Early expression of mature αß TCR in CD4-CD8- T cell progenitors enables MHC to drive development of T-ALL bearing NOTCH mutations.

During normal T cell development in mouse and human, a low-frequency population of immature CD4-CD8- double-negative (DN) thymocytes expresses early, mature αß T cell antigen receptor (TCR). We report that these early αß TCR+ DN (EADN) cells are DN3b-DN4 stage and require CD3δ but not major histocompatibility complex (MHC) for their generation/detection. When MHC - is present, however, EADN cells can respond to it, displaying a degree of coreceptor-independent MHC reactivity not typical of mature, conventional αß T cells. We found these data to be connected with observations that EADN cells were susceptible to T cell acute lymphoblastic leukemia (T-ALL) transformation in both humans and mice. Using the OT-1 TCR transgenic system to model EADN-stage αß TCR expression, we found that EADN leukemogenesis required MHC to induce development of T-ALL bearing NOTCH1 mutations. This leukemia-driving MHC requirement could be lost, however, upon passaging the tumors in vivo, even when matching MHC was continuously present in recipient animals and on the tumor cells themselves. These data demonstrate that MHC:TCR signaling can be required to initiate a cancer phenotype from an understudied developmental state that appears to be represented in the mouse and human disease spectrum.

Non-syndromic single-suture craniosynostosis in triplets.

INTRODUCTION: Craniosynostosis is the premature fusion of one or more cranial sutures. The cause of non-syndromic craniosynostosis has been attributed to a complex interaction among genetic, epigenetic, and environmental factors. Increased concordance rates in monozygotic twins support a genetic etiology while a concordance rate less than 100% suggests environmental and/or epigenetic influences. Here, we describe the first reported occurrence of all three children in a triplet set with non-syndromic single-suture craniosynostosis. CASE REPORT: The dichorionic triamniotic triplets were the product of a non-consanguineous marriage delivered at 35 weeks' gestation by a 38-year-old mother and consisted of a monochorionic-diamniotic pair (A and B) and a fraternal triplet (C). Three-dimensional computed tomography scans confirmed sagittal synostosis in A and B and metopic synostosis in C. All patients underwent endoscopic strip craniectomy and were discharged on the second postoperative day with helmet orthoses. Comparative genetic hybridization (CGH) and whole-exome sequencing (WES) failed to identify pathogenic copy number variants or gene mutations, respectively. DISCUSSION AND CONCLUSION: The results of the genetic testing suggest the possibility of a rare variant contributing to the risk of midline craniosynostosis shared among the triplets, with potential modifiers at other genetic loci affecting the phenotype. We speculate mutations at loci within non-coding regions not captured by our genetic analysis may have been involved. Moreover, epigenetic factors as well as environmental factors including, but not limited to, in utero head constraint could have contributed to the observed phenotype.

Atypical Presentation of a Pediatric Cerebellar Ganglioglioma.

BACKGROUND/AIMS: Gangliogliomas (GGs) are rare central nervous system tumors occurring primarily in the supratentorial compartment with infratentorial instances most often involving the brain stem. Infratentorial GGs typically present with signs and symptoms of increased intracranial pressure (ICP), cranial nerve deficits, or focal cerebellar findings; rarely, these tumors have been associated with focal seizures. METHODS: In this report, we describe an atypical presentation of a cerebellar GG in a 20-month-old male who initially presented with syncope and emesis in the absence of electrographic evidence of seizures, radiographic evidence of hydrocephalus, or elevated ICP. The epidemiology, radiographic, and pathological findings as well as the treatment of these tumors are also discussed. RESULTS: After gross total resection, the patient experienced full resolution of all his preoperative symptoms without the development of new neurological deficits. CONCLUSIONS: Unlike their supratentorial counterparts, infratentorial GGs do not commonly present with seizures although rare reports exist in the literature of seizures attributed to cerebellar GG. Moreover, cerebellar GGs may produce nonspecific symptoms in the absence of concrete diagnostic findings. Such a presentation should prompt further neurological evaluation. Most cases of isolated cerebellar GG can be successfully treated with surgical resection and carry a favorable prognosis.

Heterozygosity for transmembrane activator and calcium modulator ligand interactor A144E causes haploinsufficiency and pneumococcal susceptibility in mice.

BACKGROUND: The B-cell receptor transmembrane activator and calcium modulator ligand interactor (TACI) is important for T-independent antibody responses. One in 200 blood donors are heterozygous for the TACI A181E mutation. OBJECTIVE: We sought to investigate the effect on B-cell function of TACI A181E heterozygosity in reportedly healthy subjects and of the corresponding TACI A144E mutation in mice. METHODS: Nuclear factor κB (NF-κB) activation was measured by using the luciferase assay in 293T cells cotransfected with wild-type and mutant TACI. TACI-driven proliferation, isotype switching, and antibody responses were measured in B cells from heterozygous TACI A144E knock-in mice. Mouse mortality was monitored after intranasal pneumococcal challenge. RESULTS: Levels of natural antibodies to the pneumococcal polysaccharide component phosphocholine were significantly lower in A181E-heterozygous than TACI-sufficient Swedish blood donors never immunized with pneumococcal antigens. Although overexpressed hTACI A181E and mTACI A144E acted as dominant-negative mutations in transfectants, homozygosity for A144E in mice resulted in absent TACI expression in B cells, indicating that the mutant protein is unstable when naturally expressed. A144E heterozygous mice, such as TACI+/- mice, expressed half the normal level of TACI on their B cells and exhibited similar defects in a proliferation-inducing ligand-driven B-cell activation, antibody responses to TNP-Ficoll, production of natural antibodies to phosphocholine, and survival after intranasal pneumococcal challenge. CONCLUSION: These results suggest that TACI A181E heterozygosity results in TACI haploinsufficiency with increased susceptibility to pneumococcal infection. This has important implications for asymptomatic TACI A181E carriers.

Tail-anchored Protein Insertion in Mammals: FUNCTION AND RECIPROCAL INTERACTIONS OF THE TWO SUBUNITS OF THE TRC40 RECEPTOR.

The GET (guided entry of tail-anchored proteins)/TRC (transmembrane recognition complex) pathway for tail-anchored protein targeting to the endoplasmic reticulum (ER) has been characterized in detail in yeast and is thought to function similarly in mammals, where the orthologue of the central ATPase, Get3, is known as TRC40 or Asna1. Get3/TRC40 function requires an ER receptor, which in yeast consists of the Get1/Get2 heterotetramer and in mammals of the WRB protein (tryptophan-rich basic protein), homologous to yeast Get1, in combination with CAML (calcium-modulating cyclophilin ligand), which is not homologous to Get2. To better characterize the mammalian receptor, we investigated the role of endogenous WRB and CAML in tail-anchored protein insertion as well as their association, concentration, and stoichiometry in rat liver microsomes and cultured cells. Functional proteoliposomes, reconstituted from a microsomal detergent extract, lost their activity when made with an extract depleted of TRC40-associated proteins or of CAML itself, whereas in vitro synthesized CAML and WRB together were sufficient to confer insertion competence to liposomes. CAML was found to be in ∼5-fold excess over WRB, and alteration of this ratio did not inhibit insertion. Depletion of each subunit affected the levels of the other one; in the case of CAML silencing, this effect was attributable to destabilization of the WRB transcript and not of WRB protein itself. These results reveal unanticipated complexity in the mutual regulation of the TRC40 receptor subunits and raise the question as to the role of the excess CAML in the mammalian ER.

Calcium-Modulating Cyclophilin Ligand Is Essential for the Survival of Activated T Cells and for Adaptive Immunity.

Calcium-modulating cyclophilin ligand (CAML) is an endoplasmic reticulum resident protein that is widely expressed. Although it has been demonstrated to participate in the tail-anchored protein insertion pathway, its physiological role in the mature immune system is unknown. In this work, we show that mature, peripheral T cells require CAML for survival specifically following TCR-induced activation. In this study, we examined mature T cells from spleen and lymph nodes of tamoxifen-inducible CAML knockout mice (tCAML(-/-)). Whereas CAML-deficient T cells were able to express the early activation markers CD25 and CD69, and produce IL-2 normally upon stimulation, deficient cells proliferated less and died. Cells did not require CAML for entry into the S phase of the cell cycle, thus implicating its survival function at a relatively late step in the T cell activation sequence. In addition, CAML was required for homeostatic proliferation and for Ag-dependent cell killing in vivo. These results demonstrate that CAML critically supports T cell survival and cell division downstream of T cell activation.

Impaired B cell receptor signaling is responsible for reduced TACI expression and function in X-linked immunodeficient mice.

Immune response to T cell independent type 2 (TI-2) Ags, such as bacterial polysaccharides, is severely impaired in X-linked immunodeficient (XID) mice. In this study, we investigated the involvement of a proliferation-inducing ligand (APRIL) or BAFF and their receptors in the unresponsiveness of XID mouse to TI-2 Ags. We discovered that whereas serum BAFF levels were increased, the expression of the APRIL and BAFF receptor transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) was severely reduced in XID B cells. Moreover, B cells from XID mouse were unable to secrete Igs in response to APRIL or BAFF. In correlation with reduced TACI expression and impaired TACI function, APRIL or BAFF did not activate the classical NF-κB pathway in XID cells. Also correlating with the unaltered expression of BAFF receptor, BAFF stimulation induced the activation of the alternative NF-κB pathway in XID cells. Moreover, activation of MAPK pathway was ablated in APRIL-stimulated XID cells. Prestimulation of XID B cells with the TLR9 agonist, CpG led to a significant increase in TACI expression and restored TACI-mediated functions. CpG prestimulation also restored TACI-mediated signaling in APRIL- or BAFF-stimulated XID B cells. Finally, immunization of XID mouse with the prototype TI-2 Ag NP-Ficoll induced IgG and IgM Abs when CpG was given with NP-Ficoll. Collectively, these results suggest that reduced TACI expression is responsible for the unresponsiveness of XID mouse to TI-2 Ags and BCR activation controls TACI expression.

Efficient B cell responses to Borrelia hermsii infection depend on BAFF and BAFFR but not TACI.

T cell-independent antibody responses develop rapidly, within 3 to 4 days, and are critical for preventing blood-borne pathogens from evolving into life-threatening infections. The interaction of BAFF, also known as BLyS, with its receptors BAFFR and TACI on B cells is critical for B cell homeostasis and function. Using a synthetic polysaccharide antigen, it has previously been shown that TACI is critical for T cell-independent antibody responses. To examine the role of BAFFR and TACI in T cell-independent antibody responses to an active infection, we utilized the Borrelia hermsii infection system. In this infection system, T cell-independent responses mediated by the B1b cell subset are critical for controlling bacteremia. We found that B1b cells express BAFFR and TACI and that the surface expression of both receptors is upregulated on B1b cells following exposure to whole B. hermsii cells. Surprisingly, we found that TACI(-/-) mice are not impaired either in specific antibody responses to B. hermsii or in controlling B. hermsii bacteremia. In contrast, TACI-deficient mice immunized with heat-killed type 3 serotype pneumococcus cells are impaired in generating pneumococcal polysaccharide-specific responses and succumb to challenge with live type 3 serotype pneumococcus, indicating that TACI is required for T cell-independent antibody responses to bacterial-associated polysaccharides. Although we have found that TACI is dispensable for controlling B. hermsii infection, mice deficient in BAFFR or BAFF exhibit impairment in B. hermsii-specific IgM responses and clearance of bacteremia. Collectively, these data indicate a disparity in the roles for TACI and BAFFR in primary T cell-independent antibody responses to bacterial pathogens.

The anticancer drug Dp44mT inhibits T-cell activation and CD25 through a copper-dependent mechanism.

The di-2-pyridylketone thiosemicarbazone Dp44mT is a metal-chelating compound that has been demonstrated to have potent activity as an anticancer agent. Here we report that it also has a dramatic inhibitory effect on T-cell activation in vitro. We found that 10 nM Dp44mT (IC(50) 3.2 nM) prevented the up-regulation of surface CD25, and completely suppressed the activation and proliferation of splenic T cells isolated from Mus musculus that were stimulated with either T-cell receptor (TCR) cross-linking antibodies or phorbol ester plus ionomycin. In contrast, Dp44mT had no adverse effects on the survival of resting T cells. In addition, T cells stimulated in the presence of Dp44mT maintained the ability to up-regulate CD69 surface expression and secrete interleukin-2. Consistent with these observations, Dp44mT did not inhibit multiple canonical signals downstream of the TCR, including the nuclear factor of activated T cells. The effects of Dp44mT were easily mitigated by addition of nontoxic copper chelators or N-acetylcysteine, indicating a role for copper and reactive oxygen species in its actions. Together, these findings suggest that Dp44mT may serve as a potent immunosuppressive agent that could complicate its use as a cancer therapeutic agent, but might have utility in the treatment of autoimmunity.

Essential role for CAML in follicular B cell survival and homeostasis.

Calcium-modulating cyclophilin ligand (CAML) is a ubiquitously expressed protein that is important during thymopoiesis. However, whether it serves a function in mature lymphocytes is unknown. In this article, we show that CAML is essential for survival of peripheral follicular (Fo) B cells. Conditional deletion of CAML in CD19-Cre transgenic mice caused a significant reduction in Fo cell numbers and increased rates of homeostatic proliferation. CAML-deficient Fo cells showed increased cellular turnover and normal proliferative ability. Although CAML-deficient Fo cells responded to AgR stimulation and to B cell activating factor, they displayed decreased survival and increased apoptosis following stimulation with LPS and IL-4 in vitro. Failure to survive was not due to aberrant B cell development in the absence of CAML, because induced deletion of the gene in mature cells resulted in a similar phenotype. These data establish an essential and ongoing role for CAML in the long-term survival of mature B cells.

Cutting edge: regulation of TLR4-driven B cell proliferation by RP105 is not B cell autonomous.

Mechanistic understanding of RP105 has been confounded by the fact that this TLR homolog has appeared to have opposing, cell type-specific effects on TLR4 signaling. Although RP105 inhibits TLR4-driven signaling in cell lines and myeloid cells, impaired LPS-driven proliferation by B cells from RP105(-/-) mice has suggested that RP105 facilitates TLR4 signaling in B cells. In this article, we show that modulation of B cell proliferation by RP105 is not a function of B cell-intrinsic expression of RP105, and identify a mechanistic role for dysregulated BAFF expression in the proliferative abnormalities of B cells from RP105(-/-) mice: serum BAFF levels are elevated in RP105(-/-) mice, and partial BAFF neutralization rescues aberrant B cell proliferative responses in such mice. These data indicate that RP105 does not have dichotomous effects on TLR4 signaling and emphasize the need for caution in interpreting the results of global genetic deletion.

Vertebral Artery to Middle Cerebral Artery Bypass for Flow Augmentation.

BACKGROUND AND IMPORTANCE: Extracranial-intracranial bypass remains an enduring procedure for a select group of patients suffering from steno-occlusive cerebrovascular disease. Although the superficial temporal artery (STA) to middle cerebral artery (MCA) bypass is most familiar among neurosurgeons, particular circumstances preclude the use of an STA donor. In such cases, alternative revascularization strategies must be pursued. CLINICAL PRESENTATION: A 63-year-old female presented with symptoms of hemodynamic insufficiency and was found to have left common carotid artery occlusion at the origin. She experienced progressive watershed ischemia and pressure-dependent fluctuations in her neurological examination despite maximum medical therapy. The ipsilateral STA was unsuitable for use as a donor vessel. We performed an extracranial vertebral artery (VA) to MCA bypass with a radial artery interposition graft. CONCLUSION: This technical case description and accompanying surgical video review the relevant anatomy and surgical technique for a VA-MCA bypass. The patient was ultimately discharged home at her preoperative neurological baseline with patency of the bypass. The VA can serve as a useful donor vessel for cerebral revascularization procedures in pathologies ranging from malignancies of the head and neck to cerebral aneurysms and cerebrovascular steno-occlusive disease.

Cyclosporin A impairs the secretion and activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat).

The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knock-out mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients.

TACI deficiency impairs sustained Blimp-1 expression in B cells decreasing long-lived plasma cells in the bone marrow.

Deficiencies in transmembrane activator and CAML interactor (TACI) result in common variable immune deficiency, a syndrome marked by recurrent infections with encapsulated microorganisms, impaired production of antibodies, and lymphoproliferation. How TACI promotes antibody production and inhibits lymphoproliferation is not understood. To answer this question, we studied the generation of immunity to protein antigens in both TACI-deficient and TACI-proficient mice. We show that TACI promotes sustained Blimp-1 expression by B cells responding to antigen, which in turn limits B-cell clonal expansion and facilitates differentiation of long-lived antibody-secreting cells. Short-term IgG secretion occurs independently of TACI as DNA double-strand breaks associated with isotype class switching induce Blimp-1 transiently, independently of TACI. Our results showing that TACI induces and maintains Blimp-1 provide, for the first time, a unified molecular and cellular mechanism explaining the primary features of common variable immune deficiency, exquisite vulnerability to infection with encapsulated organisms, lymphoproliferation, and hypogammaglobulinemia.

Transradial flow diversion with an aberrant right subclavian artery.

Transradial access for diagnostic and therapeutic neurointerventional procedures has gained popularity due to a decreased incidence of access site complications and improved patient comfort compared with transfemoral access.1-4 An aberrant right subclavian artery is an aortic arch variant characterized by a right subclavian artery that arises directly from the arch as the most distal great vessel. Transradial access with an aberrant right subclavian artery is anatomically challenging due to the predilection of the catheter system to collapse into the descending aorta. In this (video 1), we describe a step-by-step technique for transradial access in a patient with an aberrant right subclavian artery undergoing endovascular flow diversion for a left superior hypophyseal artery aneurysm. Particular emphasis is placed on the technique for accessing the proximal arch and aortic valve as well as distal catheter navigation while avoiding prolapse into the descending aorta. neurintsurg;15/11/1164/V1F1V1Video 1 .

A persistent trigeminal artery demonstrates cerebrovascular embryologic development.

Background: Cerebrovascular embryologic development is characterized by the presence of four well-described carotid-vertebrobasilar (VB) anastomoses. As the fetal hindbrain matures and the VB system develops, these connections involute, yet some may persist into adulthood. The persistent primitive trigeminal artery (PPTA) is the most common of these anastomoses. In this report, we describe a unique variant of the PPTA and a four-way division of the VB circulation. Case Description: A female in her 70s presented with a Fisher Grade 4 subarachnoid hemorrhage. Catheter angiography revealed a fetal origin of the left posterior cerebral artery (PCA) giving rise to a left P2 aneurysm which was coiled. A PPTA arose from the left internal carotid artery and supplied the distal basilar artery (BA) including the superior cerebellar arteries bilaterally and the right but not left PCA. The mid-BA was atretic and the anterior inferior cerebellar artery-posterior inferior cerebellar artery complexes were fed solely from the right vertebral artery. Conclusion: Our patient's cerebrovascular anatomy represents a unique variant of the PPTA not well described in the literature. This demonstrates how hemodynamic capture of the distal VB territory by a PPTA is sufficient to prevent fusion of the BA.

Hemodynamic outcomes of stenting for vertebrobasilar insufficiency in patients with a low flow state.

INTRODUCTION: The Vertebrobasilar Flow Evaluation and Risk of Transient Ischemic Attack and Stroke (VERiTAS) study determined patients with low flow in their vertebrobasilar (VB) system are at increased risk of recurrent stroke. Endovascular interventions such as angioplasty and stenting are reserved for patients with refractory symptoms; however, few series to date have demonstrated either hemodynamic or clinical outcomes in this high-risk patient group. We present our combined institutional series of patients with symptomatic VB atherosclerotic disease and low-flow state who underwent angioplasty and stenting. METHODS: Retrospective chart review of patients undergoing angioplasty and stenting for symptomatic VB atherosclerotic disease at two institutions was performed. Clinical and radiographical outcomes were collected including flow rates using quantitative MRA (QMRA) pre- and post-stenting. RESULTS: Seventeen patients underwent angioplasty and stenting for symptomatic VB atherosclerotic disease and met VERiTAS low-flow state criteria. There were four cases (23.5%) of periprocedural stroke, two of which were minor and transient. The stent was placed intracranially in 82.4% of patients. Basilar and bilateral posterior cerebral artery (PCA) flows significantly improved post-stenting (p < 0.05) and normalized based upon VERiTAS criteria in all patients. Fourteen patients had delayed QMRA at mean follow-up 20 months demonstrating appropriate patency and flow post-stenting. Two patients (10%) had recurrent stroke, one from medication nonadherence and in-stent thrombosis, and the other from a procedural dissection that subsequently became symptomatic. CONCLUSIONS: Our series demonstrates angioplasty and stenting significantly improve intracranial flow over long-term. Angioplasty and stenting may improve the natural history of low-flow VB atherosclerotic disease.

Conditional deletion of calcium-modulating cyclophilin ligand causes deafness in mice.

Calcium-modulating cyclophilin ligand (Caml) is a ubiquitously expressed cytoplasmic protein that is involved in multiple signaling and developmental pathways. An observation in our laboratory of a protein-protein interaction between Caml and the cytoplasmic region of Cadherin23 led us to speculate that Caml might be important in the inner ear and play a role in the development and/or function of hair cells. To address this question, we generated a mouse line in which Caml expression was eliminated in Atoh1-expressing cells of the inner ear upon administration of tamoxifen. Tamoxifen was administered immediately after birth to neonates to assess the effect of loss of Caml in the inner ear during postnatal development. Hearing in treated animals was tested by auditory brain stem response (ABR) analysis and cochlear pathology was evaluated by light microscopy. Lack of Caml expression in the inner ear leads to severe loss of cochlear hair cells and complete deafness. Elucidating the role of Caml in the inner ear will aid our understanding of the molecular pathways important for auditory development and function.