An HLA-E-targeted TCR bispecific molecule redirects T cell immunity against Mycobacterium tuberculosis.

Peptides presented by HLA-E, a molecule with very limited polymorphism, represent attractive targets for T cell receptor (TCR)-based immunotherapies to circumvent the limitations imposed by the high polymorphism of classical HLA genes in the human population. Here, we describe a TCR-based bispecific molecule that potently and selectively binds HLA-E in complex with a peptide encoded by the inhA gene of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans. We reveal the biophysical and structural bases underpinning the potency and specificity of this molecule and demonstrate its ability to redirect polyclonal T cells to target HLA-E-expressing cells transduced with mycobacterial inhA as well as primary cells infected with virulent Mtb. Additionally, we demonstrate elimination of Mtb-infected cells and reduction of intracellular Mtb growth. Our study suggests an approach to enhance host T cell immunity against Mtb and provides proof of principle for an innovative TCR-based therapeutic strategy overcoming HLA polymorphism and therefore applicable to a broader patient population.

Pediatric neuromodulation for drug-resistant epilepsy: Survey of current practices, techniques, and outcomes across US epilepsy centers.

Neuromodulation via Responsive Neurostimulation (RNS) or Deep Brain Stimulation (DBS) is an emerging treatment strategy for pediatric drug-resistant epilepsy (DRE). Knowledge gaps exist in patient selection, surgical technique, and perioperative care. Here, we use an expert survey to clarify practices. Thirty-two members of the Pediatric Epilepsy Research Consortium were surveyed using REDCap. Respondents were from 17 pediatric epilepsy centers (missing data in one): Four centers implant RNS only while 13 implant both RNS and DBS. Thirteen RNS programs commenced in or before 2020, and 10 of 12 DBS programs began thereafter. The busiest six centers implant 6-10 new RNS devices per year; all DBS programs implant <5 annually. The youngest RNS patient was 3 years old. Most centers (11/12) utilize MP2RAGE and/or FGATIR sequences for planning. Centromedian thalamic nuclei were the unanimous target for Lennox-Gastaut syndrome. Surgeon exposure to neuromodulation occurred mostly in clinical practice (14/17). Clinically significant hemorrhage (n = 2) or infection (n = 3) were rare. Meaningful seizure reduction (>50%) was reported by 81% (13/16) of centers. RNS and DBS are rapidly evolving treatment modalities for safe and effective treatment of pediatric DRE. There is increasing interest in multicenter collaboration to gain knowledge and facilitate dialogue. PLAIN LANGUAGE SUMMARY: We surveyed 32 pediatric epilepsy centers in USA to highlight current practices of intracranial neuromodulation. Of the 17 that replied, we found that most centers are implanting thalamic targets in pediatric drug-resistant epilepsy using the RNS device. DBS device is starting to be used in pediatric epilepsy, especially after 2020. Different strategies for target identification are enumerated. This study serves as a starting point for future collaborative research.

Instability of the HLA-E peptidome of HIV presents a major barrier to therapeutic targeting.

Naturally occurring T cells that recognize microbial peptides via HLA-E, a nonpolymorphic HLA class Ib molecule, could provide the foundation for new universal immunotherapeutics. However, confidence in the biological relevance of putative ligands is crucial, given that the mechanisms by which pathogen-derived peptides can access the HLA-E presentation pathway are poorly understood. We systematically interrogated the HIV proteome using immunopeptidomic and bioinformatic approaches, coupled with biochemical and cellular assays. No HIV HLA-E peptides were identified by tandem mass spectrometry analysis of HIV-infected cells. In addition, all bioinformatically predicted HIV peptide ligands (>80) were characterized by poor complex stability. Furthermore, infected cell elimination assays using an affinity-enhanced T cell receptor bispecific targeted to a previously reported HIV Gag HLA-E epitope demonstrated inconsistent presentation of the peptide, despite normal HLA-E expression on HIV-infected cells. This work highlights the instability of the HIV HLA-E peptidome as a major challenge for drug development.

Bilateral Rasmussen Encephalitis: Good Outcome Following Hemispherotomy.

BACKGROUND: Bilateral Rasmussen encephalitis is a rare variant of a debilitating, typically unihemispheric disease with limited treatment options. Few cases with bilateral histopathology have been reported, all with poor seizure control following surgery. Here we report a favorable outcome following hemispherotomy in a four-year-old male with biopsy-confirmed bilateral disease. CASE: The patient presented with right hemispheric focal seizures with behavioral arrest and over a year progressed to left lower extremity clonic seizures, epilepsia partialis continua, and loss of ambulation, with transient response to steroids and tacrolimus. Histopathology confirmed bilateral disease. The patient developed super-refractory status epilepticus and underwent right functional hemispherotomy 4.5 years after initial presentation. In a 2.5-year follow-up period, an Engel 1D outcome classification was observed with substantially improved quality of life. CONCLUSION: Previous reports of bilateral Rasmussen encephalitis describe universally poor outcomes, and hemispherotomy is often considered contraindicated. However, hemispherotomy in a patient with bilateral Rasmussen encephalitis may have a good outcome if seizures are unihemispheric.

A water-soluble manganese(II) octanediaoate/phenanthroline complex acts as an antioxidant and attenuates alpha-synuclein toxicity.

The overproduction of reactive oxygen species (ROS) induces oxidative stress, a well-known process associated with aging and several human pathologies, such as cancer and neurodegenerative diseases. A large number of synthetic compounds have been described as antioxidant enzyme mimics, capable of eliminating ROS and/or reducing oxidative damage. In this study, we investigated the antioxidant activity of a water-soluble 1,10-phenantroline-octanediaoate Mn2+-complex on cells under oxidative stress, and assessed its capacity to attenuate alpha-synuclein (aSyn) toxicity and aggregation, a process associated with increased oxidative stress. This Mn2+-complex exhibited a significant antioxidant potential, reducing intracelular oxidation and increasing oxidative stress resistance in S. cerevisiae cells and in vivo, in G. mellonella, increasing the activity of the intracellular antioxidant enzymes superoxide dismutase and catalase. Strikingly, the Mn2+-complex reduced both aSyn oligomerization and aggregation in human cell cultures and, using NMR and DFT/molecular docking we confirmed its interaction with the C-terminal region of aSyn. In conclusion, the Mn2+-complex appears as an excellent lead for the design of new phenanthroline derivatives as alternative compounds for preventing oxidative damages and oxidative stress - related diseases.

Efficacy of Levetiracetam and Phenobarbital as First-Line Treatment for Neonatal Seizures.

High neonatal seizure burden is associated with worsened neurodevelopmental outcomes. We compared the efficacy of initial treatment with levetiracetam vs phenobarbital for maintaining low seizure burden in a retrospective cohort of 25 neonates monitored with video electroencephalography (EEG). Video EEG tracing were reviewed and paired with medication bolus times to determine seizure burden after treatment. Initial cumulative dose of phenobarbital was 20 mg/kg in all but 1 case; initial cumulative dose of levetiracetam ranged from 50 to 100 mg/kg. Eleven of 17 (65%) patients sustained seizure burden <10% following initial treatment with levetiracetam, compared with 5 of 8 (63%) with phenobarbital. Thirteen (76%) patients treated with levetiracetam had sustained seizure burden <20% compared with 6 (75%) treated with phenobarbital. The phenobarbital group showed a larger absolute reduction in average seizure burden in the hour before and after treatment (-24.3  vs -14.2 minutes/h). Six of 17 (35%) patients treated with levetiracetam remained seizure free after initial treatment, compared with 2 of 8 (25%) patients treated with phenobarbital. Initial treatment with levetiracetam was associated with shorter average time to seizure freedom (15 vs 21 hours). None of these results were statistically significant. Cumulative doses of levetiracetam 100 mg/kg were well tolerated and associated with substantial decrease in seizure burden in several cases. Levetiracetam remains a promising first-line treatment for neonatal seizures; additional randomized controlled trials evaluating the effects of high-dose levetiracetam on seizure burden and long-term outcomes are warranted.

Structure-guided stabilization of pathogen-derived peptide-HLA-E complexes using non-natural amino acids conserves native TCR recognition.

The nonpolymorphic class Ib molecule, HLA-E, primarily presents peptides from HLA class Ia leader peptides, providing an inhibitory signal to NK cells via CD94/NKG2 interactions. Although peptides of pathogenic origin can also be presented by HLA-E to T cells, the molecular basis underpinning their role in antigen surveillance is largely unknown. Here, we solved a co-complex crystal structure of a TCR with an HLA-E presented peptide (pHLA-E) from bacterial (Mycobacterium tuberculosis) origin, and the first TCR-pHLA-E complex with a noncanonically presented peptide from viral (HIV) origin. The structures provided a molecular foundation to develop a novel method to introduce cysteine traps using non-natural amino acid chemistry that stabilized pHLA-E complexes while maintaining native interface contacts between the TCRs and different pHLA-E complexes. These pHLA-E monomers could be used to isolate pHLA-E-specific T cells, with obvious utility for studying pHLA-E restricted T cells, and for the identification of putative therapeutic TCRs.

Modulating the Blood-Brain Barrier: A Comprehensive Review.

The highly secure blood-brain barrier (BBB) restricts drug access to the brain, limiting the molecular toolkit for treating central nervous system (CNS) diseases to small, lipophilic drugs. Development of a safe and effective BBB modulator would revolutionise the treatment of CNS diseases and future drug development in the area. Naturally, the field has garnered a great deal of attention, leading to a vast and diverse range of BBB modulators. In this review, we summarise and compare the various classes of BBB modulators developed over the last five decades-their recent advancements, advantages and disadvantages, while providing some insight into their future as BBB modulators.

Incoming HIV virion-derived Gag Spacer Peptide 2 (p1) is a target of effective CD8+ T cell antiviral responses.

Persistence of HIV through integration into host DNA in CD4+ T cells presents a major barrier to virus eradication. Viral integration may be curtailed when CD8+ T cells are triggered to kill infected CD4+ T cells through recognition of histocompatibility leukocyte antigen (HLA) class I-bound peptides derived from incoming virions. However, this has been reported only in individuals with "beneficial" HLA alleles that are associated with superior HIV control. Through interrogation of the pre-integration immunopeptidome, we obtain proof of early presentation of a virion-derived HLA-A∗02:01-restricted epitope, FLGKIWPSH (FH9), located in Gag Spacer Peptide 2 (SP2). FH9-specific CD8+ T cell responses are detectable in individuals with primary HIV infection and eliminate HIV-infected CD4+ T cells prior to virus production in vitro. Our data show that non-beneficial HLA class I alleles can elicit an effective antiviral response through early presentation of HIV virion-derived epitopes and also demonstrate the importance of SP2 as an immune target.

Unexpected pain with electrocortical stimulation in a teenager with temporal encephalocele.

Temporal lobe encephalocele has emerged as a potentially unrecognized cause of drug-resistant temporal lobe epilepsy (TLE) that can be effectively treated with epilepsy surgery. Here we present a case in which a 17-year-old male with drug-resistant epilepsy and left temporal encephalocele underwent workup for epilepsy surgery, and experienced unexpected pain with electrocortical stimulation. Stimulation of stereo-EEG electrodes in the left temporal pole resulted in severe, unilateral left-sided facial pain due to inadvertent stimulation of the trigeminal nerve. Stereo-EEG showed seizure onset adjacent to encephalocele with no involvement of mesial temporal structures. A temporal pole resection sparing the mesial temporal structures and repair of the sphenoid bone defect was performed. The patient experienced post-operative seizure freedom, with no loss of language function or sensory deficits in the distribution of the trigeminal nerve. This case highlights temporal encephalocele as a potentially overlooked cause of TLE and underscores the anatomical proximity of the trigeminal nerve to the temporal pole, an important consideration for surgical planning.

Unpaid Professional Work at Home and Work-Life Interference among Employees with Care Responsibilities.

Employees with caregiving responsibilities often experience work-life interference (WLI), particularly when caring for either disabled persons and/or children. This study examines sample of 288 working Australians from the AWALI national survey data, who care for at least one family member or friend with long-term physical or mental illness, disability, or aging-related problems. We investigated the role of unpaid work at home in predicting WLI, based on a model that included indirect association via inferred causes for working unpaid hours at home and a conditional direct relationship based on number of children. The findings supported our prediction that unpaid work at home is positively associated with WLI but its effect is moderated by number of children. There was a conditional direct effect where employees with care responsibilities experienced a stronger relationship between unpaid hours and WLI when having more children. Further, when the perceived reason for unpaid work was excessively demanding work, the relationship with WLI was stronger. Implications for workers with multiple caregiving responsibilities are discussed.

Synthesis and optimisation of P3 substituted vinyl sulfone-based inhibitors as anti-trypanosomal agents.

A series of lysine-based vinyl sulfone peptidomimetics were synthesised and evaluated for anti-trypanosomal activity against bloodstream forms of T. brucei. This focused set of compounds, varying in the P3 position, were accessed in a divergent manner from a common intermediate (ammonium salt 8). Several P3 analogues exhibited sub-micromolar EC50 values, with thiourea 14, urea 15 and amide 21 representing the most potent anti-trypanosomal derivatives of the series. In order to establish an in vitro selectivity index the most active anti-trypanosomal compounds were also assessed for their impact on cell viability and cytotoxity effects in mammalian cells. Encouragingly, all compounds only reduced cellular metabolic activity in mammalian cells to a modest level and little, or no cytotoxicity, was observed with the series.

Immune-Mobilizing Monoclonal T Cell Receptors Mediate Specific and Rapid Elimination of Hepatitis B-Infected Cells.

BACKGROUND AND AIMS: Therapies for chronic hepatitis B virus (HBV) infection are urgently needed because of viral integration, persistence of viral antigen expression, inadequate HBV-specific immune responses, and treatment regimens that require lifelong adherence to suppress the virus. Immune mobilizing monoclonal T Cell receptors against virus (ImmTAV) molecules represent a therapeutic strategy combining an affinity-enhanced T Cell receptor with an anti-CD3 T Cell-activating moiety. This bispecific fusion protein redirects T cells to specifically lyse infected cells expressing the target virus-derived peptides presented by human leukocyte antigen (HLA). APPROACH AND RESULTS: ImmTAV molecules specific for HLA-A*02:01-restricted epitopes from HBV envelope, polymerase, and core antigens were engineered. The ability of ImmTAV-Env to activate and redirect polyclonal T cells toward cells containing integrated HBV and cells infected with HBV was assessed using cytokine secretion assays and imaging-based killing assays. Elimination of infected cells was further quantified using a modified fluorescent hybridization of viral RNA assay. Here, we demonstrate that picomolar concentrations of ImmTAV-Env can redirect T cells from healthy and HBV-infected donors toward hepatocellular carcinoma (HCC) cells containing integrated HBV DNA resulting in cytokine release, which could be suppressed by the addition of a corticosteroid in vitro. Importantly, ImmTAV-Env redirection of T cells induced cytolysis of antigen-positive HCC cells and cells infected with HBV in vitro, causing a reduction of hepatitis B e antigen and specific loss of cells expressing viral RNA. CONCLUSIONS: The ImmTAV platform has the potential to enable the elimination of infected cells by redirecting endogenous non-HBV-specific T cells, bypassing exhausted HBV-specific T cells. This represents a promising therapeutic option in the treatment of chronic hepatitis B, with our lead candidate now entering trials.

Molecular Basis for Synaptotagmin-1-Associated Neurodevelopmental Disorder.

At neuronal synapses, synaptotagmin-1 (syt1) acts as a Ca2+ sensor that synchronizes neurotransmitter release with Ca2+ influx during action potential firing. Heterozygous missense mutations in syt1 have recently been associated with a severe but heterogeneous developmental syndrome, termed syt1-associated neurodevelopmental disorder. Well-defined pathogenic mechanisms, and the basis for phenotypic heterogeneity in this disorder, remain unknown. Here, we report the clinical, physiological, and biophysical characterization of three syt1 mutations from human patients. Synaptic transmission was impaired in neurons expressing mutant variants, which demonstrated potent, graded dominant-negative effects. Biophysical interrogation of the mutant variants revealed novel mechanistic features concerning the cooperative action, and functional specialization, of the tandem Ca2+-sensing domains of syt1. These mechanistic studies led to the discovery that a clinically approved K+ channel antagonist is able to rescue the dominant-negative heterozygous phenotype. Our results establish a molecular cause, basis for phenotypic heterogeneity, and potential treatment approach for syt1-associated neurodevelopmental disorder.

Microfluidics in Haemostasis: A Review.

Haemostatic disorders are both complex and costly in relation to both their treatment and subsequent management. As leading causes of mortality worldwide, there is an ever-increasing drive to improve the diagnosis and prevention of haemostatic disorders. The field of microfluidic and Lab on a Chip (LOC) technologies is rapidly advancing and the important role of miniaturised diagnostics is becoming more evident in the healthcare system, with particular importance in near patient testing (NPT) and point of care (POC) settings. Microfluidic technologies present innovative solutions to diagnostic and clinical challenges which have the knock-on effect of improving health care and quality of life. In this review, both advanced microfluidic devices (R&D) and commercially available devices for the diagnosis and monitoring of haemostasis-related disorders and antithrombotic therapies, respectively, are discussed. Innovative design specifications, fabrication techniques, and modes of detection in addition to the materials used in developing micro-channels are reviewed in the context of application to the field of haemostasis.

TCRs with Distinct Specificity Profiles Use Different Binding Modes to Engage an Identical Peptide-HLA Complex.

The molecular rules driving TCR cross-reactivity are poorly understood and, consequently, it is unclear the extent to which TCRs targeting the same Ag recognize the same off-target peptides. We determined TCR-peptide-HLA crystal structures and, using a single-chain peptide-HLA phage library, we generated peptide specificity profiles for three newly identified human TCRs specific for the cancer testis Ag NY-ESO-1157-165-HLA-A2. Two TCRs engaged the same central peptide feature, although were more permissive at peripheral peptide positions and, accordingly, possessed partially overlapping peptide specificity profiles. The third TCR engaged a flipped peptide conformation, leading to the recognition of off-target peptides sharing little similarity with the cognate peptide. These data show that TCRs specific for a cognate peptide recognize discrete peptide repertoires and reconciles how an individual's limited TCR repertoire following negative selection in the thymus is able to recognize a vastly larger antigenic pool.