Respiratory and sleep outcomes in children with SMA treated with nusinersen - real world experience.

It is unclear how improvements in peripheral motor function in children with spinal muscular atrophy (SMA), treated with nusinersen, translate into clinically significant respiratory/sleep outcomes. A retrospective chart review of SMA children at the Sydney Children's Hospital Network was undertaken looking at 2 years before and after receiving their first dose of nusinersen. Polysomnography (PSG), spirometry and clinical data were collected and analysed using paired and unpaired t-tests for PSG parameters and generalised estimating equations for longitudinal lung function data. Forty-eight children (10 Type 1, 23 Type 2, 15 Type 3) at mean age 6.98 yrs (SD 5.25) for nusinersen initiation were included. There was a statistically significant improvement in oxygen nadir during sleep in individuals post nusinersen (mean of 87.9% to 92.3% (95%CI 1.24 - 7.63, p = 0.01)). Based on clinical and PSG findings, 6/21 patients (5 Type 2, 1 Type 3) ceased nocturnal NIV post nusinersen. Non-significant improvements were demonstrated in mean slope for FVC% predicted, FVC Z-score and mean FVC% predicted. Within 2 years of commencing nusinersen, stabilisation of respiratory outcomes occurred. Whilst some of the SMA type 2/3 cohort ceased NIV, there were no statistically significant improvements lung function and most PSG parameters.

Changes in long term peripheral nerve biophysical properties in childhood cancer survivors following neurotoxic chemotherapy.

OBJECTIVE: In the context of increasing numbers of childhood cancer survivors (CCS), this study aimed to enhance understanding of the biophysical basis for long term chemotherapy induced peripheral neuropathy from different chemotherapy agents in CCS. METHODS: Detailed cross-sectional neurophysiological examination, using median nerve axonal excitability studies, alongside clinical assessments, in 103 long term CCS (10.5 ± 0.6 years post-treatment). RESULTS: Cisplatin treated CCS (n = 16) demonstrated multiple sensory axonal excitability changes including increased threshold (P < 0.05), alterations in depolarising and hyperpolarising threshold electrotonus (P < 0.05) and reduction in resting and minimum IV slope (P < 0.01). Vincristine treated CCS (n = 73) were comparable to controls, except for prolonged distal motor latency (P = 0.001). No differences were seen in the non-neurotoxic chemotherapy group (n = 14). Abnormalities were more evident in the cisplatin subgroup with greater clinical neuropathy manifestations. CONCLUSION: Persistent long term changes in axonal biophysical properties vary with different chemotherapy agents, most evident after cisplatin exposure. Longitudinal studies of nerve function during chemotherapy treatment are required to further evaluate these differences and their mechanistic basis. SIGNIFICANCE: This study provides a unique biophysical perspective for persistent cisplatin related neurotoxicity in children, previously under recognised.

MSTO1 mutations cause mtDNA depletion, manifesting as muscular dystrophy with cerebellar involvement.

MSTO1 encodes a cytosolic mitochondrial fusion protein, misato homolog 1 or MSTO1. While the full genotype-phenotype spectrum remains to be explored, pathogenic variants in MSTO1 have recently been reported in a small number of patients presenting with a phenotype of cerebellar ataxia, congenital muscle involvement with histologic findings ranging from myopathic to dystrophic and pigmentary retinopathy. The proposed underlying pathogenic mechanism of MSTO1-related disease is suggestive of impaired mitochondrial fusion secondary to a loss of function of MSTO1. Disorders of mitochondrial fusion and fission have been shown to also lead to mitochondrial DNA (mtDNA) depletion, linking them to the mtDNA depletion syndromes, a clinically and genetically diverse class of mitochondrial diseases characterized by a reduction of cellular mtDNA content. However, the consequences of pathogenic variants in MSTO1 on mtDNA maintenance remain poorly understood. We present extensive phenotypic and genetic data from 12 independent families, including 15 new patients harbouring a broad array of bi-allelic MSTO1 pathogenic variants, and we provide functional characterization from seven MSTO1-related disease patient fibroblasts. Bi-allelic loss-of-function variants in MSTO1 manifest clinically with a remarkably consistent phenotype of childhood-onset muscular dystrophy, corticospinal tract dysfunction and early-onset non-progressive cerebellar atrophy. MSTO1 protein was not detectable in the cultured fibroblasts of all seven patients evaluated, suggesting that pathogenic variants result in a loss of protein expression and/or affect protein stability. Consistent with impaired mitochondrial fusion, mitochondrial networks in fibroblasts were found to be fragmented. Furthermore, all fibroblasts were found to have depletion of mtDNA ranging from 30 to 70% along with alterations to mtDNA nucleoids. Our data corroborate the role of MSTO1 as a mitochondrial fusion protein and highlight a previously unrecognized link to mtDNA regulation. As impaired mitochondrial fusion is a recognized cause of mtDNA depletion syndromes, this novel link to mtDNA depletion in patient fibroblasts suggests that MSTO1-deficiency should also be considered a mtDNA depletion syndrome. Thus, we provide mechanistic insight into the disease pathogenesis associated with MSTO1 mutations and further define the clinical spectrum and the natural history of MSTO1-related disease.

Sleeping Beauty transposon screen identifies signaling modules that cooperate with STAT5 activation to induce B-cell acute lymphoblastic leukemia.

Signal transducer and activator of transcription 5 (STAT5) activation occurs frequently in human progenitor B-cell acute lymphoblastic leukemia (B-ALL). To identify gene alterations that cooperate with STAT5 activation to initiate leukemia, we crossed mice expressing a constitutively active form of STAT5 (Stat5b-CA) with mice in which a mutagenic Sleeping Beauty transposon (T2/Onc) was mobilized only in B cells. Stat5b-CA mice typically do not develop B-ALL (<2% penetrance); in contrast, 89% of Stat5b-CA mice in which the T2/Onc transposon had been mobilized died of B-ALL by 3 months of age. High-throughput sequencing approaches were used to identify genes frequently targeted by the T2/Onc transposon; these included Sos1 (74%), Kdm2a (35%), Jak1 (26%), Bmi1 (19%), Prdm14 or Ncoa2 (13%), Cdkn2a (10%), Ikzf1 (8%), Caap1 (6%) and Klf3 (6%). Collectively, these mutations target three major cellular processes: (i) the Janus kinase/STAT5 pathway (ii) progenitor B-cell differentiation and (iii) the CDKN2A tumor-suppressor pathway. Transposon insertions typically resulted in altered expression of these genes, as well as downstream pathways including STAT5, extracellular signal-regulated kinase (Erk) and p38. Importantly, expression of Sos1 and Kdm2a, and activation of p38, correlated with survival, further underscoring the role these genes and associated pathways have in B-ALL.

Spinal muscular atrophy: molecular mechanisms.

Spinal muscular atrophy (SMA) is a relatively common autosomal recessive neuromuscular disorder characterised by muscle weakness and atrophy due to degeneration of motor neurons of the spinal cord and cranial motor nuclei. The clinical phenotype incorporates a wide spectrum. No effective treatment is currently available and patients may experience severe physical disability which is often life limiting. The most common type of SMA is caused by homozygous disruption of the survival motor neuron 1 (SMN1) gene by deletion, conversion or mutation and results in insufficient levels of survival motor neuron (SMN) protein in motor neurons. While diagnosis is usually achieved by genetic testing, an illustrative clinical case is described that highlights the molecular and diagnostic complexities. While there is an emerging picture concerning the function of the SMN protein and the molecular pathophysiological mechanisms underpinning the disease, a number of substantial issues remain unresolved. The selective vulnerability of the motor neuron and the site and timing of the primary pathogenesis are not yet determined. Utilising the organisation of the SMN genomic region, recent advances have identified a number of potential therapeutic targets. As such, this review incorporates discussion of the clinical manifestations, molecular genetics, diagnosis, mechanisms of disease pathogenesis and development of novel treatment strategies.

Combined endovascular and open surgery for four-vessel cerebrovascular occlusive disease.

PURPOSE: To report a case of bilateral simultaneous percutaneous transluminal angioplasty and stenting of the intracranial vertebral arteries prior to staged bilateral carotid endarterectomy. CASE REPORT: A 68-year-old man presented with a 3-month history of recurrent, intermittent left-sided weakness and diplopia. Imaging defined bilateral 80% to 99% internal carotid artery stenoses and >90% stenoses of both distal vertebral arteries at the level of the foramen magnum. Bilateral intracranial vertebral artery stenting was performed, followed by staged carotid endarterectomies. No complications occurred, and the patient recovered uneventfully from all 3 procedures. He remains symptom- and event-free 20 months later. CONCLUSIONS: Our initial success in this case indicates a role for percutaneous transluminal angioplasty and stenting as an alternative to open surgery for intracranial vertebral artery stenosis.

Membrane localization of Raf assists engagement of downstream effectors.

We have previously described a small molecule-directed protein dimerization strategy, using coumermycin to juxtapose Raf fusion proteins containing the coumermycin-binding domain GyrB. Oligomerization of cytoplasmically localized Raf-GyrB fusion proteins leads to an increase in the kinase activity of both Raf and its substrate Mek. Surprisingly, more distal targets, such as Erk1 and Erk2, are not activated using this approach. Here we report that coumermycin-induced oligomerization of a membrane-localized Raf-GyrB fusion protein potently activated Erk1 and Erk2, up-regulated Fos protein levels, and induced expression of many immediate-early response genes. Thus, both membrane localization and oligomerization of Raf-GyrB are required to target Raf signals to downstream effectors. The ability to activate the entire Raf signal transduction cascade conditionally, using coumermycin-induced oligomerization, should prove useful for dissecting Raf-mediated effects on gene expression and cellular differentiation.

Regulation of L-selectin-mediated rolling through receptor dimerization.

L-selectin binding activity for its ligand expressed by vascular endothelium is rapidly and transiently increased after leukocyte activation. To identify mechanisms for upregulation and assess how this influences leukocyte/endothelial cell interactions, cell-surface dimers of L-selectin were induced using the coumermycin-GyrB dimerization strategy for cross-linking L-selectin cytoplasmic domains in L-selectin cDNA-transfected lymphoblastoid cells. Coumermycin- induced L-selectin dimerization resulted in an approximately fourfold increase in binding of phosphomanan monoester core complex (PPME), a natural mimic of an L-selectin ligand, comparable to that observed after leukocyte activation. Moreover, L-selectin dimerization significantly increased (by approximately 700%) the number of lymphocytes rolling on vascular endothelium under a broad range of physiological shear stresses, and significantly slowed their rolling velocities. Therefore, L-selectin dimerization may explain the rapid increase in ligand binding activity that occurs after leukocyte activation and may directly influence leukocyte migration to peripheral lymphoid tissues or to sites of inflammation. Inducible oligomerization may also be a common mechanism for rapidly upregulating the adhesive or ligand-binding function of other cell-surface receptors.

Control of B cell development by Ras-mediated activation of Raf.

Cell fate commitment in a variety of lineages requires signals conveyed via p21ras. To examine the role of p21ras in the development of B lymphocytes, we generated transgenic mice expressing a dominant-negative form of Ras in B lymphocyte progenitors, using a novel transcriptional element consisting of the Emu enhancer and the lck proximal promoter. Expression of dominant-negative Ras arrests B cell development at a very early stage, prior to formation of the pre-B cell receptor. Furthermore, an activated form of Raf expressed in the same experimental system could both drive the maturation of normal pro-B cells and rescue development of progenitors expressing dominant-negative Ras. Hence p21ras normally regulates early development of B lymphocytes by a mechanism that involves activation of the serine/threonine kinase Raf.

Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization.

The Raf-1 serine/threonine kinase is a key component of the MAP kinase cascade, regulating both proliferation and commitment to cell fate. Raf activation is stimulated following its translocation to the plasma membrane, a process that ordinarily requires interaction with the membrane-localized GTPase, Ras-GTP. To investigate the mechanisms underlying Raf activation, we have developed a coumermycin-induced chemical dimerization method. We find that dimerization is by itself sufficient, in the absence of any membrane components, both to activate a modified Raf protein and to stimulate the MAP kinase cascade appropriately. As Ras-GTP-induced membrane localization increases the effective intracellular Raf concentration, our results indicate that homotypic oligomerization may ordinarily act to promote Raf activation in vivo.

Myocardial cell damage by fatty acid ethyl esters.

Fatty acid ethyl ester (FAEE), a myocardial metabolite of ethanol, causes mitochondrial dysfunction in vitro in rabbits. We investigated the effect of these esters on rat heart mitochondria in vitro and in vivo. In vitro studies were conducted to investigate the binding of ethyl oleate (FAEE) to mitochondria and their capacity to hydrolyze these FAEE. In vivo effects of ethyl esters were studied by the direct transfer of [3H]oleate into the myocardium. Mitochondria were prepared from the myocardium of injected rats, and the amount of [3H]oleate bound to them was determined. In another in vivo study, 50 microliters of 50 microM cold oleic acid ethyl ester was injected into the rat myocardium and the histopathological changes induced by oleic acid ethyl ester were examined by light microscopy. Our results show that fatty acid ethyl ester can bind to myocardial mitochondria in vitro as well as in vivo and the mitochondria can hydrolyze FAEE to fatty acid, which is a known uncoupler of oxidative phosphorylation. Of the total ethyl [3H] oleate injected, 8 microM [3H]oleate and 1 microM ethyl [3H]oleate was bound to the mitochondria. Significant myocardial cell damage was first observed on day 4 and markedly increased on day 30 after ethyl ester injection, with cells showing gross deformation and enlargement. However, no significant histopathological changes were observed in the myocardial tissue on day 2 after injection. Our results suggest that the FAEE may damage the myocardial cells as well as the mitochondria and may provide a metabolic link between ethanol abuse and myocardial dysfunction.

Immunohistochemical characterization of immune cell composition and cytokine receptor expression in human coronary atherectomy tissue.

BACKGROUND: Prior histologic studies have examined smooth muscle cell, macrophage and thrombus constituents of atherosclerotic coronary atherectomy specimens. Lymphocytes and mononuclear leukocytes are also detectable in atherosclerotic surgical pathology specimens utilizing immunocytochemical techniques. METHODS: In order to quantify the histological contribution of cytokine receptor-expressing immunocompetent cells to human coronary artery stenoses, 30 directional atherectomy catheter biopsy specimens (wet weight < or = 10 mg) from 16 patients were snap frozen (-70 degrees C) for quantitative immunocytochemical studies. Following computer-assisted quantification of total intimal nuclei per tissue section (mean 297 +/- 177; cell density 7 +/- 5/10(4) microns 2), monoclonal antibody cytochemistry was used to identify the percentage of these cells expressing antigenic clusters of differentiation (CD) characteristic of T-lymphocytes, B-lymphocytes and monocytes. Identification of alpha (low affinity) and beta (intermediate affinity) interleukin-2 receptors on intimal cells was accomplished using a three-step streptavidin-biotin method. RESULTS: A significant percentage of intimal cells were of lymphocytic (11 +/- 13%) or monocytic (12 +/- 14%) origin, with helper T-cells (9 +/- 12%) outnumbering both suppressor T-cells (2 +/- 4%) and B-lymphocytes (1 +/- 2%). Interleukin-2 receptors were noted on 9 +/- 12% of intimal cells, including cells with a vascular smooth muscle phenotype. CONCLUSIONS: These quantitative immunocytochemical data conclusively demonstrate that lymphocytes and monocytes account for over 20% of coronary plaque cells obtained by in-vivo atherectomy, and that helper (CD4) T-cells predominate over suppressor (CD8) T-cells and B-lymphocytes. Variable interleukin-2 receptor subtype expression occurs in mononuclear leukocytes infiltrating chronic human atheroma. By applying these techniques, the therapeutic effects of cytotoxic agents on selectively targeted cytokine receptor-expressing cells may now be evaluated in vivo in small human directional coronary atherectomy specimens.

Histomorphometric and biochemical correlates of arterial procollagen gene expression during vascular repair after experimental angioplasty.

BACKGROUND: To determine the transcriptional, biochemical, and histomorphometric correlates of neointimal procollagen accumulation during arterial repair after balloon angioplasty of atherogenic vessels, rabbit iliac artery collagen content and the induction of alpha 1(I) and alpha 1(III) procollagen mRNA were assessed in normal vessels and at 2, 7, and 30 days after angioplasty. METHODS AND RESULTS: Quantitative iliac artery histomorphometric neointimal collagen analysis was performed using a specific picrosirius red stain under polarized light. Arterial cross-sectional area reduction, total cellularity, and vascular smooth muscle cell density (per 10(4) mu2 of neointima) were quantified in routine and immunohistochemically stained sections (alpha-actin and RAM-11), from which biochemical concentrations of tissue protein, RNA, and DNA were also measured. Collagen comprised 0.23 +/- 0.1 mg/mg of total protein in the normal vessel wall and did not increase in vessels studied 2 and 7 days after angioplasty (0.26 +/- 0.06, 0.28 +/- 0.05 mg/mg of protein, P = NS). By 30 days after angioplasty, > 50% of the protein concentration was collagen (0.55 +/- 0.11 mg/mg of protein, P = .02). Collagen-positive histological staining also increased significantly from 17 +/- 2% of the neointima at day 2 to 32 +/- 5% by day 30 (P = .01). The transcript regulatory signal for alpha 1(I) procollagen mRNA was induced 2 days after angioplasty, peaking at 7 days for both alpha 1(I) and alpha 1(III), and returning to control levels 30 days after angioplasty. A significant luminal cross-sectional area reduction of the arterial wall was confirmed both by angiography and histomorphometry (P = .01). This was not associated with a significant change in alpha-actin (+) vascular smooth muscle cell density (38 +/- 7 nuclei per 10(4) mu2 at day 2 and at day 30) or tissue DNA concentration (P = NS). CONCLUSIONS: We conclude that procollagen genes are transcriptionally activated early (2 to 7 days) after angioplasty vessel injury and that collagen subsequently constitutes a major biochemical and histological component of the proliferative neointima by 30 days after angioplasty. Alterations in pathways regulating procollagen metabolism may also contribute to the accumulation of extracellular matrix and growth of the neointima in the late repair phase after vessel wall injury.

Ligand-induced IFN gamma receptor tyrosine phosphorylation couples the receptor to its signal transduction system (p91).

Herein we report that interferon-gamma (IFN gamma) induces the rapid and reversible tyrosine phosphorylation of the IFN gamma receptor. Using a panel of receptor intracellular domain mutants, we show that a membrane-proximal LPKS sequence (residues 266-269) is required for ligand-induced tyrosine kinase activation and/or kinase-receptor association and biological responsiveness, and a functionally critical membrane-distal tyrosine residue (Y440) is a target of the activated enzyme. The biological significance of Y440 phosphorylation was demonstrated by showing that a receptor-derived nonapeptide corresponding to receptor residues 436-444 and containing phosphorylated Y440 bound specifically to p91, blocked p91 phosphorylation and inhibited the generation of an active p91-containing transcription factor complex. In contrast, nonphosphorylated wild-type, phosphorylated mutant, or phosphorylated irrelevant peptides did not. Moreover, the phosphorylated Y440-containing peptide did not interact with a related but distinct latent transcription factor (p113) which is activatible by IFN alpha but not IFN gamma. These results thus document the specific and inducible association of p91 with the phosphorylated IFN gamma receptor and thereby elucidate the mechanism by which ligand couples the IFN gamma receptor to its signal transduction system.

Pulmonary endothelial nitric oxide production is developmentally regulated in the fetus and newborn.

To define the role of endothelial nitric oxide (NO) in developmental changes in pulmonary vascular resistance and oxygen responsiveness, we determined the ontogeny of endothelial NO production and of oxygen modulation of that process in pulmonary arteries from fetal and newborn lambs. NO production was assessed by measuring endothelium-dependent arterial guanosine 3',5'-cyclic monophosphate synthesis. Basal NO rose two-fold from late gestation to 1 wk of age and another 1.6-fold from 1 to 4 wk. Acetylcholine-stimulated NO also increased 1.6-fold from 1 to 4 wk. The maturational rise in NO was evident at high Po2 in vitro, and it was not modified by L-arginine. This suggests that the developmental increase may alternatively involve enhanced calcium-calmodulin-mediated mechanisms, increased expression of NO synthase, or greater availability of required cofactor(s). With an acute decline in Po2 in vitro from 680 to 150 or 40 mmHg, there was 50-88% attenuation of basal and acetylcholine-stimulated NO late in the third trimester and in the newborn but not early in the third trimester. Parallel studies of mesenteric endothelium revealed postnatal increases in basal and stimulated NO but no decline in NO at lower Po2. Ontogenic changes in endothelial NO production and in oxygen modulation of that process may be involved in the maturational decrease in vascular resistance and the development of oxygen responsiveness in the pulmonary circulation.

Oxygen modulation of pulmonary arterial prostacyclin synthesis is developmentally regulated.

To better understand the role of prostacyclin [prostaglandin (PG) I2] in oxygen mediation of vasomotor tone in the developing lung, we determined the ontogeny of the direct effects of acute changes in oxygen on in vitro PGI2 synthesis and adenosine 3',5'-cyclic monophosphate (cAMP) production in intrapulmonary arteries from fetal and newborn lambs. In the absence of varying oxygen, PGI2 synthesis increased 6.9-fold from early to late in the third trimester, and it rose an additional 3.2-fold from late gestation to 1 wk of age, and another 2.1-fold from 1 to 4 wk. PGE2 synthesis similarly rose 4.9-fold during the third trimester, but it then fell 69% from late gestation to 1 wk of age and remained unchanged postnatally. Paralleling the developmental increase in PGI2 synthesis, basal cAMP production rose 6.2-fold from the early third trimester to 4 wk of age. In contrast, PGI2-stimulated cAMP production was similar in all age groups. With an acute decline in PO2 in vitro from 680 to 40 mmHg, PGI2 and PGE2 synthesis in fetal arteries fell 33-46 and 39-55%, respectively. In contrast, they were increased by 9-145% and 44-130%, respectively, at lower PO2 in arteries from newborn lambs. Basal cAMP production was altered by decreased oxygen in a similar manner, falling by 35-39% in fetal arteries yet increasing by 21-47% in the newborn. PGI2-stimulated cAMP production, however, was not affected by oxygen at all ages except in the early third trimester. Thus there is a dramatic developmental increase in pulmonary arterial PGI2 synthesis that causes a marked maturational rise in cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of cellular responsiveness to interferon-gamma (IFN gamma) induced by overexpression of inactive forms of the IFN gamma receptor.

Herein, we report that overexpression of either human or murine interferon-gamma (IFN-gamma) receptors lacking their entire intracellular domains in cells bearing functionally active IFN gamma receptors ablates responsiveness to homologous ligand in a dominant negative manner. Unresponsiveness could also be induced in murine cells overexpressing murine IFN gamma receptor mutants that either lack 39 COOH-terminal amino acids or contain an alanine substitution for a functionally critical tyrosine. Overexpression of the full-length receptor did not alter cellular responsiveness to IFN gamma. The inhibitory activities of the receptor mutants were dose-dependent, generalizable to a variety of cellular responses, and specific. Cells expressing 100:1 ratios of mutant to wild-type receptor were unresponsive to IFN gamma even at doses 30,000 times greater than that required to induce a maximal response in wild-type cells. These results provide an example of a dominant negative mutation that effects the complete inactivation of a transmembrane receptor lacking a kinase domain and suggest a more general utility for dominant negative mutations in the study of cytokine receptor function.

The biology and biochemistry of interferon-gamma and its receptor.

Interferon-gamma (IFN gamma) is an important immunomodulatory cytokine produced by activated T cells and NK cells that plays a pivotal role in promoting host defense. IFN gamma is distinguished from IFN alpha and IFN beta by its ability to regulate a number of immune functions. IFN gamma induces its biologic effects by interacting with a specific IFN gamma receptor expressed at the cell surface. Recently, IFN gamma receptors have been purified from human and murine cells and their cDNAs cloned and expressed. This work has revealed that IFN gamma receptors are 90 kDa, single chain glycoproteins that bind ligand with high affinity in a species specific manner. There appears to be only a single type of IFN gamma receptor that is expressed on nearly all cell types. Whereas this single polypeptide is sufficient to confer ligand binding and processing activity to transfected cells, a second, as yet undefined, component is required to form a functionally active IFN gamma receptor. The identity of this second component is currently being investigated. In addition, recent work has revealed novel structure-function relationships that exist within the IFN gamma receptor's intracellular domain. This work has shown that distinct portions of the intracellular domain are differentially responsible for mediating different biologic activities of the receptor.