients: 52 (51%) men and 50 (49%) women, with a median age of 46.5 years. The demyelination mimicked a variety of conditions with a picture of encephalitis/encephalomyelitis being the most common. At the same time other patterns were less frequently reported such as MS, NMOSD and even MOGAD. Longitudinally extensive transverse myelitis (LETM) was the most frequently reported pattern of spinal cord involvement. CONCLUSION: A growing body of literature has shown an association between SARS-CoV-2 infection and the development of different types of CNS demyelination. Although causality cannot readily be inferred, this review may suggest a probable causal relationship, through a para-infectious or post-infectious immune-mediated etiology in COVID-19 patients. This relationship needs to be clarified in future research.

A systematic review of cases of CNS demyelination following COVID-19 vaccination.

BACKGROUND: Since the emergency use approval of different types of COVID-19 vaccines, several safety concerns have been raised regarding its early and delayed impact on the nervous system. OBJECTIVE: This study aims to systematically review the reported cases of CNS demyelination in association with COVID-19 vaccination, which has not been performed, to our knowledge. METHODS: A systematic review was performed by screening published articles and preprints of cases of CNS demyelination in association with COVID-19 vaccines in PubMed, SCOPUS, EMBASE, Google Scholar, Ovid and medRxiv databases, until September 30, 2021. This study followed PRISMA guidelines. Descriptive findings of reported cases were reviewed and stratified by demographic and clinical findings, diagnostic work-up, management, and overall outcome. RESULTS: A total of 32 cases were identified, with female predominance (68.8%) and median age of 44 years. Eleven cases were reported after Pfizer vaccine, 8 following AstraZeneca vaccine, 6 following Moderna, 5 following Sinovac/ Sinopharm vaccines, and one following each of Sputnik and Johnson&Johnson vaccines. The majority of cases (71.8%) occurred after the first dose of the vaccine, with neurological symptoms manifesting after a median of 9 days. The most common reported presentations were transverse myelitis (12/32) and MS-like pictures (first diagnosis or a relapse) in another 12/32 cases, followed by ADEM- like (5/32), and NMOSD- like (3/32) presentations. History of a previous immune-mediated disease was reported in 17/32 (53.1%) cases. The mRNA-based vaccines resulted in the greatest number of demyelinating syndromes (17/32), followed by viral vector vaccines (10/32), and inactivated vaccines (5/32). Most MS-like episodes (9/12) were triggered by mRNA-based vaccines, while TM occurred following both viral vector and mRNA-based vaccines. Management included high dose methylprednisolone, PLEX, IVIg, or a combination of those, with a favorable outcome in the majority of case; marked/complete improvement (25/32) or stabilized/ partial recovery in the remaining cases. CONCLUSION: This systematic review identified few cases of CNS demyelination following all types of approved COVID-19 vaccines so far. Clinical presentation was heterogenous, mainly following the first dose, however, half of the reported cases had a history of immune-mediated disease. Favorable outcome was observed in most cases. We suggest long-term post-marketing surveillance for these cases, to assess for causality, and ensure the safety of COVID-19 vaccines.

Reviewing a plethora of oxidative-type reactions catalyzed by whole cells of Streptomyces species.

Selective oxidation reactions represent a challenging task for conventional organic chemistry. Whole-cell biocatalysis provides a very convenient, easy to apply method to carry out different selective oxidation reactions including chemo-, regio-, and enantio-selective reactions. Streptomyces species are important biocatalysts as they can catalyze these selective reactions very efficiently owing to the wide diversity of enzymes and enzymatic cascades in their cell niche. In this review, we present and analyze most of the examples reported to date of oxidative reactions catalyzed by Streptomyces species as whole-cell biocatalysts. We discuss 33 different Streptomyces species and strains and the role they play in different oxidative reactions over the past five decades. The oxidative reactions have been classified into seven categories that include: hydroxylation of steroids/non-steroids, asymmetric sulfoxidations, oxidation of aldehydes, multi-step oxidations, oxidative cleavage, and N-oxidations. The role played by Streptomyces species as recombinant hosts catalyzing bio-oxidations has also been highlighted.

Defining the Importance of Age-Related Changes in Drug Clearance to Optimizing Aminoglycoside Dosing Regimens for Adult Patients with Cystic Fibrosis.

BACKGROUND AND OBJECTIVE: The number of adults living with cystic fibrosis (CF) has increased and will continue to do so with the approval of cystic fibrosis transmembrane conductance regulator (CFTR) modulators. Because systemic aminoglycosides are commonly administered for CF pulmonary exacerbations, we sought to define optimized dosing regimens using a population pharmacokinetic modeling and simulation approach. METHODS: Adult CF patients admitted for pulmonary exacerbation, receiving at least 72 h of systemic gentamicin, tobramycin, or amikacin, with measured concentrations were included. Covariates [e.g., age, weight, creatinine clearance (CRCL)] were screened. Population modeling was completed using Monolix, and simulations were conducted in R. Simulated exposures were calculated using noncompartmental analysis. Once-daily fixed (10 mg/kg) and exposure-matched dosing (i.e., 15, 10, 7.5, 6 mg/kg for ages 20, 30, 40, and 50 years, respectively) strategies were compared. First-24 h exposures were evaluated for each strategy according to the probability of target attainment (PTA) (ratio of peak plasma concentrations relative to the minimum inhibitory concentration [Cmax/MIC] or ratio of the area under the concentration-time curve to MIC [AUC/MIC]) and the probability of toxic exposure (PTE) (trough concentration, Ctrough > 2 mg/l). RESULTS: Forty-eight adult patients (55% female) were included. A one-compartment model best fit the data. Estimates for volume of distribution (V) and clearance (CL) were 22 l and 5.57 l/h, respectively. Weight significantly modified CL and V. Age significantly modified CL and was more influential than CRCL. PTA was > 90% at MICs ≤ 1 mg/l for fixed doses of 10 mg/kg and for exposure-matched doses at MIC ≤ 1 mg/l. Exposure-matched dosing reduced PTE roughly 50% in patients aged 40 and 50 years vs. fixed dosing. CONCLUSIONS: Exposure-matching maintained PTA at MICs ≤ 1 mg/l while reducing toxicity risk in older patients compared to fixed dosing. Confirmatory studies are needed.

Method Matters: Effect of Purification Technology on Neutrophil Phenotype and Function.

Neutrophils are the most abundant leukocytes in human blood and the first cells responding to infection and injury. Due to their limited ex vivo lifespan and the impossibility to cryopreserve or expand them in vitro, neutrophils need to be purified from fresh blood for immediate use in experiments. Importantly, neutrophil purification methods may artificially modify the phenotype and functional characteristics of the isolated cells. The aim of this study was to expose the effects of 'classical' density-gradient purification versus the more expensive but faster immunomagnetic isolation on neutrophil phenotype and functionality. We found that in the absence of inflammatory stimuli, density-gradient-derived neutrophils showed increased polarization responses as well as enhanced release of reactive oxygen species (ROS), neutrophil extracellular traps (NETs) and granular proteins compared to cells derived from immunomagnetic isolation, which yields mostly quiescent neutrophils. Upon exposure to pro-inflammatory mediators, immunomagnetic isolation-derived neutrophils were significantly more responsive in polarization, ROS production, phagocytosis, NETosis and degranulation assays, in comparison to density-gradient-derived cells. We found no difference in chemotactic response in Multiscreen and under-agarose migration assays, but Boyden assays showed reduced chemotaxis of immunomagnetic isolation-derived neutrophils. Finally, we confirmed that density-gradient purification induces artificial activation of neutrophils, evidenced by e.g. higher expression of CD66b, formyl peptide receptor 1 (FPR1) and CD35, and the appearance of a separate neutrophil population expressing surface molecules atypical for neutrophils (e.g. CXCR3, MHC-II and CD14). Based on these results, we recommend using immunomagnetic separation of neutrophils for studying neutrophil polarization, phagocytosis, ROS production, degranulation and NETosis, whereas for Boyden chemotaxis assays, the density-gradient purification is more suitable.

From ELISA to Immunosorbent Tandem Mass Spectrometry Proteoform Analysis: The Example of CXCL8/Interleukin-8.

With ELISAs one detects the ensemble of immunoreactive molecules in biological samples. For biomolecules undergoing proteolysis for activation, potentiation or inhibition, other techniques are necessary to study biology. Here we develop methodology that combines immunosorbent sample preparation and nano-scale liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) for proteoform analysis (ISTAMPA) and apply this to the aglycosyl chemokine CXCL8. CXCL8, the most powerful human chemokine with neutrophil chemotactic and -activating properties, occurs in different NH2-terminal proteoforms due to its susceptibility to site-specific proteolytic modification. Specific proteoforms display up to 30-fold enhanced activity. The immunosorbent ion trap top-down mass spectrometry-based approach for proteoform analysis allows for simultaneous detection and quantification of full-length CXCL8(1-77), elongated CXCL8(-2-77) and all naturally occurring truncated CXCL8 forms in biological samples. For the first time we demonstrate site-specific proteolytic activation of CXCL8 in synovial fluids from patients with chronic joint inflammation and address the importance of sample collection and processing.

Cognitive functions in Egyptian neuromyelitis optica spectrum disorder.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune demyelinating disease of the central nervous system, characterized by optic neuritis and longitudinally extensive transverse myelitis. Magnetic resonance imaging abnormalities may be observed in various brain regions of NMOSD patients. Only a few studies have addressed the cognitive functions in NMOSD, but none among Egyptian patients. OBJECTIVE: To investigate cognitive performance in a cohort of 20 Egyptian patients with NMOSD. DESIGN: Observational, prospective study. PATIENTS: We studied 20 Egyptian patients with NMOSD and compared them with 18 healthy Egyptian controls matched for age, sex, and educational level. MAIN OUTCOME MEASURE: We applied an Arabic translation of MOCA and BICAMS Tests for Multiple Sclerosis. RESULTS: Cognitive performance was significantly worse in the NMOSD group than in healthy controls for CVLT (P = 0.0099), SDMT (P = 0.0112), BVSMT (P = 0.019) and BICAMS in total (P = 0.0014). Patients with a later disease onset performed worse in MOCA and BVSMT. CONCLUSIONS: This study confirms the concept of cognitive involvement in NMOSD among Egyptian patients. Information processing speed was the function most commonly impaired.