Comparative transcriptional analysis identifies genes associated with the attenuation of Theileria parva infected cells after long-term in vitro culture.

Autologous administration of attenuated Theileria parva-infected cells induces immunity to T. parva in cattle. The mechanism of attenuation, however, is largely unknown. Here, we used RNA sequencing of pathogenic and attenuated T. parva-infected T-cells to elucidate the transcriptional changes underpinning attenuation. We observed differential expression of several host genes, including TRAIL, PD-1, TGF-ß and granzymes that are known to regulate inflammation and proliferation of infected cells. Importantly, many genes linked with the attenuation of the related T. annulata-infected cells were not dysregulated in this study. Furthermore, known T. parva antigens were not dysregulated in attenuated relative to pathogenic cells, indicating that attenuation is not due to enhanced immunogenicity. Overall this study suggests that attenuation is driven by a decrease in proliferation and restoration of the inflammatory profile of T. parva-infected cells. Additionally, it provides a foundation for future mechanistic studies of the attenuation phenotype in Theileria-infected cells.

Immune Responses and Pathogenesis following Experimental SARS-CoV-2 Infection in Domestic Cats.

Several reports demonstrated the susceptibility of domestic cats to SARS-CoV-2 infection. Here, we describe a thorough investigation of the immune responses in cats after experimental SARS-CoV-2 inoculation, along with the characterization of infection kinetics and pathological lesions. Specific pathogen-free domestic cats (n = 12) were intranasally inoculated with SARS-CoV-2 and subsequently sacrificed on DPI (days post-inoculation) 2, 4, 7 and 14. None of the infected cats developed clinical signs. Only mild histopathologic lung changes associated with virus antigen expression were observed mainly on DPI 4 and 7. Viral RNA was present until DPI 7, predominantly in nasal and throat swabs. The infectious virus could be isolated from the nose, trachea and lungs until DPI 7. In the swab samples, no biologically relevant SARS-CoV-2 mutations were observed over time. From DPI 7 onwards, all cats developed a humoral immune response. The cellular immune responses were limited to DPI 7. Cats showed an increase in CD8+ cells, and the subsequent RNA sequence analysis of CD4+ and CD8+ subsets revealed a prominent upregulation of antiviral and inflammatory genes on DPI 2. In conclusion, infected domestic cats developed a strong antiviral response and cleared the virus within the first week after infection without overt clinical signs and relevant virus mutations.

Extremely high unbound flucloxacillin concentrations in a patient with acute septic renal failure during continuous venovenous hemofiltration: Therapeutic drug monitoring to optimize treatment.

A 75-year-old female orthopedic patient with spondylodiscitis was admitted to the intensive care unit, where she developed severe acute renal injury (AKI) due to a Staphylococcus aureus bloodstream infection. Continuous venovenous hemofiltration (CVVH) was initiated as renal replacement therapy. According to physician experience and based on (inter)national guidelines and the severity of the infection, treatment with intravenous (IV) flucloxacillin at an initial continuous dose of 9 g/24h was started. The dose was increased to 12 g/24h because endocarditis could not be excluded. Therapeutic drug monitoring (TDM) was used to monitor flucloxacillin levels which are related to antibiotic efficacy and toxicity. Total and unbound flucloxacillin concentrations were measured following 24 hours of continuous infusion at three time points before regional citrate anticoagulation (RCA)-CVVH was initiated, at three time points in plasma, pre-filter, and post-filter, and in ultrafiltrate samples during RCA-CVVH treatment and 1 day following cessation of CVVH treatment. Extremely high total (up to 299.8 mg/L) and unbound (up to 155.1 mg/L) flucloxacillin concentrations were found in the plasma. This led to a dose decrease to 6 g/24h and subsequently to 3 g/24h. Antimicrobial target attainment against S. aureus was achieved by dosing IV flucloxacillin based on TDM. Based on these findings, we conclude that current dosing guidelines for flucloxacillin during renal replacement therapy need revision. We suggest a starting dose of 4 g/24h, which should be adjusted based on the TDM of the unbound flucloxacillin concentration.

Association of Levetiracetam Concentration With Seizure Frequency in Pregnant Women With Epilepsy.

BACKGROUND AND OBJECTIVES: Pharmacologic treatment of epilepsy in pregnant women is balancing between risks for the mother and fetus. Levetiracetam (LEV) is considered to be safe during pregnancy because of its low teratogenic potential and lack of drug-drug interaction with other antiseizure medications (ASMs). Recent studies have shown decline of ASM concentrations during pregnancy because of physiologically based pharmacokinetic changes. In this study, we established this decrease in LEV concentration during pregnancy. In addition, we aimed at investigating the effect of the low LEV levels during pregnancy and developing a target value for the level during pregnancy. METHODS: Pregnant patients using levetiracetam were studied in this retrospective cohort study. Blood samples were monthly collected through venous puncture or the dried blood spot method. ASM serum concentrations were determined at least 6 months before conception and for each month of pregnancy. Seizure frequency and ASM dosages during pregnancy were obtained from patient records. Patients were divided into 2 groups: a seizure-free group and a non-seizure-free group, which contained pregnancies in which the mother had experienced an epileptic seizure more than 12 months and less than 12 months before pregnancy, respectively. RESULTS: We found decreased concentration/dose ratios in 29 pregnancies throughout all months of pregnancy. In the non-seizure-free group, it was found that low LEV concentrations were associated with seizure increase frequency (p = 0.022). For this group, the cutoff value with the highest sum of sensitivity and specificity was 0.466. DISCUSSION: All in all, we recommend therapeutic drug monitoring for all pregnant patients on LEV as the concentrations of LEV significantly decrease throughout most months of pregnancy. However, this decrease in LEV concentration was only significantly correlated with seizure deterioration in patients who had a seizure in the year preceding the pregnancy. Therefore, we suggest more careful monitoring of non-seizure-free patients as they are at higher risk for experiencing an increase of seizure frequency. For this group, we advise physicians to keep LEV concentration above 65% of the preconceptional concentration. For seizure-free patients, we recommend an LEV threshold value of approximately 46% of the preconceptional concentration.

Evaluation of two anti-seizure medication strategies in refractory epilepsy patients from a tertiary center with complementary insights from data visualization.

OBJECTIVE: To evaluate the healthcare resources in a tertiary center related to exclusive use of non-enzyme inducing anti-seizure medications relative to concomitant use of enzyme-inducing anti-seizure medications in patients with refractory epilepsy. METHODS: In this retrospective case-time-control study, we compared the effects of two anti-seizure medication strategies: exclusively non-inducing anti-seizure medications (NIND) or a combination of NIND and inducing anti-seizure medications (IND+). The primary outcome parameter was the number of consultations with relevant healthcare professionals in our tertiary center, assessed with a negative binomial regression model, adjusting for several covariates like blood drug level and time interval (TI). Results from statistical models were visualized to explore the contribution of all covariates on the outcome in the total population and in subgroups. RESULTS: From the 21538 patients with refractory epilepsy referred to our center 1648 patients met the inclusion criteria. The regression model showed that the IND + strategy was significantly associated with fewer consultations compared to the NIND strategy (p < 0.001), reflected in an incidence risk ratio (IRR) of 0.844 (0.799-0.890). Visualization of subgroups, defined by anti-seizure medications strategy, revealed patterns in contribution of blood drug level measurements on the outcome. Although sex was not included as a covariate in the regression model, as it was eliminated by the backward-elimination approach, visualization of this subgroup showed differences in effects of blood drug level and TI. CONCLUSION: For patients with refractory epilepsy in our tertiary center, treatment following the IND + strategy is associated with fewer consultations with healthcare professionals compared to the NIND strategy. Comprehensive visualization of the results facilitated the exploration of effects of covariates across subgroups.

Patient-reported outcomes and function after reinsertion of the triangular fibrocartilage complex by open surgery.

AIMS: Patients with a triangular fibrocartilage complex (TFCC) injury report ulnar-sided wrist pain and impaired function. The surgical procedure of TFCC reinsertion aims to improve function in patients with this injury in whom conservative treatment has failed. The purpose of this study was to investigate the outcomes of open TFCC reinsertion. METHODS: The study involved 274 patients who underwent open repair of the TFCC between December 2013 and December 2018. The patients completed the Patient-Rated Wrist Evaluation (PRWE) questionnaire, and scored pain and function using a visual analogue scale (VAS). Range of motion (ROM) was assessed by experienced hand therapists. RESULTS: Clinically significant improvements were reported in pain, function, and grip strength in 220 patients (80%) three and 12 months postoperatively. CONCLUSION: These data will help surgeons to make decisions about the outcomes of open repair of the TFCC and to counsel patients appropriately. Level of evidence: III Cite this article: Bone Joint J 2021;103-B(4):711-717.

Efficacy and tolerability of brivaracetam in patients with intellectual disability and epilepsy.

Patients with intellectual disability (ID) are often excluded from clinical trials, and little is known about the best approach to treat their epilepsy. Brivaracetam (BRV) is a new antiepileptic drug (AED) for adjunctive treatment in patients with focal-onset seizures with or without secondary generalization. We analyzed the efficacy and tolerability of BRV in patients with ID and epilepsy who either had or had not previously received treatment with levetiracetam (LEV). Data on efficacy and tolerability were retrospectively collected. After the initial start of BRV in our tertiary epilepsy center, we analyzed medical records at 0, 3, 6 and 12 months of follow-up. 116 patients were included (mean age = 34.9 years, 44% female). All had complete data of 3-month follow-up, 76 of 6-month follow-up, and 39 patients of 1-year follow-up. Median starting dose of BRV was 50.0 mg/day and the mean number of concomitant AEDs was 2.6. Seizure reduction and no side effects were reported in more than half of all patients. The most reported side effects were somnolence, dizziness and aggression. Retention rates for BRV were 84.4%, 75.5% and 58.1% after 3, 6 and 12 months, respectively. Seizure reduction and side effects did not differ significantly between the groups with or without previous LEV treatment. We demonstrate that BRV is effective and well tolerated in patients with epilepsy and ID, even in those where previous LEV treatment failed.

Therapeutic Drug Monitoring of Antiepileptic Drugs in Women with Epilepsy Before, During, and After Pregnancy.

During pregnancy, the pharmacokinetics of an antiepileptic drug is altered because of changes in the clearance capacity and volume of distribution. These changes may have consequences for the frequency of seizures during pregnancy and fetal exposure to antiepileptic drugs. In 2009, a review was published providing guidance for the dosing and therapeutic drug monitoring of antiepileptic drugs during pregnancy. Since that review, new drugs have been licensed and new information about existing drugs has been published. With this review, we aim to provide an updated narrative overview of changes in the pharmacokinetics of antiepileptic drugs in women during pregnancy. In addition, we aim to formulate advice for dose modification and therapeutic drug monitoring of antiepileptic drugs. We searched PubMed and the available literature on the pharmacokinetic changes of antiepileptic drugs and seizure frequency during pregnancy published between January 2007 and September 2018. During pregnancy, an increase in clearance and a decrease in the concentrations of lamotrigine, levetiracetam, oxcarbazepine's active metabolite licarbazepine, topiramate, and zonisamide were observed. Carbamazepine clearance remains unchanged during pregnancy. There is inadequate or no evidence for changes in the clearance or concentrations of clobazam and its active metabolite N-desmethylclobazam, gabapentin, lacosamide, perampanel, and valproate. Postpartum elimination rates of lamotrigine, levetiracetam, and licarbazepine resumed to pre-pregnancy values within the first few weeks after pregnancy. We advise monitoring of antiepileptic drug trough concentrations twice before pregnancy. This is the reference concentration. We also advise to consider dose adjustments guided by therapeutic drug monitoring during pregnancy if the antiepileptic drug concentration decreases 15-25% from the pre-pregnancy reference concentration, in the presence of risk factors for convulsions. If the antiepileptic drug concentration changes more than 25% compared with the reference concentration, dose adjustment is advised. Monitoring of levetiracetam, licarbazepine, lamotrigine, and topiramate is recommended during and after pregnancy. Monitoring of clobazam, N-desmethylclobazam, gabapentin, lacosamide, perampanel, and zonisamide during and after pregnancy should be considered. Because of the risk of teratogenic effects, valproate should be avoided during pregnancy. If that is impossible, monitoring of both total and unbound valproate is recommended. More research is needed on the large number of unclear pregnancy-related effects on the pharmacokinetics of antiepileptic drugs.

Mycobacterium bovis prevalence affects the performance of a commercial serological assay for bovine tuberculosis in African buffaloes.

The endemic presence of bovine tuberculosis (BTB) in African buffaloes in South Africa has severe consequences for BTB control in domestic cattle, buffalo ranching and wildlife conservation, and poses a potential risk to public health. This study determined the BTB prevalence in free-ranging buffaloes in two game reserves and assessed the influence of the prevalence of mycobacterial infections on the performance of a commercial cattle-specific serological assay for BTB (TB ELISA). Buffaloes (n = 997) were tested with the tuberculin skin test and TB ELISA; a subset (n = 119) was tested longitudinally. Culture, PCR and sequencing were used to confirm infection with M. bovis and/or non-tuberculous mycobacteria (NTM). Prevalence of BTB, but not NTM, influenced the TB ELISA performance. Multiple testing did not increase test confidence. The findings strongly illustrate the need for development of novel assays that can supplement existing assays for a more comprehensive testing scheme for BTB in African buffaloes.

Natural Exon Skipping Sets the Stage for Exon Skipping as Therapy for Dystrophic Epidermolysis Bullosa.

Dystrophic epidermolysis bullosa (DEB) is a devastating blistering disease affecting skin and mucous membranes. It is caused by pathogenic variants in the COL7A1 gene encoding type VII collagen, and can be inherited dominantly or recessively. Recently, promising proof-of-principle has been shown for antisense oligonucleotide (AON)-mediated exon skipping as a therapeutic approach for DEB. However, the precise phenotypic effect to be anticipated from exon skipping, and which patient groups could benefit, is not yet clear. To answer these questions, we studied new clinical and molecular data on seven patients from the Dutch EB registry and reviewed the literature on COL7A1 exon skipping variants. We found that phenotypes associated with dominant exon skipping cannot be distinguished from phenotypes caused by other dominant DEB variants. Recessive exon skipping phenotypes are generally relatively mild in the spectrum of recessive DEB. Therefore, for dominant DEB, AON-mediated exon skipping is unlikely to ameliorate the phenotype. In contrast, the overall severity of phenotypes associated with recessive natural exon skipping pivots toward the milder end of the spectrum. Consequently, we anticipate AON-mediated exon skipping for recessive DEB caused by bi-allelic null variants should lead to a clinically relevant improvement of this devastating phenotype.

Comparative proteomics identified immune response proteins involved in response to vaccination with heat-inactivated Mycobacterium bovis and mycobacterial challenge in cattle.

There is an imperative need for effective control of bovine tuberculosis (BTB) on a global scale and vaccination of cattle may prove to be pivotal in achieving this. The oral and parenteral use of a heat-inactivated Mycobacterium bovis (M. bovis) vaccine has previously been found to confer partial protection against BTB in several species. A role for complement factor C3 has been suggested in wild boar, but the exact mechanism by which this vaccine provides protection remains unclear. In the present study, a quantitative proteomics approach was used to analyze the white blood cell proteome of vaccinated cattle in comparison to unvaccinated controls, prior (T0) and in response to vaccination, skin test and challenge (T9 and T12). The Fisher's exact test was used to compare the proportion of positive reactors to standard immunological assays for BTB (the BOVIGAM® assay, IDEXX TB ELISA and skin test) between the vaccinated and control groups. Using reverse-phase liquid-chromatography tandem mass spectrometry (RP-LC-MS/MS), a total of 12,346 proteins were identified with at least two peptides per protein and the Chi2-test (P = 0.05) determined 1,222 to be differentially represented at the key time point comparisons. Gene ontology (GO) analysis was performed in order to determine the biological processes (BPs), molecular functions (MFs) and cell components (CCs) the proteins formed part of. The analysis was focused on immune system BPs, specifically. GO analysis revealed that the most overrepresented proteins in immune system BPs, were kinase activity and receptor activity molecular functions and extracellular, Golgi apparatus and endosome cell components and included complement factor C8α and C8ß as well as toll-like receptors 4 (TLR4) and 9 (TLR9). Proteins of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) (JAK-STAT) and protein kinase C (PKC) signaling pathways were furthermore found to potentially be involved in the immune response elicited by the inactivated vaccine. In conclusion, this study provides a first indication of the role of several immune system pathways in response to the heat-inactivated M. bovis vaccine and mycobacterial challenge.

Review on the relevance of therapeutic drug monitoring of levetiracetam.

Therapeutic Drug Monitoring (TDM) of anti-epileptic drugs (AEDs) is not routinely performed, although this can guide the dosage regimen to achieve greater efficacy and safety. Levetiracetam (LEV) has been introduced as an AED with an almost perfect pharmacokinetic (PK) profile. Nonetheless, recent research challenges this statement and therefore we aimed to explore factors that modify LEV PK. Age and enzyme-inducing drugs (EIDs) appear to be major factors influencing the PK profile of LEV. Therefore, 30-50% lower dosages should be used in the elderly (> 65 years of age) and the dosing regimen should be guided by monitoring SDC (TDM). In contrast, higher LEV dosages are necessary in children aged between 2 months and 12 years (compared to adults) due to a 30-70% increase of LEV clearance (CL). Higher dosages are also required if a patient receives EIDs, again due to a higher CL of LEV (range 24-60%). This could also be true for pregnant women. LEV TDM is currently not common in the clinical setting due to the wide therapeutic range and the low prevalence of side-effects. However, LEV dose should on the one hand be increased in certain physiological situations (pregnancy, neonates) and patients on EIDs (especially carbamazepine). On the other hand, dose reductions are necessary when the LEV CL is impaired (elderly). Nevertheless, current data to support regular LEV TDM are lacking. Prospective research is needed to explore the importance of LEV TDM in elected patient groups; i.e. neonates, elderly, patients on EIDs and pregnant women.

Experimental Mycobacterium bovis infection in three white rhinoceroses (Ceratotherium simum): Susceptibility, clinical and anatomical pathology.

Tuberculosis caused by Mycobacterium bovis is endemic in the African buffalo (Syncerus caffer) population in the Kruger National Park and other conservation areas in South Africa. The disease has been diagnosed in a total of 21 free ranging or semi-free ranging wildlife species in the country with highly variable presentations in terms of clinical signs as well as severity and distribution of tuberculous lesions. Most species are spillover or dead-end hosts without significant role in the epidemiology of the disease. White rhinoceroses (Ceratotherium simum) are translocated from the Kruger National Park in substantial numbers every year and a clear understanding of their risk to manifest overt tuberculosis disease and to serve as source of infection to other species is required. We report the findings of experimental infection of three white rhinoceroses with a moderately low dose of a virulent field isolate of Mycobacterium bovis. None of the animals developed clinical signs or disseminated disease. The susceptibility of the white rhinoceros to bovine tuberculosis was confirmed by successful experimental infection based on the ante mortem isolation of M. bovis from the respiratory tract of one rhinoceros, the presence of acid-fast organisms and necrotizing granulomatous lesions in the tracheobronchial lymph nodes and the detection of M. bovis genetic material by PCR in the lungs of two animals.

Maternal homocysteine and related B vitamins as risk factors for low birthweight.

OBJECTIVE: We designed a large prospective study to explore the relationship between maternal homocysteine concentrations and related B vitamins and birthweight. STUDY DESIGN: Blood was sampled from pregnant women at 30-34 weeks of gestation and their newborn infants (n = 366). RESULTS: Concentrations of all analytes were higher in umbilical cord compared with maternal samples. Birthweight was related negatively to maternal homocysteine (r = -0.12) but not related to maternal cobalamin, methylmalonic acid, and folate (r = 0.02, r = 0.06, and r = 0.04, respectively). Regression analysis revealed smoking (beta = -313; 95% confidence interval [CI], -479 to -149), gestational age (beta = 150; 95% CI, 118-182), female sex (beta = -146; 95% CI, -256 to -35), and parity (beta = 104; 95% CI, 37-171) as strong determinants of birthweight. Maternal homocysteine, cobalamin, methylmalonic acid, and folate were not determinants of birthweight in multivariate analysis. CONCLUSION: Maternal homocysteine and B vitamins are not related to birthweight in a multivariate model that was adjusted for potential confounders.

Effect of low-intensity acenocoumarol on frequency and severity of migraine attacks.

OBJECTIVE: To investigate the effect of low-intensity acenocoumarol treatment (target INR 1.5 to 2.0) on the frequency and severity of migraine attacks. BACKGROUND: The positive effect of anticoagulation on migraine has been described in case reports and observational studies. METHODS: We conducted a randomized, open, crossover study in migraine patients. After a run-in period of 8 weeks, all patients received acenocoumarol or propranolol during a period of 12 weeks and, after a washout period of 2 weeks, propranolol or acenocoumarol during a second period of 12 weeks. RESULTS: Nineteen patients fulfilling the criteria were included. In 12 patients with complete data collection, only one good responder could be noted. In the other patients, treatment with low-intensity acenocoumarol did not show improvement of migraine symptoms compared with the run-in period. Treatment with propranolol showed a trend towards improvement compared with the run-in period. No serious adverse events were observed. CONCLUSIONS: Overall, low-intensity acenocoumarol treatment has no prophylactic effect in migraine patients.

A thromboembolic predisposition and the effect of anticoagulants on migraine.

OBJECTIVE: To investigate the presence of thromboembolic risk factors and the effect of low-dose acenocoumarol therapy on migraine in patients who spontaneously reported a reduction of their migraine attacks during previous therapeutic use of anticoagulants. BACKGROUND: The positive effect of anticoagulants on migraine has been described in case reports and observational studies. It remains unclear whether this concerns only a select group of migraineurs with certain common characteristics. METHODS: In 4 migraineurs with a self-reported reduction of attack frequency during previous use of anticoagulants (international normalization ratio [INR], 2.5:4.0), the presence of thromboembolic risk factors and the effect of low-dose acenocoumarol therapy (INR, 1.5:2.0) on migraine attacks were prospectively investigated in an open study. RESULTS: All patients had one or more thromboembolic risk factors. Two patients, both with factor V Leiden heterozygosity, experienced a clear improvement of migraine during low-dose acenocoumarol therapy. CONCLUSIONS: Our findings support the hypothesis that migraine, as a phenotype, has different underlying mechanisms, amongst which a thromboembolic tendency. In this group of patients, oral anticoagulants may be a suitable form of migraine prophylaxis, but this needs further clinical investigation.