Developing evidence-based guidelines for the safety of symptomatic drugs in multiple sclerosis during pregnancy and breastfeeding: A systematic review and Delphi consensus.

BACKGROUND: Multiple sclerosis (MS) is frequently diagnosed in people of reproductive age, many of whom will become pregnant following diagnosis. Although many women report an improvement in symptoms and relapses during pregnancy, symptoms such as fatigue and spasticity are commonly reported and can worsen. Prescribing medications during pregnancy and breastfeeding presents unique challenges and guidance on the use of symptomatic therapies is limited. OBJECTIVES: This paper aims to provide a consensus on the current evidence base to facilitate informed decision-making and optimise pre-conception counselling. METHODS: A list of most commonly prescribed medications for symptom management in MS was created using pregnancy and MS-related READ codes in the Welsh GP Dataset, followed by a review by MS neurologists. RESULTS: A final list of 24 medications was generated for review. Searches were performed on each medication, and evidence graded using standardised criteria. Evidence-based recommendations were developed and distributed to experts in the field and revised according to feedback using modified Delphi criteria. CONCLUSIONS: Our guidelines provide evidence-based recommendations on the safety of symptomatic therapies during pregnancy and breastfeeding for general practitioners and specialist teams working with people with MS who are hoping to embark on pregnancy or are currently pregnant. Individual risk-benefit ratios should be considered during pre-conception counselling to optimise symptom burden and minimise harm to both parent and child.

Microbial profile, antimicrobial resistance, and molecular characterization of diabetic foot infections in a university hospital.

INTRODUCTION: Diabetic foot infections (DFIs) are among the most severe complications of diabetes. The aim of this study was to determine the etiological pathogens of DFIs in different Wagner's and IDSA/IWGDF grades, and to assess their antimicrobial susceptibility pattern together with molecular characterization of antibiotic resistance genes. METHODS: A prospective study was conducted on 120 DFI patients at Main Alexandria University Hospital, Egypt. The aerobic and anaerobic etiological pathogens were determined using semi-quantitative culture and PCR respectively. The antimicrobial susceptibility pattern was done according to Clinical Laboratory Standards Institute guidelines. Detection of carbapenemases and class-1 integron genes was carried out by polymerase chain reaction (PCR). RESULTS: A total of 178 (124 aerobic, 54 anaerobic) pathogens were identified from patients with DFI, with an average of 1.82 isolates/subject. Among aerobic pathogens, Gram-negative predominated (98/124; 79%), of which Pseudomonas spp. and Proteus spp. were the most common. MRSA constituted more than 50% of Gram-positive isolates. Polymicrobial infection was found in 42 (42.9%) subjects. The proportion of Gram-negative bacteria and anaerobes increased with increased DFI grades and severity. Multidrug and extensively drug resistant isolates were observed in 86 patients (87.7%). PCR identified carbapenemases genes in 14 (11.7%) and class 1 integron in 28 (23.3%) DFI cases. Vancomycin, teicoplanin, linezolid were the most effective antimicrobial agents against Gram-positive pathogens, while colistin, imipenem, meropenem, and piperacillin-tazobactam were effective against Gram-negative pathogens. CONCLUSIONS: Multidrug and extensively drug resistant Gram-negative bacteria were the dominant pathogens among all DFI severity grades. However, the proportion of Gram-positive bacteria decreased with the severity of infection. The clinical role of our relatively high rate of anaerobes should be investigated. The results found in this study could be beneficial for designing future empiric antimicrobial protocols in relation to the severity of DFIs.

Challenging inflammatory process at molecular, cellular and in vivo levels via some new pyrazolyl thiazolones.

The work reported herein describes the synthesis of a new series of anti-inflammatory pyrazolyl thiazolones. In addition to COX-2/15-LOX inhibition, these hybrids exerted their anti-inflammatory actions through novel mechanisms. The most active compounds possessed COX-2 inhibitory activities comparable to celecoxib (IC50 values of 0.09-0.14 µM) with significant 15-LOX inhibitory activities (IC50s 1.96 to 3.52 µM). Upon investigation of their in vivo anti-inflammatory activities and ulcerogenic profiles, these compounds showed activity patterns equivalent or more superior to diclofenac and/or celecoxib. Intriguingly, the most active compounds were more effective than diclofenac in suppressing monocyte-to-macrophage differentiation and inflammatory cytokine production by activated macrophages, as well as their ability to induce macrophage apoptosis. The latter finding potentially adds a new dimension to the previously reported anti-inflammatory mechanisms of similar compounds. These compounds were effectively docked into COX-2 and 15-LOX active sites. Also, in silico predictions confirmed the appropriateness of these compounds as drug-like candidates.

Rehabilitation Before and After Autologous Haematopoietic Stem Cell Transplantation (AHSCT) for Patients With Multiple Sclerosis (MS): Consensus Guidelines and Recommendations for Best Clinical Practice on Behalf of the Autoimmune Diseases Working Party, Nurses Group, and Patient Advocacy Committee of the European Society for Blood and Marrow Transplantation (EBMT).

Autologous haematopoietic stem cell transplantation (AHSCT) is increasingly used to treat people with multiple sclerosis (MS). Supported by an evolving evidence base, AHSCT can suppress active inflammation in the central nervous system and induce long-term changes in immune cell populations, thereby stabilizing, and, in some cases, reversing disability in carefully selected MS patients. However, AHSCT is an intensive chemotherapy-based procedure associated with intrinsic risks, including profound cytopenia, infection, and organ toxicity, accompanied by an on-going degree of immuno-compromise and general deconditioning, which can be associated with a transient increase in functional impairment in the early stages after transplantation. Although international guidelines and recommendations have been published for clinical and technical aspects of AHSCT in MS, there has been no detailed appraisal of the rehabilitation needed following treatment nor any specific guidelines as to how this is best delivered by hospital and community-based therapists and wider multidisciplinary teams in order to maximize functional recovery and quality of life. These expert consensus guidelines aim to address this unmet need by summarizing the evidence-base for AHSCT in MS and providing recommendations for current rehabilitation practice along with identifying areas for future research and development.

Novel pyrazine based anti-tubercular agents: Design, synthesis, biological evaluation and in silico studies.

TB continues to be a leading health threat despite the availability of powerful anti-TB drugs. We report herein the design and synthesis of various hybrid molecules comprising pyrazine scaffold and various formerly identified anti-mycobacterial moieties. Thirty-one compounds were screened in vitro for their activity against Mycobacterium tuberculosis H37Rv strain using MABA assay. The results revealed that six compounds (8a, 8b, 8c, 8d, 14b and 18) displayed significant activity against Mtb with MIC values ≤6.25 µg/ml versus 6.25 µg/ml for pyrazinamide. The most active compounds were then assessed for their in vitro cytotoxicity against PBMC normal cell line using MTT assay and showed SI > 200. Several in silico studies have been carried out for target fishing of the novel compounds such as shape-based similarity, pharmacophore mapping and inverse docking. Based on this multi-step target fishing study, we suggest that pantothenate synthetase could be the possible target responsible for the action of these compounds. The most active compounds were then successfully docked into the active site of pantothenate synthetase enzyme with favorable binding interactions. In addition, in silico prediction of physicochemical, ADMET and drug-like properties were also determined indicating that compounds 8b, 8c and 8d are promising candidates for the development of new anti-TB agents with enhanced activity and better safety profile.

Effect of remote ischaemic preconditioning on walking in people with multiple sclerosis: double-blind randomised controlled trial.

BACKGROUND: Remote ischaemic preconditioning (RIPC) is the exposure of body parts to brief periods of circulatory occlusion and reperfusion. Recent studies have also shown that RIPC can improve exercise performance in healthy individuals. OBJECTIVE: This study aimed to assess the effect of RIPC on walking in people with multiple sclerosis (MS). METHODS: This was a double-blind randomised controlled clinical trial. We used three cycles of RIPC delivered by occluding the upper arm with a blood pressure (BP) cuff inflated to a pressure of 30 mm Hg above the systolic BP. In patients in the sham intervention group, the BP cuff was inflated only to 30 mm Hg below diastolic BP. Outcome measures included the Six-Minute Walk Test (6MWT), gait speed, the Borg rate of perceived exertion (RPE) scale, the tolerability of the RIPC using a Numerical Rating Scale for discomfort from 0 to 10, and adverse events. We identified responders meeting the minimal clinically important difference (MCID) established in the literature in each group. RESULTS: Seventy-five participants completed the study (RIPC: 38 and Sham: 37). The distance walked during the 6MWT improved by 1.9% in the sham group and 5.7% in the RIPC group (p=0.012). The number of responders meeting MCID criteria in the RIPC group was significantly greater compared with the sham intervention group. No serious adverse events occurred. CONCLUSION: Single cycle of RIPC resulted in immediate improvement in walking distances during 6MWT in people with MS. TRIAL REGISTRATION NUMBERS: NCT03153553.

Autologous haematopoietic stem cell therapy for multiple sclerosis: a review for supportive care clinicians on behalf of the Autoimmune Diseases Working Party of the European Society for Blood and Marrow Transplantation.

PURPOSE OF REVIEW: In this review, we summarize the recently published literature that demonstrates the efficacy and safety of autologous haematopoietic stem cell therapy (AHSCT) in multiple sclerosis (MS) and highlight the importance of supportive care required for the safe and well-tolerated delivery of AHSCT. RECENT FINDINGS: MS is an autoimmune inflammatory and degenerative disorder of the central nervous system (CNS). In the majority of patients, the illness runs a relapsing remitting course (RRMS), culminating in a secondary progressive phase with gradual accumulation of fixed disabilities. Currently available disease-modifying therapies suppress CNS inflammation but have a limited effect on preventing disease progression for which there remains no effective therapy. Over the last two decades, there has been increasing evidence that AHSCT is a highly effective therapeutic strategy for treatment-resistant inflammatory types of MS, especially RRMS. Concerns about the safety of AHSCT in MS, usually a nonlife-threatening disease, have previously limited its use. However, AHSCT can now be delivered safely with major long-term benefits because of increasing transplant centre experience, judicious patient selection and good supportive care. SUMMARY: MS is currently the fastest growing indication for AHSCT in Europe. Supportive care before, during and after the transplant period is key to the successful delivery of AHSCT.

Association of diabetic foot ulcers with chronic vascular diabetic complications in patients with type 2 diabetes.

CONTEXT: Diabetes mellitus is a common disease which is prevalent globally, presenting with chronic complications and constitutes a major risk to the patient. Diabetic foot ulcers are the single biggest risk factor for non-traumatic lower limb amputations in persons with diabetes. We aimed to screen for the chronic vascular diabetic complications in patients with diabetic foot ulcers (DFUs) and to assess the association of diabetic foot ulcers with these complications in the study group. SUBJECTS AND METHODS: This cross-sectional study included 180 type 2 diabetic patients (aged 30-70 years) with diabetic foot ulcers who attended the Outpatient Clinic of Diabetes in Alexandria Main University Hospital. Full diabetic foot examination was done to all study subjects. DFUs were assessed using University of Texas Diabetic Wound Classification System. HbA1c, LDL-C, serum creatinine, and urinary albumin creatinine ratio (ACR) were measured for all study subjects. Estimated glomerular filtration rate (eGFR) was calculated using CKD-EPI equation. Fundus examination was done for all study subjects. RESULTS: The prevalence of diabetic kidney disease (DKD) and diabetic retinopathy (DR) was 86.1% and 90% respectively among the study group. 86.7% of patients had neuropathic DFUs, 11.1% of them had ischemic DFUs and 2.2% had neuro-ischemic DFUs. Regarding diabetic peripheral neuropathy (DPN) and peripheral arterial disease (PAD) as risk factors for developing DFU, the prevalence of both of them respectively was 82% and 20% among the study group. There was statistically significant association between both DKD, DR and peripheral neuropathy. There was also statistically significant association between both DKD, DR and peripheral arterial disease (PAD). CONCLUSION: Chronic vascular diabetic complications are common among type 2 diabetic patients with diabetic foot ulcers. There is statistically significant association between these complications and diabetic peripheral neuropathy (DPN) and peripheral arterial disease (PAD).

Assessment of comorbid mild cognitive impairment and depression in patients with type 2 diabetes mellitus.

CONTEXT: Depression, mild cognitive impairment, and dementia are highly prevalent chronic conditions associated with social, medical, and economic burdens. Although there are several epidemiological studies that have reported the prevalence of mild cognitive impairment or depressive syndrome in elderly diabetic population little is known about the comorbidity of these conditions. We aimed to study the prevalence of comorbid mild cognitive impairment (MCI) and depression in patients with Type 2 diabetes mellitus and its relation to glycemic control. MATERIALS AND METHODS: the present work was carried on 400 patients with T2DM. History taking, physical examination, laboratory investigations (with special emphasis on glycemic profile and lipid profile parameters) were done for every patient. Assessment of anxiety and depression using the HADS score and assessment of mild cognitive impairment using MoCA score were done. RESULTS: 76% of studied patients had depression of varying degrees while 56.8% of studied patients had MCI. Decreased level of HDL-cholesterol and increased HADS anxiety score were significant predictors of depression. On the other hand, increased level of total cholesterol, decreased level of HDL-cholesterol, increased HADS depression score and decreased MoCA score were significant predictors of anxiety. HDL-cholesterol HADS anxiety score, FBG, and duration of DM were the significant predictors of MCI. CONCLUSION: Increased level of total cholesterol, decreased level of HDL-cholesterol, increased HADS depression score and decreased MoCA score were significant predictors of anxiety. HDL-cholesterol, HADS anxiety score, FBG, and duration of DM were the significant predictors of MCI.

Mechanical Thrombectomy for Acute Ischemic Stroke in Pregnancy.

Intravenous recombinant tissue-plasminogen-activator (rtPA) and mechanical-thrombectomy (MT) are currently the only approved treatments for acute ischemic stroke. Their effectiveness was demonstrated in several clinical trials, and is therefore standard of care. Pregnant women were not included in these studies and consequently the effectiveness and safety in this group are unclear. We present a rare case of a patient in the third-trimester of pregnancy that underwent MT. A 29-year-old woman of 39 weeks' gestation presented with left facial-paresis, hemiparesis, and neglect. Her CT-Angiogram showed a large occlusive thrombus within the right M1-M2 segments. During pregnancy she had developed thrombocytopenia. There was initial treatment decision dilemma. In view of her history of thrombocytopenia, there was concern about administering rtPA due to the risk of bleeding. As the thrombus was large, rtPA may also be ineffective. MT was proposed by the Stroke Physician as the preferred treatment option. A concern from the Interventional-Radiologist was the risk of exposure to radiation and contrast agents. As the patient had a disabling stroke at a young age, decision was made to proceed with MT which started 141 minutes after symptom onset. The clot was aspirated without complications. Final check angiogram showed complete resolution of flow within the right middle cerebral artery territory. The patient underwent elective uncomplicated Caesarean-section 5 days later delivering a healthy new born. Severe stroke in pregnancy is rare, but has grave consequences for both mother and infant. Timely decision-making is crucial. Our case demonstrates that MT can be provided safely and effectively in the third trimester of pregnancy.

Shooting three inflammatory targets with a single bullet: Novel multi-targeting anti-inflammatory glitazones.

In search for effective multi-targeting drug ligands (MTDLs) to address low-grade inflammatory changes of metabolic disorders, we rationally designed some novel glitazones-like compounds. This was achieved by incorporating prominent pharmacophoric motifs from previously reported COX-2, 15-LOX and PPARγ ligands. Challenging our design with pre-synthetic docking experiments on PPARγ showed encouraging results. In vitro tests have identified 4 compounds as simultaneous partial PPARγ agonist, potent COX-2 antagonist (nanomolar IC50 values) and moderate 15-LOX inhibitor (micromolar IC50 values). We envisioned such outcome as a prototypical balanced modulation of the 3 inflammatory targets. In vitro glucose uptake assay defined six compounds as insulin-sensitive and the other two as insulin-independent glucose uptake enhancers. Also, they were able to induce PPARγ nuclear translocation in immunohistochemical analysis. Their anti-inflammatory potential has been translated to effective inhibition of monocyte to macrophage differentiation, suppression of LPS-induced inflammatory cytokine production in macrophages, as well as significant in vivo anti-inflammatory activity. Ligand co-crystallized PPARγ X-ray of one of MTDLs has identified new clues that could serve as structural basis for its partial agonism. Docking of the most active compounds into COX-2 and 15-LOX active sites, pinpointed favorable binding patterns, similar to those of the co-crystallized ligands. Finally, in silico assessment of pharmacokinetics, physicochemical properties, drug-likeness and ligand efficiency indices was performed. Hence, we anticipate that the prominent biological profile of such series will rationalize relevant anti-inflammatory drug development endeavors.

Click chemistry inspired copper sulphide nanoparticle-based fluorescence assay of kanamycin using DNA aptamer.

A highly selective and sensitive fluorescence assay for kanamycin has been developed that depends on complementation of two splits of DNA aptamer. One DNA split was labeled with CuS nanoparticle and the other was decorated with biotin, which enabled coupling with streptavidin magnesphere paramagnetic particles (PMPs). Complementation of the two-aptamer splits happened only in the presence of kanamycin and the subsequent sandwich was separated via a magnet. The released Cu(II) was reduced to Cu(I) by sodium ascorbate and finally catalyzed the click reaction between fluorogenic 3-azido-7-hydroxycoumarin and propargyl alcohol to afford the corresponding fluorescent 1,4-disubstituted-1,2,3-triazole. The fluorescence signal produced (λex. = 365 nm, λem. = 470 nm) was dependent on kanamycin concentration. Fluorescence signal amplification was found to be in good linear relationship with the logarithm of kanamycin concentration in the range of 0.04-20 nM. Furthermore, the proposed assay showed a good reproducibility, high selectivity and low detection limits for kanamycin determination. In addition, the capability of the proposed method to detect kanamycin in biological samples with satisfactory results was demonstrated.

Novel click modifiable thioquinazolinones as anti-inflammatory agents: Design, synthesis, biological evaluation and docking study.

Click chemistry was used to synthesize a new series of thioquinazolinone molecules equipped with propargyl moiety,1,2,3-triazolyl and isoxazolyl rings. Our design was based on merging pharmacophores previously reported to exhibit COX-2 inhibitory activities to a thioquinazolinone-privileged scaffold. The synthesized compounds were subjected to in vitro cyclooxygenase COX-1/COX-2 and 15-LOX inhibition assays. Compounds 2c, 3b, 3h, 3j, and 3k showed COX-2 inhibition with IC50 (µM) 0.18, 0.19, 0.11, 0.16 and 0.17 respectively. These values were compared to celecoxib (IC50 0.05 µM), diclofenac (IC50 0.8 µM) and indomethacin (IC50 0.49 µM) reference drugs. They also showed 15-LOX inhibition with IC50 (µM) 6.21, 4.33, 7.62, 5.21 and 3.98 respectively. These values were compared with Zileuton (IC50 2.41 µM) and Meclofenamate sodium (IC50 5.64 µM) as positive controls. These compounds were further challenged by PMA-induced THP-1 differentiation assay where compounds 2c and 3j inhibited monocyte to macrophage differentiation efficiently with IC50 values of 4.78 µM and 5.63 µM, respectively, compared to that of diclofenac sodium (4.86 µM). On the other hand, 3h demonstrated a significantly increased potency compared to diclofenac in this assay (IC50 = 0.13 µM). The same compounds exhibited significant in vivo anti-inflammatory effect as indicated by the formalin-induced rat-paw edema test. Docking experiments of compounds 2c, 3b, 3h, 3j, and 3k into COX-2 binding pocket have been conducted, where strong binding interactions have been identified and effective overall docking scores have been recorded. Their drug-likeness has been assessed using Molinspiration, Molsoft and Pre-ADMET software products.

Depletion of recombination-specific cofactors by the C-terminal mutant of the activation-induced cytidine deaminase causes the dominant negative effect on class switch recombination.

Activation-induced cytidine deaminase (AID) is essential for class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes. Studies on in vitro mutagenized AID as well as its mutations in human patients with hyper-IgM (HIGM)-syndrome type II revealed that C-terminal AID mutations were defective in CSR whereas their DNA cleavage and SHM activities remained intact. The C-terminal mutants of AID were speculated to exert the dominant negative effect on wild-type (WT) AID whereas its mechanism remains unknown. We generated the JP41 (R190X) mutation in one allele and a null mutation on the other allele in a mouse B cell line (CH12F3-2A) using CRISPR/Cas9 genome-editing tools and studied the effect of JP41 expression on the function of exogenously introduced WT AID fused with estrogen receptor (AIDER) in AIDJP41/∆/AIDER CH12F3-2A cells. We found that JP41 expression strongly suppressed not only CSR but also Igh/c-Myc chromosomal translocations by AIDER. We showed that the dominant negative effect is not evident at the DNA cleavage step but obvious at both deletional and inversional recombination steps. We also confirmed the dominant negative effect of other C-terminal mutants, JP8Bdel (R183X) and P20 (34-aa insertion at residue 182) in AID-deficient spleen B cells. Finally, we showed that the expression of JP41 reduced the binding of AIDER with its cofactors (hnRNP L, SERBP1 and hnRNP U). Together, these data indicate that dominant negative effect of JP41 on CSR is likely due to the depletion of the CSR-specific RNA-binding proteins from WT AID.

Synthesis of Some 1,4,6-Trisubstituted-2-oxo-1,2-dihydropyridine-3-carbonitriles and Their Biological Evaluation as Cytotoxic and Antimicrobial Agents.

A series of novel 1,4,6-trisubstituted-2-oxo-1,2-dihydropyridine-3-carbonitriles supported with some functionalities reported to contribute to significant chemotherapeutic potential were synthesized and evaluated for their antimicrobial and/or cytotoxic activities. Thirteen compounds exhibited cytotoxic potential against a panel of three human tumor cell lines. Compounds 15, 23, and 24 proved to be the most active agents with a broad spectrum of cytotoxic activity. Analog 24 was considered as the most active cytotoxic agent, being 2.5 times more active than doxorubicin against the colon HT29 carcinoma cell line. Seventeen compounds were able to exert a variable antimicrobial profile, among which analogs 15, 20, 21, 23, and 24 were prominently active. The highest antimicrobial potential was displayed by analog 24, being equipotent to ampicillin against Staphylococcus aureus and Escherichia coli, together with a considerable antifungal activity comparable with clotrimazole. Collectively, compounds 15, 23, and 24 could be considered as possible dual antimicrobial-anticancer candidates.

Isolation, purification and characterisation of transglutaminase from rosemary (Rosmarinus officinalis L.) leaves.

BACKGROUND: Rosemary (Rosmarinus officinalis L.) is a spice and medicinal herb widely used around the world of the natural antioxidants, and it has been widely accepted as one of the spices with the highest antioxidant activity. Transglutaminase (EC 2.3.2.13: TGase) is an enzyme capable of catalysing acyl transfer reactions by introducing covalent cross-links between proteins, as well as peptides and various primary amines. TGase activity in plants was first observed in pea seedlings, and subsequently found in organs of both lower and higher plants. Recently. TGase has captured researchers' interest due to its attractive potential application in food industries. Therefore, the objectives of this study are isolation and purification of TGase from new plant source rosemary (Rosmarinus officinalis L.) leaves at the laboratory scale. Moreover, investigation of the biochemical properties of the purified TGase to provide a suitable TGase enzyme for food industry applications are in focus. MATERIAL AND METHODS: Rosemary (Rosmarinus officinalis L.) leaves was used as a new plant source to TGase. The biochemical characteristics of the crude and purified enzyme were determined. RESULTS: Rosemary (Rosmarinus officinalis L.) TGase was purified to homogeneity by successive three purification steps including ammonium sulfate precipitatation, ion exchange chromatography on DEAE-Sephadex A-50 column and Size exclusion column chromatography on Sephadex G-100 column. Under experimental conditions. 20-30% of ammonium sulfate saturation in the enzyme solution had a high yield of enzyme activity could be obtained. The purified enzyme from the Sephadex G-100 column had 21.35% yield with increased about 7.31 in purification fold. Rosemary TGase exhibited optimum activity at pH 7.0 and 55°C for the catalytic reaction of hydroxylarnine and Z-Gln-Gly. The purified TGase almost maintained full activity after incubation for 15 ruin up to 60°C and it was completely inactivated at 85°C. The rosemary TGase was stimulated at 2-6 rnM CaCl2 concentrations while it lost about 5-20% from its activity by increasing CaCl2 concentration. Sodium chloride (2-14%) shows no noticeable inhibition of the purified TGase activity. Mg+2, Ba+2 were acivited by the purified TGase while it was str ongly inhibited by Fe+2, moderately by Cir2 and Mn+2. CONCLUSION: This paper reports on the purification and characterisation of TGase from newly isolated plant, rosemary (Rosmarinus officinalis L.) leaves. Finding results of the TGase properties make this enzyme a good candidate for application in the food industry. However, additional work is required to increase activity yield during extraction and purification for commercial scale of TGase from this plant.

Increased levels of interleukins 2 and 17 in the cerebrospinal fluid of patients with idiopathic intracranial hypertension.

IIH is a condition of raised intracranial pressure of unknown pathogenesis, which is most commonly seen in young overweight women. This study was designed to confirm and extend previous reports by our and other groups showing increased inflammatory cytokine expression in patients with IIH. We analyzed the concentrations of 14 cytokines (IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17, IL-22, IL-23, IFNγ, TNFα, TGFß, and osteopontin) in the serum and cerebrospinal fluid (CSF) of 17 patients with IIH and 53 patients with other neurological conditions. Patients with IIH had highly elevated IL-2, IL-4, IL-10, IL-17 and IFNγ in the CSF compared to patients with multiple sclerosis or non-organic/non-inflammatory neurological conditions. No significant differences were seen between serum cytokine levels in four patient groups (IIH - multiple sclerosis - inflammatory neurological conditions - non-organic/non-inflammatory neurological conditions) and there were no correlations between serum and CSF cytokine levels. In IIH, levels of IL-2, IL-8 and IL-17 were significantly higher in CSF than serum; levels of IL-1ß, IL-4, IL-22, IFNγ and TNFα were significantly higher in serum than CSF. For most cytokines, the CSF/serum cytokine ratio was significantly higher than the CSF/serum albumin ratio, indicating intrathecal synthesis of these cytokines in IIH. We conclude that IIH is associated with elevated levels of IL-17 and IL-2 in the CSF, suggesting the involvement of these cytokines in disease pathogenesis.

Concurrence of multiple sclerosis and amyotrophic lateral sclerosis in patients with hexanucleotide repeat expansions of C9ORF72.

BACKGROUND: Crossover in the pathogenic mechanisms of amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) has been described but is poorly understood. A GGGGCC hexanucleotide repeat expansion of C9ORF72 has recently been identified in a significant proportion of patients with ALS. METHODS: In approximately 650 patients diagnosed with ALS from the North of England we identified seven patients who initially presented with MS. DNA obtained from five patients with MS-ALS and 215 patients with MS alone was screened for the C9ORF72 expansion. Post-mortem material was examined from two patients with MS-ALS. Gene expression profiling was performed on lymphoblastoid cells and levels of CXCL10 were measured in cerebrospinal fluid (CSF) from patients with ALS with and without the C9ORF72 expansion and controls. RESULTS: Concurrence of MS and ALS is higher than expected in our population. The C9ORF72 expansion was identified in 80% of patients with MS-ALS but not in those with MS alone. In the presence of preceding MS, C9ORF72-ALS was more rapidly progressive. MetaCore analysis identified alteration of the NF-кB pathway in C9ORF72-ALS and non-C9ORF72-ALS. NF-кB activation is associated with increased expression of the neuroprotective cytokine CXCL10 but, in C9ORF72-ALS, CXCL10 is downregulated and CSF levels are reduced. CONCLUSIONS: We propose that MS-associated neuroinflammation may affect penetrance and progression of the C9ORF72 expansion. In particular, the NF-кB pathway is activated in MS and appears to be dysfunctional in C9ORF72-ALS. Aberrant downregulation of CXCL10 may explain the predisposition of C9ORF72 expansion carriers to develop ALS in the context of MS and NF-кB activation, and offers a potential therapeutic target.

Utilization of salt whey from Egyptian Ras (cephalotyre) cheese in microbial milk clotting enzymes production.

BACKGROUND: Microbial milk-clotting enzymes are valued as calf rennet substitutes in the cheese industry. The worldwide increase of cheese production coupled with a reduced supply of calf rennet has prompted a search for calf rennet substitutes, including microbial and plant rennets. However, most plant rennets have proved unsuitable because they impart a bitter taste to the cheese. Microbial rennet appears to be more promising because its production is cheaper, biochemical diversity is greater, and genetic modification is easier. Most cheese manufacturing facilities in Egypt perform land spreading of salt whey. However, this practice increases the chloride levels of soil, and elevates the risk of crop damage. One possible application for salt whey is to use it as a whole medium for growth and production of milk clotting enzyme from fungi. MATERIAL AND METHODS: Mucor pusillus QM 436 was identified to produce the highest milk-clotting activity during screening of 19 fungal strains. Salted whey results from Ras (Cephalotyre) cheese manufacture as a whole medium for growth of Mucor pusillus QM 436 and production of the enzyme. RESULTS: The milk-clotting enzyme from Mucor pusillus QM 436 was purified to 7.14-fold with 54.4% recovery by precipitation in ammonium sulfate, ethanol and fractionated by gel filtration on Sephadex G-100. The enzyme was active in the pH range 5.5-7.5 and was inactivated completely by heating 5 min at 70°C and 30 min at 65°C. The highest level of enzyme activity was obtained at 60°C, pH 5.5. A positive and proportional relationship occurred in the presence of CaCl2 in milk, with inhibition which occurred in the presence of NaCl. CONCLUSIONS: The high level of milk-clotting activity coupled with a low level of thermal stability suggested that the milk-clotting enzyme from Mucor pusillus QM 436 should be considered as a potential substitute for calf rennet.

Phosphatase and tensin homologue/protein kinase B pathway linked to motor neuron survival in human superoxide dismutase 1-related amyotrophic lateral sclerosis.

Gene expression profiling has been used previously with spinal cord homogenates and laser capture microdissected motor neurons to determine the mechanisms involved in neurodegeneration in amyotrophic lateral sclerosis. However, while cellular and animal model work has focused on superoxide dismutase 1-related amyotrophic lateral sclerosis, the transcriptional profile of human mutant superoxide dismutase 1 motor neurons has remained undiscovered. The aim of this study was to apply gene expression profiling to laser captured motor neurons from human superoxide dismutase 1-related amyotrophic lateral sclerosis and neurologically normal control cases, in order to determine those pathways dysregulated in human superoxide dismutase 1-related neurodegeneration and to establish potential pathways suitable for therapeutic intervention. Identified targets were then validated in cultured cell models using lentiviral vectors to manipulate the expression of key genes. Microarray analysis identified 1170 differentially expressed genes in spinal cord motor neurons from superoxide dismutase 1-related amyotrophic lateral sclerosis, compared with controls. These genes encoded for proteins in multiple functional categories, including those involved in cell survival and cell death. Further analysis determined that multiple genes involved in the phosphatidylinositol-3 kinase signalling cascade were differentially expressed in motor neurons that survived the disease process. Functional experiments in cultured cells and primary motor neurons demonstrate that manipulating this pathway by reducing the expression of a single upstream target, the negative phosphatidylinositol-3 kinase regulator phosphatase and tensin homology, promotes a marked pro-survival effect. Therefore, these data indicate that proteins in the phosphatidylinositol-3 kinase pathway could represent a target for therapeutic manipulation in motor neuron degeneration.