Phase II study of ketogenic diets in relapsing multiple sclerosis: safety, tolerability and potential clinical benefits.

BACKGROUND: Dietary changes impact human physiology and immune function and have potential as therapeutic strategies. OBJECTIVE: Assess the tolerability of a ketogenic diet (KD) in patients with relapsing multiple sclerosis (MS) and define the impact on laboratory and clinical outcome metrics. METHODS: Sixty-five subjects with relapsing MS enrolled into a 6-month prospective, intention-to-treat KD intervention. Adherence was monitored with daily urine ketone testing. At baseline, fatigue, depression and quality of life (QoL) scores were obtained in addition to fasting adipokines and MS-related clinical outcome metrics. Baseline metrics were repeated at 3 and/or 6 months on-diet. RESULTS: Eighty-three percent of participants adhered to the KD for the study duration. Subjects exhibited significant reductions in fat mass and showed a nearly 50% decline in self-reported fatigue and depression scores. MS QoL physical health (67±16 vs 79±12, p<0.001) and mental health (71±17 vs 82±11, p<0.001) composite scores increased on-diet. Significant improvements were noted in Expanded Disability Status Scale scores (2.3±0.9 vs 1.9±1.1, p<0.001), 6-minute walk (1631±302 vs 1733±330 ft, p<0.001) and Nine-Hole Peg Test (21.5±3.6 vs 20.3±3.7 s, p<0.001). Serum leptin was lower (25.5±15.7 vs 14.0±11.7 ng/mL, p<0.001) and adiponectin was higher (11.4±7.8 vs 13.5±8.4 µg/mL, p=0.002) on the KD. CONCLUSION: KDs are safe and tolerable over a 6-month study period and yield improvements in body composition, fatigue, depression, QoL, neurological disability and adipose-related inflammation in persons living with relapsing MS. TRIAL REGISTRATION INFORMATION: Registered on ClinicalTrials.gov under registration number NCT03718247, posted on 24 October 2018. First patient enrolment date: 1 November 2018. Link: https://clinicaltrials.gov/ct2/show/NCT03718247?term=NCT03718247&draw=2&rank=1.

Mutations in SCN3A cause early infantile epileptic encephalopathy.

OBJECTIVE: Voltage-gated sodium (Na+ ) channels underlie action potential generation and propagation and hence are central to the regulation of excitability in the nervous system. Mutations in the genes SCN1A, SCN2A, and SCN8A, encoding the Na+ channel pore-forming (α) subunits Nav1.1, 1.2, and 1.6, respectively, and SCN1B, encoding the accessory subunit ß1 , are established causes of genetic epilepsies. SCN3A, encoding Nav1.3, is known to be highly expressed in brain, but has not previously been linked to early infantile epileptic encephalopathy. Here, we describe a cohort of 4 patients with epileptic encephalopathy and heterozygous de novo missense variants in SCN3A (p.Ile875Thr in 2 cases, p.Pro1333Leu, and p.Val1769Ala). METHODS: All patients presented with treatment-resistant epilepsy in the first year of life, severe to profound intellectual disability, and in 2 cases (both with the variant p.Ile875Thr), diffuse polymicrogyria. RESULTS: Electrophysiological recordings of mutant channels revealed prominent gain of channel function, with a markedly increased amplitude of the slowly inactivating current component, and for 2 of 3 mutants (p.Ile875Thr and p.Pro1333Leu), a leftward shift in the voltage dependence of activation to more hyperpolarized potentials. Gain of function was not observed for Nav1.3 variants known or presumed to be inherited (p.Arg1642Cys and p.Lys1799Gln). The antiseizure medications phenytoin and lacosamide selectively blocked slowly inactivating over transient current in wild-type and mutant Nav1.3 channels. INTERPRETATION: These findings establish SCN3A as a new gene for infantile epileptic encephalopathy and suggest a potential pharmacologic intervention. These findings also reinforce the role of Nav1.3 as an important regulator of neuronal excitability in the developing brain, while providing additional insight into mechanisms of slow inactivation of Nav1.3. Ann Neurol 2018;83:703-717.

Sperm function during incubation with oestrus oviductal fluid differs in bulls with different fertility.

Spermatozoa undergo several modifications in the oviduct before acquiring fertilising capacity. Although spermatozoa are exposed to similar conditions in the oviduct, the speed of the response varies with the male and the state of the spermatozoa. We hypothesised that spermatozoa from bulls with different fertility may differ in their ability to respond to oviductal fluid (ODF). Frozen-thawed spermatozoa from four bulls were incubated with oestrus oviductal fluid (OODF) for 6h. Sperm kinematics, tyrosine phosphorylation, phosphorylation patterns, capacitation and acrosome reaction were analysed at hourly intervals. The amplitude of lateral head displacement (ALH) and straightness coefficient (STR) were higher (P<0.05) in bulls with higher fertility compared with those with lower fertility, at 1-4h of incubation. At 4h of incubation and onwards, spermatozoa from bulls with higher fertility showed a lower degree (P<0.05) of tyrosine phosphorylation and higher degree of capacitation and acrosome reaction. At least five tyrosine-phosphorylated sperm proteins were detected in all bulls. However, the expression of two phosphorylated sperm proteins (183 and 109 kDa) was upregulated in bulls with lower fertility. It may be concluded that cryopreserved spermatozoa from high- and low- fertile bulls differ in their ability to respond to OODF. This may help in developing tools for assessing fertility of bulls, once validated in more animals.

IL-33 is related to innate immune activation and sensitization to HDM in mild steroid-free asthma.

BACKGROUND: IL-33 represents a potential link between the airway epithelium and induction of a Th2-type inflammatory response in asthma. However, the association with markers of eosinophilic airway inflammation has not previously been reported in patients with steroid-free asthma. AIM: To describe the relationship between airway IL-33 and markers of eosinophilic airway inflammation, as well potential triggers of IL-33, in mild, steroid-free asthma. METHODS: IL-33 mRNA expression and IL-33 immunoreactivity were measured in bronchial biopsies from patients with asthma untreated with inhaled steroids and healthy individuals. Furthermore, fractional exhaled nitric oxide (FeNO) and eosinophils in sputum and BAL were measured, as well as airway hyperresponsiveness to mannitol and methacholine. Epithelial integrity was assessed by computerized image analysis on haematoxylin-stained sections, and TLR mRNA expression by PCR. RESULTS: A total of 23 patients with asthma and 10 healthy individuals were examined (age: 24 years (20-40); females: 53%). The level of IL-33 mRNA expression was significantly higher in patients with asthma compared to healthy individuals (Median (IQR) 1.12 (0.78) vs. 0.86, P = 0.04). There was a positive correlation between IL-33 mRNA expression and the level of FeNO (r = 0.56, P = 0.01), whereas there was no association with airway or blood eosinophils. IL-33 expression was unrelated to loss of epithelial integrity, but correlated with an increased expression of TLR2 and TLR4 (TLR2: r = 0.47, P = 0.04; TLR4: 0.68, P < 0.001), as well allergy to house dust mites (HDMs). CONCLUSION: In mild untreated asthma, the expression of IL-33 mRNA in bronchial mucosa is related to innate immune activation and allergic sensitization to HDM, rather than epithelial damage, and correlates with FeNO. These findings suggest that in mild allergic asthma, IL-33 may represent a link between innate immune activation and FeNO production.

Airway responsiveness to mannitol in asthma is associated with chymase-positive mast cells and eosinophilic airway inflammation.

BACKGROUND: Airway hyperresponsiveness (AHR) to inhaled mannitol is associated with indirect markers of mast cell activation and eosinophilic airway inflammation. It is unknown how AHR to mannitol relates to mast cell phenotype, mast cell function and measures of eosinophilic inflammation in airway tissue. We compared the number and phenotype of mast cells, mRNA expression of mast cell-associated genes and number of eosinophils in airway tissue of subjects with asthma and healthy controls in relation to AHR to mannitol. METHODS: Airway hyperresponsiveness to inhaled mannitol was measured in 23 non-smoking, corticosteroid-free asthmatic individuals and 10 healthy controls. Mast cells and eosinophils were identified in mucosal biopsies from all participants. Mast cells were divided into phenotypes based on the presence of chymase. mRNA expression of mast cell-associated genes was measured by real-time PCR. RESULTS: The proportion of submucosal MCTC was higher in asthmatic individuals with AHR to mannitol compared with asthmatic individuals without AHR (median: 40.3% vs. 18.7%, P = 0.03). Increased submucosal MCTC numbers were associated with increased levels of mRNA for thymic stromal lymphopoietin (TSLP) and CPA3 in asthmatics. Reactivity to mannitol correlated significantly with eosinophils in submucosa (r(s): 0.56, P = 0.01). CONCLUSION: Airway hyperresponsiveness to inhaled mannitol is associated with an altered submucosal mast cell profile in asthmatic individuals. This mast cell profile is associated with increased levels of TSLP and CPA3. The degree of AHR to mannitol is correlated with the degree of eosinophilic inflammation in the airway submucosa.

Osteopontin binds and modulates functions of eosinophil-recruiting chemokines.

BACKGROUND: Allergic asthma is characterized by eosinophilic inflammation and airway obstruction. There is also an increased risk of pulmonary infection caused by Streptococcus pneumoniae, in particular during severe asthma where high levels of the glycoprotein, osteopontin (OPN), are present in the airways. Eosinophils can be recruited by chemokines activating the receptor CCR3 including eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26, RANTES/CCL5, and MEC/CCL28. In addition to inducing chemotaxis, several of these molecules have defensin-like antibacterial properties. This study set out to elucidate the functional consequences of OPN binding to eosinophil-recruiting chemokines. METHODS: Antibacterial activities of the chemokines were investigated using viable count assays and electron microscopy. Binding studies were performed by means of surface plasmon resonance. The potential interference of OPN with antibacterial, receptor-activating, and lipopolysaccharide-neutralizing abilities of these chemokines was investigated. RESULTS: We found that OPN bound all eosinophil-recruiting chemokines with high affinity except for CCL5. The eosinophil-recruiting chemokines all displayed bactericidal activity against S. pneumoniae, but only CCL26 and CCL28 retained high antibacterial activity in the presence of sodium chloride at physiologic concentrations. Preincubation of the chemokines with OPN strongly inhibited their antibacterial activity against S. pneumoniae but did not affect their ability to activate CCR3. All chemokines investigated showed LPS-neutralizing activity that was impaired by OPN only in the case of CCL24. CONCLUSIONS: The data suggest that OPN may impair host defense activities of the chemokines without affecting their eosinophil-recruiting properties. This could be one mechanism explaining the increased vulnerability to acquire pneumococcal infection in parallel with sustained allergic inflammation in asthma.

Comparison of DNA Fragmentation Assay in Frozen-Thawed Cat Epididymal Sperm.

DNA fragmentation of frozen-thawed feline epididymal sperm from corpus and cauda regions was evaluated by three different techniques. The DNA fragmentation index (DFI) was compared between techniques: the sperm chromatin structural assay (SCSA(®) ), acridine orange staining techniques (AOT) and the sperm chromatin dispersion (SCD). There were significant differences in DFI among the techniques (p < 0.05) with no correlations. Only DFI values obtained from SCD revealed a significantly higher DFI in corpus compared with cauda spermatozoa (p < 0.05). The discrepancy between techniques might be due to the sensitivity of each technique, differences in severity of DNA damaged that can be detected. The difference in DFI between epididymal regions from SCD technique might indicate different maturational stages of spermatozoa, with less chromatin condensation of spermatozoa in corpus compared with cauda epididymis.

Mapping of TLR5 and TLR7 in central and distal human airways and identification of reduced TLR expression in severe asthma.

BACKGROUND: The toll-like receptors, TLR5 and TLR7, have recently been proposed in asthma immunopathogenesis. While supporting data come from animal or in vitro studies, little is known about TLR5 and TLR7 expression in human asthmatic airways. METHODS: Advanced immunohistochemical mapping of TLR5 and TLR7 was performed on bronchial and transbronchial biopsies from healthy individuals and patients with moderate and severe asthma. RESULTS: TLR5 was identified in multiple structural cells; bronchial epithelium, alveolar type II pneumocytes, plasma cells, macrophages and neutrophils. Contrary to bronchial TLR5, which had a basolateral expression, alveolar TLR5 had polarized apical localization. Patients with severe asthma had decreased total and epithelial TLR5 expression compared to controls and moderate asthmatics (P < 0.001). TLR7 expression was found in several structural cells and asthma-related immune cells. Whereas TLR7 expression was decreased in severe asthmatics (P < 0.001), nerve-associated TLR7 increased (P = 0.035). Within the asthma groups, both TLR5 and TLR7 expression correlated with multiple lung function parameters. CONCLUSIONS: Our results reveal broad expression patterns of TLR5 and TLR7 in the lung and that the expression is decreased in severe asthma. Hence, severe asthmatics may suffer from insufficient TLR signalling during viral or bacterial infections leading to poor and impaired defence mechanisms.

Risk of seizure recurrence after achieving initial seizure freedom on the ketogenic diet.

OBJECTIVE: Few studies have examined the long-term sustainability of complete seizure freedom on the ketogenic diet (KD). The purpose of this study was to describe the risk of seizure recurrence in children who achieved at least 1 month of seizure freedom on the KD, and to assess clinical features associated with sustained seizure freedom. METHODS: Records of patients initiated on the KD at The Children's Hospital of Philadelphia (CHOP) from 1991 to 2009 were reviewed. Subjects who attained seizure freedom for at least 1 month within 2 years were included in the study. Seizure frequency was recorded based on caregiver-reported seizure diaries as unchanged, improved, or worse compared to baseline. Those patients with seizure freedom ≥1 year were compared to those with seizure freedom <1 year in terms of demographics, age of seizure onset, number of antiepileptic drugs (AEDs) prior to KD, and epilepsy classification. RESULTS: Of 276 patients initiated on the KD, 65 patients (24%) attained seizure freedom for a minimum of 1 month. The majority of these patients had daily seizures. The median time to seizure freedom after KD initiation was 1.5 months. Seizures recurred in 53 patients (82%), with a median time to seizure recurrence of 3 months. However, seizure frequency after initial recurrence remained far less than baseline. No clinical features were identified as risk factors for seizure recurrence. SIGNIFICANCE: Seizure recurrence on the KD after 1 month of seizure freedom most often occurred as occasional breakthrough seizures and not a return to baseline seizure frequency. This study provides evidence to support the continued use of the KD in patients with initial seizure freedom even after breakthrough seizures. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.

Diagnosis and treatment of growth hormone deficiency in children on the ketogenic diet: A case series.

The ketogenic diet (KD) can have a negative impact on the linear growth and body composition of children. The aims of this study were to review two centers' experience with children who developed height deceleration on the KD and determine if the height deceleration was secondary to growth hormone deficiency (GHD), and if growth hormone therapy (GHT) would be effective and safe (not altering ketosis or seizure frequency). Retrospective chart reviews were performed on patients with KD referred to Endocrinology between 2013 and 2018. Seventeen children were identified. Data reviewed included: demographics, growth velocity, KD ratio, protein/calorie intake, lab results, GH dosage, Tanner stage, and seizure frequency, and endocrine recommendations. Descriptive statistics were performed. Of the 17 children referred to the Endocrine Division, seven children were growth hormone deficient and began GHT. Data were provided for six patients (2 males, 4 females; age 2-7 years at the start of KD) on the KD for >6 years and on GHT for >4 years. Growth for all patients stabilized or increased. IGF-1 z-scores normalized. GHT did not affect seizure frequency or ketosis. GHT in those with GHD can be an appropriate option allowing better growth while still maintaining ketogenic therapy and seizure control. PLAIN LANGUAGE SUMMARY: The KD can be an effective treatment for difficult-to-control epilepsy and some disorders of carbohydrate metabolism. The KD can adversely affect the linear growth (height) of children. This case series reviewed six patients who had slow linear growth. It was found that all six children had growth hormone deficiency, grew better with growth hormone treatments, and that their seizures and ketone levels were not affected.

Impact of a ketogenic diet on sleep quality in people with relapsing multiple sclerosis.

BACKGROUND: Sleep disturbance in MS is common and can significantly impair overall quality of life. The ketogenic diet (KD) associates with improved sleep quality in people living with epilepsy and may have similar benefits when used within MS; however, the impact of a KD on sleep in this population remains poorly defined. METHODS: Forty-five patients with relapsing MS enrolled into a 6-month KD intervention trial and completed self-reported assessments of sleep quality and sleep disorder symptoms prior to diet initiation and while on diet, using the Epworth Sleepiness Scale (ESS) and Sleep Disorders Symptom Checklist-25 (SDS). Participants who did not complete sleep assessments at baseline and 6-months were excluded from analysis. In addition to sleep metrics, data collection included anthropometrics and MS-related fatigue scores. RESULTS: Thirty-nine of 45 (87 %) participants completed the required sleep assessments. There was a mean reduction in ESS score of 1.90 (95 % CI [-2.85, -0.94], p < 0.001). Total SDS score decreased at 6-months on KD (-4.4, 95 % CI [-7.1, -1.7], p = 0.002), with improvements noted in insomnia (-1.55, 95 % CI [-2.66, -0.43], p = 0.008), obstructive sleep apnea (-0.91, 95 % CI [-1.57, -0.25], p = 0.008), and restless leg syndrome screening scores (-1.00, 95 % CI [-1.95, -0.051], p = 0.04). Sleep duration was unchanged on KD. CONCLUSION: KD associates with improvements in daytime sleepiness, independent of sleep duration, and common comorbid sleep disorders in people living with relapsing MS. The findings herein support the benefits of KD on sleep quality and highlight the potential role of dietary therapeutics for sleep disorders in neurological disease. TRIAL REGISTRATION INFORMATION: Registered on Clinicaltrials.gov under registration number NCT03718247, posted on Oct 24, 2018. First patient enrollment date: Nov 1, 2018. Link: https://clinicaltrials.gov/ct2/show/NCT03718247?term=NCT03718247&draw=2&rank=1.

Ketogenic diet in relapsing multiple sclerosis: Patient perceptions, post-trial diet adherence & outcomes.

BACKGROUND: Ketogenic diets (KDs) are safe and tolerable in people with multiple sclerosis (MS). While many patient-reported and clinical benefits are noted, the sustainability of these diets outside of a clinical trial is unknown. AIMS: Evaluate patient perceptions of the KD following intervention, determine the degree of adherence to KDs post-trial, and examine what factors increase the likelihood of KD continuation following the structured diet intervention trial. METHODS: Sixty-five subjects with relapsing MS previously enrolled into a 6-month prospective, intention-to-treat KD intervention. Following the 6-month trial, subjects were asked to return for a 3-month post-study follow-up, at which time patient reported outcomes, dietary recall, clinical outcome measures, and laboratory values were repeated. In addition, subjects completed a survey to evaluate sustained and attenuated benefits following completion of the intervention phase of the trial. RESULTS: Fifty-two subjects (81%) returned for the 3-month post-KD intervention visit. Twenty-one percent reported continued adherence to a strict KD and an additional 37% reported adhering to a liberalized, less restrictive form of the KD. Those subjects with greater reductions in body mass index (BMI) and fatigue at 6-months on-diet were more likely to continue on KD following trial completion. Using intention-to-treat analysis, patient-reported and clinical outcomes at 3-months post-trial remained significantly improved from baseline (pre-KD), though the degree of improvement was slightly attenuated relative to outcomes at 6-months on KD. Regardless of diet type following the KD intervention, dietary patterns shifted toward greater protein and polyunsaturated fats and less carbohydrate/added sugar consumption. CONCLUSIONS: Following the 6-month KD intervention study, the majority of subjects elected to continue on KD, though many pursued a more liberal limit for carbohydrate restriction. Those who experienced a greater reduction in BMI or fatigue were more likely to continue with strict KD. The 6-month KD intervention induced persistent changes to dietary habits in the months following study completion. TRIAL REGISTRATION INFORMATION: Registered on Clinicaltrials.gov under registration number NCT03718247, posted on Oct 24, 2018. First patient enrollment date: Nov 1, 2018. Link: https://clinicaltrials.gov/ct2/show/NCT03718247?term=NCT03718247&draw=2&rank=1.

Quantification of kinetic changes in protein tyrosine phosphorylation and cytosolic Ca²âº concentration in boar spermatozoa during cryopreservation.

Protein tyrosine phosphorylation in sperm is associated with capacitation in several mammalian species. Although tyrosine phosphorylated proteins have been demonstrated in cryopreserved sperm, indicating capacitation-like changes during cryopreservation, these changes have not yet been quantified objectively. We monitored tyrosine phosphorylation, intracellular calcium and sperm kinematics throughout the cryopreservation process, and studied the relationships among them in boar spermatozoa. Sperm kinetics changed significantly during cryopreservation: curvilinear velocity, average path velocity and straight line velocity all decreased significantly (P < 0.05). While the percentage of sperm with high intracellular calcium declined (P < 0.05), global phosphorylation increased significantly (P < 0.01). Specifically, cooling to 5 °C induced phosphorylation in the spermatozoa. After cooling, a 32-kDa protein not observed in fresh semen appeared and was consistently present throughout the cryopreservation process. While the level of expression of this phosphoprotein decreased after addition of the second extender, frozen-thawed spermatozoa showed an increased expression. The proportion of sperm cells with phosphorylation in the acrosomal area also increased significantly (P < 0.05) during cryopreservation, indicating that phosphorylation might be associated with capacitation-like changes. These results provide the first quantitative evidence of dynamic changes in the subpopulation of boar spermatozoa undergoing tyrosine phosphorylation during cryopreservation.

Endometriosis as a prognostic factor for cancer survival.

Studies have shown an increased risk of malignancies in women with endometriosis. Little is known about the impact of endometriosis on cancer survival. We investigated whether the survival after a diagnosis of a malignancy differs in women with a previously diagnosed endometriosis compared to other women. Women with a first time diagnosis of a malignancy in 1969-2005, were identified using the National Swedish Cancer Register (NSCR). By use of the National Swedish Patient Register (NSPR) we identified all women with a diagnosis of endometriosis during the same period and linked these patients with the data from the NSCR. The cohort comprised 4,278 women with endometriosis and a malignancy, and 41,831 randomly selected matched women without endometriosis. Cox regression was used for all calculations to obtain crude and adjusted cause specific mortality rates, measured as hazard ratios (HR) with 95% confidence intervals (CI). A total of 46,109 women entered the study. There was a statistically significant better survival for women with endometriosis for all malignancies combined (HR=0.92) and for breast cancer (HR=0.86) and ovarian cancer (HR=0.81) specifically. For breast cancer the survival enhancing effect in women with endometriosis decreased with increasing parity. There was poorer survival in malignant melanoma for women with endometriosis (HR=1.52). The survival in a malignancy is better in women with a previously diagnosed endometriosis compared to women without endometriosis especially for breast and ovarian cancers. The prognosis of malignant melanoma is poorer in women with endometriosis.

Alveolar mast cells shift to an FcεRI-expressing phenotype in mild atopic asthma: a novel feature in allergic asthma pathology.

BACKGROUND: A unique feature of alveolar mast cells is their low high-affinity IgE receptor (FcεRI) expression. Recent discoveries in uncontrolled asthma suggest that the appearance of FcεRI-expressing alveolar mast cells may be a novel disease-specific feature of allergic asthma. This study investigates whether increased FcεRI-expressing alveolar mast cells are present in patients with mild allergic asthma or even in non-asthmatic allergic rhinitis patients (AR) who have developed bronchial hyperactivity (BHR). METHODS: Bronchial and alveolar tissues were obtained from healthy controls, AR patients with or without BHR, and AR patients with concurrent asthma. Samples were processed for immunohistochemical identification of MC(T) and MC(TC) and expression of FcεRI and surface-bound IgE. RESULTS: Bronchial mast cell expression of FcεRI was high in all groups. In contrast, in the alveolar tissue, the expression of FcεRI on mast cells was low in healthy controls and in the AR patient groups, whereas a high expression was present in AR patients with concurrent asthma (P = 0.006 compared to controls). The asthmatics had a 29-fold increase in numbers (P = 0.006) and a 19-fold increase in proportion (P = 0.007) of alveolar mast cells that expressed surface-bound IgE. CONCLUSIONS: The present data show that alveolar mast cells in patients with mild atopic asthma, but not atopic patients with AR, have turned into a highly FcεRI- and IgE-expressing phenotype. These data support the hypothesis that increased FcεRI expression on alveolar mast cells is a novel disease-specific feature of allergic asthma that is important for understanding asthma phenotypes and designing new therapeutic strategies.

Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate.

Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PTP and intracellular calcium ([Ca(2+)]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between P1 and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (P<0.05) in SRF-P1 compared to P1. There were no significant differences in percentages of spermatozoa with high [Ca(2+)]i between P1 and SRF-P1 in fresh as well as in frozen-thawed semen. A higher (P<0.001) proportion of spermatozoa displayed PTP during the course of cryopreservation indicating a definite effect of the cryopreservation process on sperm PTP. The proportion of spermatozoa with PTP did not differ significantly between portions of the boar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF.

Single layer centrifugation of stallion spermatozoa through Androcoll™-E does not adversely affect their capacitation-like status, as measured by CTC staining.

This study was designed to evaluate the effect of single layer centrifugation (SLC) and subsequent cold storage on stallion sperm capacitation-like status and acrosome reaction. Three stallions were included in the study, with three ejaculates per stallion. The samples were examined 4, 24 and 72 h after collection, extension and SLC, with storage at 6°C. Sperm capacitation-like status was investigated using the fluorescent dye chlortetracycline (CTC). There was no difference in capacitation-like status between colloid-selected and non-selected spermatozoa. Sperm motility decreased significantly during cold storage, whereas the proportion of apparently capacitated spermatozoa increased. There was no change in the proportion of acrosome-reacted spermatozoa. In conclusion, SLC through Androcoll™-E does not adversely affect the capacitation-like status of stallion spermatozoa, although it did increase with time during cold storage.

The ketogenic diet in treatment of two adults with prolonged nonconvulsive status epilepticus.

Prolonged status epilepticus (SE) can be refractory to conventional interventions, with high rates of subsequent morbidity and mortality. A high fat, low protein, low carbohydrate ketogenic diet (KD) has been used successfully to treat intractable epilepsy. However, its possible role in prolonged SE has not been well described. We report successful use of the KD in two adult patients with prolonged nonconvulsive SE (NCSE) refractory to multiple other interventions. Our observations suggest induction of ketosis may be a novel strategy to safely and effectively treat status in adults even after weeks to months of refractory seizures. Although there are few data regarding the use of the ketogenic diet in the treatment of adult epilepsy syndromes, it may be an option for the treatment of adults with refractory, prolonged SE.

Ketogenic diet treatment of children in the intensive care unit: Safety, tolerability, and effectiveness.

PURPOSE: The ketogenic diet (KD) is initiated emergently in the intensive care unit (ICU) for patients with super refractory status epilepticus (SRSE) and epileptic encephalopathies (EE). However, few data are available regarding safety, effectiveness, and long-term outcomes. METHODS: We performed a retrospective cohort study of consecutive patients with KD initiated in the ICU from 2010 to 2018 for SRSE and EE. We characterized time to ketosis, adverse effects, and seizure outcomes. Responders were defined as having ≥50 % reduction in seizure frequency compared to prior to KD initiation. RESULTS: We identified 29 patients. KD was initiated for SRSE in 12 patients, EE in 8 patients, and EE with SRSE in 9 patients. KD was initiated after a median of 9 days. Ketosis was achieved 2 days faster in fasted patients (p < 0.0001). All patients had at least 1 KD-related adverse effect, most often hypoglycemia, constipation, or acidosis. There was ≥50 % reduction in seizure frequency compared to prior to KD initiation by 1 week in 17/28 patients, seizure-freedom by 2 weeks in 7/28 patients, and weaned off anesthetics in 11/17 patients. All KD-responders at 1 month had continued response at 6 months. Mortality at 1 year was 24 %. There was no difference in KD response or mortality between KD indication groups. CONCLUSION: Emergent KD initiation in the ICU is feasible, safe, and often effective for SRSE and EE. Expected adverse effects were common but treatable. Morbidity and mortality in this group was high. A ≥ 50 % reduction in seizure is achieved in most responders by 1-2 weeks.

Optimal clinical management of children receiving the ketogenic diet: recommendations of the International Ketogenic Diet Study Group.

The ketogenic diet (KD) is an established, effective nonpharmacologic treatment for intractable childhood epilepsy. The KD is provided differently throughout the world, with occasionally significant variations in its administration. There exists a need for more standardized protocols and management recommendations for clinical and research use. In December 2006, The Charlie Foundation commissioned a panel comprised of 26 pediatric epileptologists and dietitians from nine countries with particular expertise using the KD. This group was created in order to create a consensus statement regarding the clinical management of the KD. Subsequently endorsed by the Practice Committee of the Child Neurology Society, this resultant manuscript addresses issues such as patient selection, pre-KD counseling and evaluation, specific dietary therapy selection, implementation, supplementation, follow-up management, adverse event monitoring, and eventual KD discontinuation. This paper highlights recommendations based on best evidence, including areas of agreement and controversy, unanswered questions, and future research.