Bi-allelic variants in neuronal cell adhesion molecule cause a neurodevelopmental disorder characterized by developmental delay, hypotonia, neuropathy/spasticity.

Cell adhesion molecules are membrane-bound proteins predominantly expressed in the central nervous system along principal axonal pathways with key roles in nervous system development, neural cell differentiation and migration, axonal growth and guidance, myelination, and synapse formation. Here, we describe ten affected individuals with bi-allelic variants in the neuronal cell adhesion molecule NRCAM that lead to a neurodevelopmental syndrome of varying severity; the individuals are from eight families. This syndrome is characterized by developmental delay/intellectual disability, hypotonia, peripheral neuropathy, and/or spasticity. Computational analyses of NRCAM variants, many of which cluster in the third fibronectin type III (Fn-III) domain, strongly suggest a deleterious effect on NRCAM structure and function, including possible disruption of its interactions with other proteins. These findings are corroborated by previous in vitro studies of murine Nrcam-deficient cells, revealing abnormal neurite outgrowth, synaptogenesis, and formation of nodes of Ranvier on myelinated axons. Our studies on zebrafish nrcamaΔ mutants lacking the third Fn-III domain revealed that mutant larvae displayed significantly altered swimming behavior compared to wild-type larvae (p < 0.03). Moreover, nrcamaΔ mutants displayed a trend toward increased amounts of α-tubulin fibers in the dorsal telencephalon, demonstrating an alteration in white matter tracts and projections. Taken together, our study provides evidence that NRCAM disruption causes a variable form of a neurodevelopmental disorder and broadens the knowledge on the growing role of the cell adhesion molecule family in the nervous system.

Alternative splicing expands the clinical spectrum of NDUFS6-related mitochondrial disorders.

PURPOSE: We describe 3 families with Charcot-Marie-Tooth neuropathy (CMT), harboring a homozygous NDUFS6 NM_004553.6:c.309+5G>A variant previously linked to fatal Leigh syndrome. We aimed to characterize clinically and molecularly the newly identified patients and understand the mechanism underlying their milder phenotype. METHODS: The patients underwent extensive clinical examinations. Exome sequencing was done in 4 affected individuals. The functional effect of the c.309+5G>A variant was investigated in patient-derived EBV-transformed lymphoblasts at the complementary DNA, protein, and mitochondrial level. Alternative splicing was evaluated using complementary DNA long-read sequencing. RESULTS: All patients presented with early-onset, slowly progressive axonal CMT, and nystagmus; some exhibited additional central nervous system symptoms. The c.309+5G>A substitution caused the expression of aberrantly spliced transcripts and negligible levels of the canonical transcript. Immunoblotting showed reduced levels of mutant isoforms. No detectable defects in mitochondrial complex stability or bioenergetics were found. CONCLUSION: We expand the clinical spectrum of NDUFS6-related mitochondrial disorders to include axonal CMT, emphasizing the clinical and pathophysiologic overlap between these 2 clinical entities. This work demonstrates the critical role that alternative splicing may play in modulating the severity of a genetic disorder, emphasizing the need for careful consideration when interpreting splice variants and their implications on disease prognosis.

Identification and Characterization of Novel Founder Mutations in NDRG1: Refining the Genetic Landscape of Charcot-Marie-Tooth Disease Type 4D in Bulgaria.

Charcot-Marie-Tooth neuropathy type 4D (CMT4D) is a rare genetic disorder of the peripheral nervous system caused by biallelic mutations in the N-Myc Downstream Regulated 1 gene (NDRG1). Patients present with an early onset demyelinating peripheral neuropathy causing severe distal muscle weakness and sensory loss, leading to loss of ambulation and progressive sensorineural hearing loss. The disorder was initially described in the Roma community due to a common founder mutation, and only a handful of disease-causing variants have been described in this gene so far. Here, we present genetic and clinical findings from a large Bulgarian cohort of demyelinating CMT patients harboring recurrent and novel variants in the NDRG1 gene. Notably, two splice-site variants are exclusive to Bulgarian Muslims and reside in ancestral haplotypes, suggesting a founder effect. Functional characterization of these novel variants implicates a loss-of-function mechanism due to shorter gene products. Our findings contribute to a deeper understanding of the genetic and clinical heterogeneity of CMT4D and highlight novel founder mutations in the ethnic minority of Bulgarian Muslims.

Linkage analysis and whole exome sequencing reveals AHNAK2 as a novel genetic cause for autosomal recessive CMT in a Malaysian family.

Charcot-Marie-Tooth (CMT) disease is a form of inherited peripheral neuropathy that affects motor and sensory neurons. To identify the causative gene in a consanguineous family with autosomal recessive CMT (AR-CMT), we employed a combination of linkage analysis and whole exome sequencing. After excluding known AR-CMT genes, genome-wide linkage analysis mapped the disease locus to a 7.48-Mb interval on chromosome 14q32.11-q32.33, flanked by the markers rs2124843 and rs4983409. Whole exome sequencing identified two non-synonymous variants (p.T40P and p.H915Y) in the AHNAK2 gene that segregated with the disease in the family. Pathogenic predictions indicated that p.T40P is the likely causative allele. Analysis of AHNAK2 expression in the AR-CMT patient fibroblasts showed significantly reduced mRNA and protein levels. AHNAK2 binds directly to periaxin which is encoded by the PRX gene, and PRX mutations are associated with another form of AR-CMT (CMT4F). The altered expression of mutant AHNAK2 may disrupt the AHNAK2-PRX interaction in which one of its known functions is to regulate myelination.

MCM3AP in recessive Charcot-Marie-Tooth neuropathy and mild intellectual disability.

Defects in mRNA export from the nucleus have been linked to various neurodegenerative disorders. We report mutations in the gene MCM3AP, encoding the germinal center associated nuclear protein (GANP), in nine affected individuals from five unrelated families. The variants were associated with severe childhood onset primarily axonal (four families) or demyelinating (one family) Charcot-Marie-Tooth neuropathy. Mild to moderate intellectual disability was present in seven of nine affected individuals. The affected individuals were either compound heterozygous or homozygous for different MCM3AP variants, which were predicted to cause depletion of GANP or affect conserved amino acids with likely importance for its function. Accordingly, fibroblasts of affected individuals from one family demonstrated severe depletion of GANP. GANP has been described to function as an mRNA export factor, and to suppress TDP-43-mediated motor neuron degeneration in flies. Thus our results suggest defective mRNA export from nucleus as a potential pathogenic mechanism of axonal degeneration in these patients. The identification of MCM3AP variants in affected individuals from multiple centres establishes it as a disease gene for childhood-onset recessively inherited Charcot-Marie-Tooth neuropathy with intellectual disability.

Novel mutations in genes causing hereditary spastic paraplegia and Charcot-Marie-Tooth neuropathy identified by an optimized protocol for homozygosity mapping based on whole-exome sequencing.

PURPOSE: Homozygosity mapping is an effective approach for detecting molecular defects in consanguineous families by delineating stretches of genomic DNA that are identical by descent. Constant developments in next-generation sequencing created possibilities to combine whole-exome sequencing (WES) and homozygosity mapping in a single step. METHODS: Basic optimization of homozygosity mapping parameters was performed in a group of families with autosomal-recessive (AR) mutations for which both single-nucleotide polymorphism (SNP) array and WES data were available. We varied the criteria for SNP extraction and PLINK thresholds to estimate their effect on the accuracy of homozygosity mapping based on WES. RESULTS: Our protocol showed high specificity and sensitivity for homozygosity detection and facilitated the identification of novel mutations in GAN, GBA2, and ZFYVE26 in four families affected by hereditary spastic paraplegia or Charcot-Marie-Tooth disease. Filtering and mapping with optimized parameters was integrated into the HOMWES (homozygosity mapping based on WES analysis) tool in the GenomeComb package for genomic data analysis. CONCLUSION: We present recommendations for detection of homozygous regions based on WES data and a bioinformatics tool for their identification, which can be widely applied for studying AR disorders.Genet Med 18 6, 600-607.

Unraveling the genetic landscape of autosomal recessive Charcot-Marie-Tooth neuropathies using a homozygosity mapping approach.

Autosomal recessive forms of Charcot-Marie-Tooth disease (ARCMT) are rare but severe disorders of the peripheral nervous system. Their molecular basis is poorly understood due to the extensive genetic and clinical heterogeneity, posing considerable challenges for patients, physicians, and researchers. We report on the genetic findings from a systematic study of a large collection of 174 independent ARCMT families. Initial sequencing of the three most common ARCMT genes (ganglioside-induced differentiation protein 1­GDAP1, SH3 domain and tetratricopeptide repeats-containing protein 2­SH3TC2, histidine-triad nucleotide binding protein 1­HINT1) identified pathogenic mutations in 41 patients. Subsequently, 87 selected nuclear families underwent single nucleotide polymorphism (SNP) genotyping and homozygosity mapping, followed by targeted screening of known ARCMT genes. This strategy provided molecular diagnosis to 22% of the families. Altogether, our unbiased genetic approach identified pathogenic mutations in ten ARCMT genes in a total of 41.3% patients. Apart from a newly described founder mutation in GDAP1, the majority of variants constitute private molecular defects. Since the gene testing was independent of the clinical phenotype of the patients, we identified mutations in patients with unusual or additional clinical features, extending the phenotypic spectrum of the SH3TC2 gene. Our study provides an overview of the ARCMT genetic landscape and proposes guidelines for tackling the genetic heterogeneity of this group of hereditary neuropathies.

Human Rab7 mutation mimics features of Charcot-Marie-Tooth neuropathy type 2B in Drosophila.

Charcot-Marie-Tooth disease type 2B (CMT2B) is an inherited axonal peripheral neuropathy. It is characterised by prominent sensory loss, often complicated by severe ulcero-mutilations of toes or feet, and variable motor involvement. Missense mutations in RAB7A, the gene encoding the small GTPase Rab7, cause CMT2B and increase Rab7 activity. Rab7 is ubiquitously expressed and is involved in degradation through the lysosomal pathway. In the neurons, Rab7 plays a role in the long-range retrograde transport of signalling endosomes in the axons. Here we developed the first animal model of CMT2B, modelling one of the mutations (L129F) in Drosophila melanogaster. Behavioural assays show that this model recapitulates several hallmarks of the human disease. Upon expression of mutant Rab7 in the sensory neurons, larvae present with a reduction of temperature and pain perception. Furthermore, the larvae exhibit a crawling defect when the mutant protein is expressed in the motor neurons. Analysis of axonal transport of Rab7 positive vesicles in sensory neurons of Drosophila larvae and in neurites of mammalian neuroblastoma cells demonstrates that mutant vesicles pause less than their wild-type counterparts. This latter finding indicates that alterations in vesicle transport might contribute to the pathomechanism of CMT2B.

Biallelic variants in COX18 cause a mitochondrial disorder primarily manifesting as peripheral neuropathy.

Defects in mitochondrial dynamics are a common cause of Charcot-Marie-Tooth disease (CMT), while primary deficiencies in the mitochondrial respiratory chain (MRC) are rare and atypical for this etiology. This study aims to report COX18 as a novel CMT-causing gene. This gene encodes an assembly factor of mitochondrial Complex IV (CIV) that translocates the C-terminal tail of MTCO2 across the mitochondrial inner membrane. Exome sequencing was performed in four affected individuals. The patients and available family members underwent thorough neurological and electrophysiological assessment. The impact of one of the identified variants on splicing, protein levels, and mitochondrial bioenergetics was investigated in patient-derived lymphoblasts. The functionality of the mutant protein was assessed using a Proteinase K protection assay and immunoblotting. Neuronal relevance of COX18 was assessed in a Drosophila melanogaster knockdown model. Exome sequencing coupled with homozygosity mapping revealed a homozygous splice variant c.435-6A>G in COX18 in two siblings with early-onset progressive axonal sensory-motor peripheral neuropathy. By querying external databases, we identified two additional families with rare deleterious biallelic variants in COX18 . All affected individuals presented with axonal CMT and some patients also exhibited central nervous system symptoms, such as dystonia and spasticity. Functional characterization of the c.435-6A>G variant demonstrated that it leads to the expression of an alternative transcript that lacks exon 2, resulting in a stable but defective COX18 isoform. The mutant protein impairs CIV assembly and activity, leading to a reduction in mitochondrial membrane potential. Downregulation of the COX18 homolog in Drosophila melanogaster displayed signs of neurodegeneration, including locomotor deficit and progressive axonal degeneration of sensory neurons. Our study presents genetic and functional evidence that supports COX18 as a newly identified gene candidate for autosomal recessive axonal CMT with or without central nervous system involvement. These findings emphasize the significance of peripheral neuropathy within the spectrum of primary mitochondrial disorders and the role of mitochondrial CIV in the development of CMT. Our research has important implications for the diagnostic workup of CMT patients.

Randomized controlled trial of liberal vs. standard fasting instructions in percutaneous cardiac procedures.

BACKGROUND: Pre-procedural fasting to reduce aspiration risk is usual care prior to surgery requiring anesthesia. Prolonged fasting, however, can result in dehydration and may adversely affect patient experience and outcomes. Previous studies suggest that providing a supplemental beverage to patients undergoing cardiac and a variety of other surgical procedures improves patients' subjective assessment of thirst and hunger and potentially decreases the need for inotrope and vasopressor therapy. Less is known, however, about the effects of ad libitum clear liquids up to 2 h prior to surgery. METHODS: Adult patients undergoing transcatheter aortic valve replacement (TAVR) or arrhythmia ablation were randomized (1:1) to ad libitum clear liquids up to 2 h prior to their procedure vs. nil per os (NPO) after midnight (control group, usual care). The primary endpoint was a composite satisfaction score that included patient-reported thirst, hunger, headache, nausea, lightheadedness, and anxiousness prior to surgery. The incidence of case-delay was recorded. Intraoperative vasopressor administration, changes in creatinine, anti-emetic use, and hospital length of stay (LOS) were recorded. Safety endpoints including aspiration were assessed. RESULTS: A total of 200 patients were randomized and 181 patients were included in the final analysis. Overall, 92% of patients were ASA class III or IV and 23% of patients had NYHA class III or IV symptoms. Groups were well balanced with no significant differences in age, sex or baseline cardiac or renal disease. The composite satisfaction score (primary endpoint) was not significantly different between groups (Ad libitum median = 12, IQR = [6, 17], vs Standard NPO median = 10, IQR = [5, 15], [95% CI = [-1, 4]). No significant differences between the two groups were observed in any of the individual survey questions (thirst, hunger, headache, nausea, lightheadedness, anxiousness). No significant differences between groups were observed for intra-operative vasopressor use, changes in creatinine, rescue anti-emetic use or hospital LOS. There were no case delays attributed to the intervention. There were no cases of suspected aspiration. CONCLUSION: No adverse events or case delays were observed in the ad libitum clears group. No significant benefit, however, was observed in patient satisfaction or any of the pre-specified secondary endpoints in patients randomized to ad libitum clear liquids up to 2 h prior to their procedure. TRIAL REGISTRATION: NCT04079543.

Tyrosyl-tRNA synthetase has a noncanonical function in actin bundling.

Dominant mutations in tyrosyl-tRNA synthetase (YARS1) and six other tRNA ligases cause Charcot-Marie-Tooth peripheral neuropathy (CMT). Loss of aminoacylation is not required for their pathogenicity, suggesting a gain-of-function disease mechanism. By an unbiased genetic screen in Drosophila, we link YARS1 dysfunction to actin cytoskeleton organization. Biochemical studies uncover yet unknown actin-bundling property of YARS1 to be enhanced by a CMT mutation, leading to actin disorganization in the Drosophila nervous system, human SH-SY5Y neuroblastoma cells, and patient-derived fibroblasts. Genetic modulation of F-actin organization improves hallmark electrophysiological and morphological features in neurons of flies expressing CMT-causing YARS1 mutations. Similar beneficial effects are observed in flies expressing a neuropathy-causing glycyl-tRNA synthetase. Hence, in this work, we show that YARS1 is an evolutionary-conserved F-actin organizer which links the actin cytoskeleton to tRNA-synthetase-induced neurodegeneration.

Interleukin-12p70 expression by dendritic cells of HIV-1-infected patients fails to stimulate gag-specific immune responses.

A variety of immune-based therapies has been developed in order to boost or induce protective CD8(+) T cell responses in order to control HIV replication. Since dendritic cells (DCs) are professional antigen-presenting cells (APCs) with the unique capability to stimulate naïve T cells into effector T cells, their use for the induction of HIV-specific immune responses has been studied intensively. In the present study we investigated whether modulation of the activation state of DCs electroporated with consensus codon-optimized HxB2 gag mRNA enhances their capacity to induce HIV gag-specific T cell responses. To this end, mature DCs were (i) co-electroporated with mRNA encoding interleukin (IL)-12p70 mRNA, or (ii) activated with a cytokine cocktail consisting of R848 and interferon (IFN)-γ. Our results confirm the ability of HxB2 gag-expressing DCs to expand functional HIV-specific CD8(+) T cells. However, although most of the patients had detectable gag-specific CD8(+) T cell responses, no significant differences in the level of expansion of functional CD8(+) T cells could be demonstrated when comparing conventional or immune-modulated DCs expressing IL-12p70. This result which goes against expectation may lead to a re-evaluation of the need for IL-12 expression by DCs in order to improve T-cell responses in HIV-1-infected individuals.

Polyelectrolyte capsules-containing HIV-1 p24 and poly I:C modulate dendritic cells to stimulate HIV-1-specific immune responses.

Polyelectrolyte microcapsules (MCs) are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells (DCs). We investigated the immune stimulatory capacity of monocyte-derived DC (Mo-DC) loaded with these MCs, containing p24 antigen from human immunodeficiency virus type 1 (HIV-1) alone [p24-containing MC (MCp24)] or with the Toll-like receptor ligand 3 (TLR3) ligand poly I:C (MCp24pIC) as a maturation factor. MO-DC, loaded with MCp24pIC, upregulated CCR7, CD80, CD83, and CD86 and produced high amounts of interleukin-12 (IL-12) cytokine, to a similar extent as MCp24 in the presence of an optimized cytokine cocktail. MO-DC from HIV-infected patients under highly active antiretroviral therapy (HAART) exposed to MCp24 together with cytokine cocktail or to MCp24pIC expanded autologous p24-specific CD4(+) and CD8(+) T-cell responses as measured by interferon-gamma (IFN-gamma) and IL-2 cytokine production and secretion. In vivo relevance was shown by immunizing C57BL/6 mice with MCp24pIC, which induced both humoral and cellular p24-specific immune responses. Together these data provide a proof of principle that both antigen and DC maturation signal can be delivered as a complex with polyelectrolyte capsules to stimulate virus-specific T cells both in vitro and in vivo. Polyelectrolyte MCs could be useful for in vivo immunization in HIV-1 and other infections.

Genetic Survey of Autosomal Recessive Peripheral Neuropathy Cases Unravels High Genetic Heterogeneity in a Turkish Cohort.

BACKGROUND AND OBJECTIVES: Inherited peripheral neuropathies (IPNs) are a group of genetic disorders of the peripheral nervous system in which neuropathy is the only or the most predominant clinical feature. The most common type of IPN is Charcot-Marie-Tooth (CMT) disease. Autosomal recessive CMT (ARCMT) is generally more severe than dominant CMT and its genetic basis is poorly understood due to high clinical and genetic diversity. Here, we report clinical and genetic findings from 56 consanguineous Turkish families initially diagnosed with CMT disease. METHODS: We initially screened the GDAP1 gene in our cohort as it is the most commonly mutated ARCMT gene. Next, whole-exome sequencing and homozygosity mapping based on whole-exome sequencing (HOMWES) analysis was performed. To understand the molecular impact of candidate causative genes, functional analyses were performed in patient primary fibroblasts. RESULTS: Biallelic recurrent mutations in the GDAP1 gene have been identified in 6 patients. Whole-exome sequencing and HOMWES analysis revealed 16 recurrent and 13 novel disease-causing alleles in known IPN-related genes and 2 novel candidate genes: 1 for a CMT-like disease and 1 for autosomal recessive cerebellar ataxia with axonal neuropathy. We have achieved a potential genetic diagnosis rate of 62.5% (35/56 families) in our cohort. Considering only the variants that meet the American College for Medical Genetics and Genomics (ACMG) classification as pathogenic or likely pathogenic, the definitive diagnosis rate was 55.35% (31/56 families). DISCUSSION: This study paints a genetic landscape of the Turkish ARCMT population and reports additional candidate genes that might help enlighten the mechanism of pathogenesis of the disease.

Efficient in vitro expansion of human immunodeficiency virus (HIV)-specific T-cell responses by gag mRNA-electroporated dendritic cells from treated and untreated HIV type 1-infected individuals.

Developing an immunotherapy to keep human immunodeficiency virus type 1 (HIV-1) replication suppressed while discontinuing highly active antiretroviral therapy (HAART) is an important challenge. In the present work, we evaluated in vitro whether dendritic cells (DC) electroporated with gag mRNA can induce HIV-specific responses in T cells from chronically infected subjects. Monocyte-derived DC, from therapy-naïve and HAART-treated HIV-1-seropositive subjects, that were electroporated with consensus codon-optimized HxB2 gag mRNA efficiently expanded T cells, secreting gamma interferon (IFN-gamma) and interleukin 2 (IL-2), as well as other cytokines and perforin, upon restimulation with a pool of overlapping Gag peptides. The functional expansion levels after 1 week of stimulation were comparable in T cells from HAART-treated and treatment-naïve patients and involved both CD4(+) and CD8(+) T cells, with evidence of bifunctionality in T cells. Epitope mapping of p24 showed that stimulated T cells had a broadened response toward previously nondescribed epitopes. DC, from HAART-treated subjects, that were electroporated with autologous proviral gag mRNA equally efficiently expanded HIV-specific T cells. Regulatory T cells did not prevent the induction of effector T cells in this system, whereas the blocking of PD-L1 slightly increased the induction of T-cell responses. This paper shows that DC, loaded with consensus or autologous gag mRNA, expand HIV-specific T-cell responses in vitro.

Sphingosine 1-phosphate lyase deficiency causes Charcot-Marie-Tooth neuropathy.

OBJECTIVE: To identify the unknown genetic cause in a nuclear family with an axonal form of peripheral neuropathy and atypical disease course. METHODS: Detailed neurologic, electrophysiologic, and neuropathologic examinations of the patients were performed. Whole exome sequencing of both affected individuals was done. The effect of the identified sequence variations was investigated at cDNA and protein level in patient-derived lymphoblasts. The plasma sphingoid base profile was analyzed. Functional consequences of neuron-specific downregulation of the gene were studied in Drosophila. RESULTS: Both patients present an atypical form of axonal peripheral neuropathy, characterized by acute or subacute onset and episodes of recurrent mononeuropathy. We identified compound heterozygous mutations cosegregating with disease and absent in controls in the SGPL1 gene, encoding sphingosine 1-phosphate lyase (SPL). The p.Ser361* mutation triggers nonsense-mediated mRNA decay. The missense p.Ile184Thr mutation causes partial protein degradation. The plasma levels of sphingosine 1-phosphate and sphingosine/sphinganine ratio were increased in the patients. Neuron-specific downregulation of the Drosophila orthologue impaired the morphology of the neuromuscular junction and caused progressive degeneration of the chemosensory neurons innervating the wing margin bristles. CONCLUSIONS: We suggest SPL deficiency as a cause of a distinct form of Charcot-Marie-Tooth disease in humans, thus extending the currently recognized clinical and genetic spectrum of inherited peripheral neuropathies. Our data emphasize the importance of sphingolipid metabolism for neuronal function.

Immune and viral correlates of "secondary viral control" after treatment interruption in chronically HIV-1 infected patients.

Upon interruption of antiretroviral therapy, HIV-infected patients usually show viral load rebound to pre-treatment levels. Four patients, hereafter referred to as secondary controllers (SC), were identified who initiated therapy during chronic infection and, after stopping treatment, could control virus replication at undetectable levels for more than six months. In the present study we set out to unravel possible viral and immune parameters or mechanisms of this phenomenon by comparing secondary controllers with elite controllers and non-controllers, including patients under HAART. As candidate correlates of protection, virus growth kinetics, levels of intracellular viral markers, several aspects of HIV-specific CD4+ and CD8+ T cell function and HIV neutralizing antibodies were investigated. As expected all intracellular viral markers were lower in aviremic as compared to viremic subjects, but in addition both elite and secondary controllers had lower levels of viral unspliced RNA in PBMC as compared to patients on HAART. Ex vivo cultivation of the virus from CD4+ T cells of SC consistently failed in one patient and showed delayed kinetics in the three others. Formal in vitro replication studies of these three viruses showed low to absent growth in two cases and a virus with normal fitness in the third case. T cell responses toward HIV peptides, evaluated in IFN-γ ELISPOT, revealed no significant differences in breadth, magnitude or avidity between SC and all other patient groups. Neither was there a difference in polyfunctionality of CD4+ or CD8+ T cells, as evaluated with intracellular cytokine staining. However, secondary and elite controllers showed higher proliferative responses to Gag and Pol peptides. SC also showed the highest level of autologous neutralizing antibodies. These data suggest that higher T cell proliferative responses and lower replication kinetics might be instrumental in secondary viral control in the absence of treatment.

Brownian dynamics of interactions between aldolase mutants and F-actin.

Previous Brownian dynamics (BD) simulations (Ouporov IG, Knull HR and Thomasson KA 1999. Biophys. J. 76: 17-27) of complex formation between rabbit aldolase and F-actin have identified three lysine residues (K288, K293 and K341) on aldolase and acidic residues (DEDE) at the N-terminus of actin as important to binding. BD simulations of computer models of aldolase mutants with any of these lysine residues replaced by alanine show reduced binding energy; the greatest effect of a single substitution is for K341A, and replacement of all three lysines greatly reduces binding. BD simulations of wild-type rabbit aldolase vs altered F-actin show that binding is decreased if any one of the four N-terminal acidic residues is replaced by alanine and binding is greatly reduced if three or more of the N-terminal acidic residues are replaced; none of the four actin residues appear more critical for binding than the others.