In vivo selection of anti-HIV-1 gene-modified human hematopoietic stem/progenitor cells to enhance engraftment and HIV-1 inhibition.

Hematopoietic stem/progenitor cell (HSPC)-based anti-HIV-1 gene therapy holds great promise to eradicate HIV-1 or to provide long-term remission through a continuous supply of anti-HIV-1 gene-modified cells without ongoing antiretroviral therapy. However, achieving sufficient engraftment levels of anti-HIV gene-modified HSPC to provide therapeutic efficacy has been a major limitation. Here, we report an in vivo selection strategy for anti-HIV-1 gene-modified HSPC by introducing 6-thioguanine (6TG) chemoresistance through knocking down hypoxanthine-guanine phosphoribosyl transferase (HPRT) expression using RNA interference (RNAi). We developed a lentiviral vector capable of co-expressing short hairpin RNA (shRNA) against HPRT alongside two anti-HIV-1 genes: shRNA targeting HIV-1 co-receptor CCR5 and a membrane-anchored HIV-1 fusion inhibitor, C46, for efficient in vivo selection of anti-HIV-1 gene-modified human HSPC. 6TG-mediated preconditioning and in vivo selection significantly enhanced engraftment of HPRT-knockdown anti-HIV-1 gene-modified cells (>2-fold, p < 0.0001) in humanized bone marrow/liver/thymus (huBLT) mice. Viral load was significantly reduced (>1 log fold, p < 0.001) in 6TG-treated HIV-1-infected huBLT mice compared to 6TG-untreated mice. We demonstrated that 6TG-mediated preconditioning and in vivo selection considerably improved engraftment of HPRT-knockdown anti-HIV-1 gene-modified HSPC and repopulation of anti-HIV-1 gene-modified hematopoietic cells in huBLT mice, allowing for efficient HIV-1 inhibition.

Covalent Targeting of Glutamate Cysteine Ligase to Inhibit Glutathione Synthesis.

Dysregulated oxidative stress plays a major role in cancer pathogenesis and some types of cancer cells are particularly vulnerable to inhibition of their cellular antioxidant capacity. Glutamate-cysteine ligase (GCL) is the first and rate-limiting step in the synthesis of the major cellular antioxidant glutathione (GSH). Developing a GCL inhibitor may be an attractive therapeutic strategy for certain cancer types that are particularly sensitive to oxidative stress. In this study, we reveal a cysteine-reactive ligand, EN25, that covalently targets an allosteric cysteine C114 on GCLM, the modifier subunit of GCL, and leads to inhibition of GCL activity. This interaction also leads to reduced cellular GSH levels and impaired cell viability in ARID1A-deficient cancer cells, which are particularly vulnerable to glutathione depletion, but not in ARID1A-positive cancer cells. Our studies uncover a novel potential ligandable site within GCLM that can be targeted to inhibit GSH synthesis in vulnerable cancer cell types.

Liquid biopsy of cerebrospinal fluid for MYD88 L265P mutation is useful for diagnosis of central nervous system lymphoma.

The current standard of diagnosing central nervous system (CNS) lymphoma is stereotactic biopsy, however the procedure has a risk of surgical complication. Liquid biopsy of the CSF is a less invasive, non-surgical method that can be used for diagnosing CNS lymphoma. In this study, we established a clinically applicable protocol for determining mutations in MYD88 in the CSF of patients with CNS lymphoma. CSF was collected prior to the start of chemotherapy from 42 patients with CNS lymphoma and matched tumor specimens. Mutations in MYD88 in 33 tumor samples were identified using pyrosequencing. Using 10 ng each of cellular DNA and cell-free DNA (cfDNA) extracted from the CSF, the MYD88 L265P mutation was detected using digital PCR. The conditions to judge mutation were rigorously determined. The median Target/Total value of cases with MYD88 mutations in the tumors was 5.1% in cellular DNA and 22.0% in cfDNA. The criteria to judge mutation were then determined, with a Target/Total value of 0.25% as the cutoff. When MYD88 mutations were determined based on these criteria, the sensitivity and specificity were 92.2% and 100%, respectively, with cellular DNA; and the sensitivity and specificity were 100% with cfDNA. Therefore, the DNA yield, mutated allele fraction, and accuracy were significantly higher in cfDNA compared with that in cellular DNA. Taken together, this study highlights the importance of detecting the MYD88 L265P mutation in cfDNA of the CSF for diagnosing CNS lymphoma using digital PCR, a highly accurate and clinically applicable method.

Survival in patients with glioblastoma at a first progression does not correlate with isocitrate dehydrogenase (IDH)1 gene mutation status.

BACKGROUNDS: Mutations in the isocitrate dehydrogenase (IDH)1 gene are favourable prognostic factors in newly diagnosed diffuse gliomas, whereas it remains controversial in the recurrent glioblastoma setting. METHODS: A total of 171 patients with newly diagnosed glioblastoma, either 'primary' glioblastoma or 'secondary' glioblastoma, treated at Kyorin University Hospital or Japanese Red Cross Medical Center from 2000 to 2015 were included. Patients with confirmed IDH1 status and O6-methylguanine-DNA methyltransferase promoter methylation status were retrospectively analysed for overall survival from the initial diagnosis (n = 147) and after the first progression (n = 122). RESULTS: IDH1 mutation but not IDH2 was noted in 19 of 147 patients with glioblastoma (12.9%). In patients with 'primary' glioblastoma (n = 136), median overall survival after the first progression was 13.5 and 10.5 months for mutant IDH1 and wild-type IDH1 glioblastoma, respectively (P = 0.747). Multivariate analysis revealed O6-methylguanine-DNA methyltransferase promoter methylation, and Karnofsky Performance status 60 or higher, were independent prognostic factors for better overall survival after the first progression. When 'primary' glioblastoma and 'secondary' glioblastoma were combined, median overall survival from the first progression was not significantly different between the mutant IDH1 group (10.1 months) and wild-type IDH1 group (10.5 months) (P = 0.559), whereas median overall survival from the initial diagnosis was significantly different (47.5 months vs.18.3 months, respectively; P = 0.035). CONCLUSIONS: These results suggest that IDH1 mutation may not be a prognostic factor for survival at the first progression of patients with 'primary' glioblastoma and pretreated 'secondary' glioblastoma, and further warrant investigation in prospective studies.

Emerging Roles of Astrocyte Kir4.1 Channels in the Pathogenesis and Treatment of Brain Diseases.

Inwardly rectifying Kir4.1 channels in astrocytes mediate spatial potassium (K+) buffering, a clearance mechanism for excessive extracellular K+, in tripartite synapses. In addition to K+ homeostasis, astrocytic Kir4.1 channels also play an essential role in regulating extracellular glutamate levels via coupling with glutamate transporters. Moreover, Kir4.1 channels act as novel modulators of the expression of brain-derived neurotrophic factor (BDNF) in astrocytes. Specifically, inhibition of astrocytic Kir4.1 channels elevates extracellular K+ and glutamate levels at synapses and facilitates BDNF expression in astrocytes. These changes elevate neural excitability, which may facilitate synaptic plasticity and connectivity. In this article, we summarize the functions and pharmacological features of Kir4.1 channels in astrocytes and highlight the importance of these channels in the treatment of brain diseases. Although further validation in animal models and human patients is required, astrocytic Kir4.1 channel could potentially serve as a novel therapeutic target for the treatment of depressive disorders and epilepsy.

Consecutive single-institution case series of primary central nervous system lymphoma treated by R-MPV or high-dose methotrexate monotherapy.

OBJECTIVE: The optimal regimen for use of high dose-methotrexate-based chemotherapy in primary central nervous system lymphoma is still under debate. We conducted a retrospective study to evaluate the treatment outcome of a combination immunochemotherapy consisting of rituximab, methotrexate, procarbazine and vincristine followed by with or without whole brain radiotherapy and consolidation cytarabine, in comparison with high dose-methotrexate monotherapy followed by full dose whole brain radiotherapy. METHODS: Newly diagnosed primary central nervous system lymphoma patients treated with either rituximab, methotrexate, procarbazine and vincristine or high dose-methotrexate in Kyorin University Hospital were identified, and the response rates and survival were compared. Toxicities, post-treatment transition of Mini-Mental State Examination, Karnofsky performance status score, Fazekas scale and prognostic factors were analysed in the rituximab, methotrexate, procarbazine and vincristine group. RESULTS: Ninety-five patients treated with rituximab, methotrexate, procarbazine and vincristine (n = 39) or high dose-methotrexate (n = 56) were analysed. The complete response/complete response unconfirmed rate was significantly higher in the rituximab, methotrexate, procarbazine and vincristine group (74.4 vs. 15.4%, P < 0.001). Accordingly, both median progression-free survival and overall survival were significantly longer in the rituximab, methotrexate, procarbazine and vincristine group (median progression-free survival: unreached vs. 14.75 months, P < 0.001) (median overall survival: unreached vs. 63.15 months, P = 0.005). Although the rate of grade 3/4 hematologic toxicities was high both during rituximab, methotrexate, procarbazine and vincristine and consolidation cytarabine, the rate of grade 3/4 infections was low, and no treatment related deaths were observed. Deterioration in Karnofsky performance status or Mini-Mental State Examination was rare, except on disease recurrence. Although whole brain radiotherapy was associated with Fazekas scale deterioration, its association with Karnofsky performance status or Mini-Mental State Examination deterioration was not significant. CONCLUSIONS: Rituximab, methotrexate, procarbazine and vincristine was apparently promising in comparison with high dose-methotrexate monotherapy with manageable toxicity in this retrospective study, and further investigation is warranted.

Down-Regulation of Astrocytic Kir4.1 Channels during the Audiogenic Epileptogenesis in Leucine-Rich Glioma-Inactivated 1 (Lgi1) Mutant Rats.

The dysfunction of astrocytic inwardly rectifying potassium (Kir) 4.1 channels, which mediate the spatial potassium-buffering function of astrocytes, is known to be involved in the development of epilepsy. Here, we analyzed the Kir4.1 expressional changes in Leucine-Rich Glioma-Inactivated 1 (Lgi1) mutant rats, which is a model of autosomal dominant lateral temporal lobe epilepsy in humans, to clarify the role of astrocytic Kir4.1 channels in Lgi1-related epileptogenesis. Priming acoustic stimulation (at postnatal day 16) conferred seizure susceptibility on Lgi1 mutant rats, which evoked audiogenic seizures with test stimulation at eight weeks. In the seizure-susceptible Lgi1 mutant rats (before test stimulation), astrocytic Kir4.1 expression was down-regulated region-specifically in the cerebral cortex, hippocampus, and amygdala. In addition, prophylactic treatments of Lgi1 mutant rats with valproic acid (VPA, 30 mg/kg and 200 mg/kg) for two weeks prevented both the development of seizure susceptibility and the down-regulation of Kir4.1 expression in astrocytes. The present study demonstrated for the first time that the astrocytic Kir4.1 expression was reduced in the Lgi1-related seizure model, suggesting that the down-regulation of Kir4.1 channels in astrocytes is involved in audiogenic epileptogenesis caused by Lgi1 mutation. In addition, VPA seemed to have a prophylactic effect on Lgi1-related seizures.

Pharmacological characterization of nicotine-induced tremor: Responses to anti-tremor and anti-epileptic agents.

We previously showed that nicotine evoked kinetic tremor by activating the inferior olive, which is implicated in the pathogenesis of essential tremor, via α7 nicotinic acetylcholine receptors. Here, we evaluated the effects of various anti-tremor and anti-epileptic agents on nicotine-induced tremor in mice to clarify the pharmacological characteristics of nicotine tremor. Drugs effective for essential tremor, propranolol, diazepam and phenobarbital, all significantly inhibited kinetic tremor induced by an intraperitoneal (i.p.) injection of nicotine (1 mg/kg). In contrast, none of the medications for Parkinson's disease, l-DOPA, bromocriptine or trihexyphenidyl, affected the nicotine tremor. Among the anti-epileptic agents examined, valproate, carbamazepine and ethosuximide, significantly inhibited nicotine-induced tremor. In addition, a selective T-type Ca2+ channel blocker, TTA-A2, also suppressed the nicotine tremor. However, neither gabapentin, topiramate, zonisamide nor levetiracetam significantly affected nicotine-induced tremor. The present results show that nicotine-induced tremor resembles essential tremor not only on the neural basis, but also in terms of the pharmacological responses to anti-tremor agents, implying that nicotine-induced tremor can serve as a model for essential tremor. In addition, it is suggested that anti-epileptic agents, which have stimulant actions on the GABAergic system or blocking actions on voltage-gated Na+ channels and T-type Ca2+ channels, can alleviate essential tremor.

Inwardly Rectifying Potassium Channel Kir4.1 as a Novel Modulator of BDNF Expression in Astrocytes.

Brain-derived neurotrophic factor (BDNF) is a key molecule essential for neural plasticity and development, and is implicated in the pathophysiology of various central nervous system (CNS) disorders. It is now documented that BDNF is synthesized not only in neurons, but also in astrocytes which actively regulate neuronal activities by forming tripartite synapses. Inwardly rectifying potassium (Kir) channel subunit Kir4.1, which is specifically expressed in astrocytes, constructs Kir4.1 and Kir4.1/5.1 channels, and mediates the spatial potassium (K⁺) buffering action of astrocytes. Recent evidence illustrates that Kir4.1 channels play important roles in bringing about the actions of antidepressant drugs and modulating BDNF expression in astrocytes. Although the precise mechanisms remain to be clarified, it seems likely that inhibition (down-regulation or blockade) of astrocytic Kir4.1 channels attenuates K⁺ buffering, increases neuronal excitability by elevating extracellular K⁺ and glutamate, and facilitates BDNF expression. Conversely, activation (up-regulation or opening) of Kir4.1 channels reduces neuronal excitability by lowering extracellular K⁺ and glutamate, and attenuates BDNF expression. Particularly, the former pathophysiological alterations seem to be important in epileptogenesis and pain sensitization, and the latter in the pathogenesis of depressive disorders. In this article, we review the functions of Kir4.1 channels, with a focus on their regulation of spatial K⁺ buffering and BDNF expression in astrocytes, and discuss the role of the astrocytic Kir4.1-BDNF system in modulating CNS disorders.

Identification of Candidate Genes for Generalized Tonic-Clonic Seizures in Noda Epileptic Rat.

The Noda epileptic rat (NER) exhibits generalized tonic-clonic seizures (GTCS). A genetic linkage analysis identified two GTCS-associated loci, Ner1 on Chr 1 and Ner3 on Chr 5. The wild-type Ner1 and Ner3 alleles suppressed GTCS when combined in double-locus congenic lines, but not when present in single-locus congenic lines. Global expression analysis revealed that cholecystokinin B receptor (Cckbr) and suppressor of tumorigenicity 5 (St5), which map within Ner1, and PHD finger protein 24 (Phf24), which maps within Ner3, were significantly downregulated in NER. De novo BAC sequencing detected an insertion of an endogenous retrovirus sequence in intron 2 of the Phf24 gene in the NER genome, and PHF24 protein was almost absent in the NER brain. Phf24 encodes a Gαi-interacting protein involved in GABAB receptor signaling pathway. Based on these findings, we conclude that Cckbr, St5, and Phf24 are strong candidate genes for GTCS in NER.

Serotonergic modulation of nicotine-induced kinetic tremor in mice.

We previously demonstrated that nicotine elicited kinetic tremor by elevating the neural activity of the inferior olive via α7 nicotinic acetylcholine (nACh) receptors. Since α7 nACh receptors reportedly facilitate synaptic monoamine release, we explored the role of 5-HT receptors in induction and/or modulation of nicotine tremor. Treatment of mice with nicotine induced kinetic tremor that normally appeared during movement. The 5-HT1A agonist, 8-hydroxydipropylaminotetraline (8-OH-DPAT), significantly enhanced nicotine-induced tremor and the action of 8-OH-DPAT was antagonized by WAY-100135 (5-HT1A antagonist). In addition, the cerebral 5-HT depletion by repeated treatment with p-chlorophenylalanine did not reduce, but rather potentiated the facilitatory effects of 8-OH-DPAT. In contrast, the 5-HT2 agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI), significantly attenuated nicotine tremor, which was antagonized by ritanserin (5-HT2 antagonist). The 5-HT3 agonist SR-57227 did not affect nicotine-induced tremor. Furthermore, when testing the direct actions of 5-HT antagonists, nicotine tremor was inhibited by WAY-100135, but was unaffected by ritanserin, ondansetron (5-HT3 antagonist) or SB-258585 (5-HT6 antagonist). These results suggest that postsynaptic 5-HT1A receptors are involved in induction of nicotine tremor mediated by α7 nACh receptors. In addition, 5-HT2 receptors have an inhibitory modulatory role in induction of nicotine tremor.

Prognostic factors for primary central nervous system lymphomas treated with high-dose methotrexate-based chemo-radiotherapy.

BACKGROUND: Primary central nervous system lymphoma (PCNSL) remains an aggressive and refractory tumor despite high-dose methotrexate-based chemo-radiotherapy. Age and performance status have been shown to be important clinical prognostic factors, however others, especially molecular factors, affecting the prognosis are still uncertain. METHODS: We investigate clinical, neuroimaging and immunohistochemical data in tissue from 41 PCNSL patients treated primarily with methotrexate-based chemo-radiotherapy and evaluate the influence of potential prognostic factors on clinical outcome as well as correlation among these factors. RESULTS: Median progression-free survival (PFS) and overall survival (OS) were 29 and 73 months, respectively. Expression of the mismatch repair (MMR) proteins, MLH1, MSH2, MSH6 and PMS2, correlated tightly with each other and high expression of MSH2 was significantly associated with better OS and PFS (P = 0.005 and P = 0.007), while methotrexate metabolism-related proteins did not affect survival. In addition, low expression of PMS2 was an independent predictor of methotrexate resistance (P = 0.039). Among neuroimaging findings, involvement of the fornix and tegmentum/velum were significantly associated with poorer OS (P < 0.001 and P = 0.013) and PFS (P = 0.014 and P = 0.043, respectively). Germinal center B cell (GCB)-PCNSL subtype as opposed to non-GCB subtype, tended toward better survival. Regarding oncogenes, cMYC-positive cases showed unfavorable OS (P = 0.046). By multivariate analysis, MSH2 and involvement of the fornix were independent predictors for both OS and PFS, whereas tegmentum/velum location and cMYC expression were significantly associated with OS. CONCLUSIONS: Although further studies are needed, these results suggest that MMR protein expression, as well as specific deep locations and cMYC expression, may be a novel prognostic and predictive markers for PCNSL.

Glycine-Binding Site Stimulants of NMDA Receptors Alleviate Extrapyramidal Motor Disorders by Activating the Nigrostriatal Dopaminergic Pathway.

Dysfunction of the N-methyl-d-aspartate (NMDA) receptor has been implicated in the pathogenesis of schizophrenia. Although agonists for the glycine-binding sites of NMDA receptors have potential as new medication for schizophrenia, their modulation of antipsychotic-induced extrapyramidal side effects (EPS) has not yet been clarified. We herein evaluated the effects of glycine-binding site stimulants of NMDA receptors on antipsychotic-induced EPS in mice and rats. d-cycloserine (DCS) and d-serine significantly improved haloperidol (HAL)-induced bradykinesia in mice, whereas glycine showed no effects. Sodium benzoate, a d-amino acid oxidase inhibitor, also attenuated HAL-induced bradykinesia. Improvements in HAL-induced bradykinesia by DCS were antagonized by the NMDA antagonist dizocilpine or nitric oxide synthase inhibitor L-NG-Nitro-l-arginine methyl ester. In addition, DCS significantly reduced HAL-induced Fos expression in the dorsolateral striatum without affecting that in the nucleus accumbens. Furthermore, a microinjection of DCS into the substantia nigra pars compacta significantly inhibited HAL-induced EPS concomitant with elevations in dopamine release in the striatum. The present results demonstrated for the first time that stimulating the glycine-binding sites of NMDA receptors alleviates antipsychotic-induced EPS by activating the nigrostriatal dopaminergic pathway, suggesting that glycine-binding site stimulants are beneficial not only for efficacy, but also for side-effect management.

Recurrent neomorphic mutations of MTOR in central nervous system and testicular germ cell tumors may be targeted for therapy.

Germ cell tumors constitute a heterogeneous group that displays a broad spectrum of morphology. They often arise in testes; however, extragonadal occurrence, in particular brain, is not uncommon, and whether they share a common pathogenesis is unknown. We performed whole exome sequencing in 41 pairs of central nervous system germ cell tumors (CNS GCTs) of various histology and their matched normal tissues. We then performed targeted sequencing of 41 selected genes in a total of 124 CNS GCTs, 65 testicular germ cell tumors (tGCTs) and 8 metastatic GCTs to the CNS. The results showed that mutually exclusive mutations of genes involved in the MAPK pathway were most common (48.4 %), typically in KIT (27.4 %), followed by those in the PI3K pathway (12.9 %), particularly in MTOR (6.5 %), among the 124 CNS GCTs. Pure germinomas and non-germinomatous germ cell tumors (NGGCTs), as well as CNS and testicular GCTs, showed similar mutational profiles, suggesting that GCTs share a common molecular pathogenesis. Mutated MTOR identified in CNS GCTs upregulated phosphorylation of the AKT pathway proteins including AKT and 4EBP1 in nutrient-deprived conditions and enhanced soft-agar colony formation; both events were suppressed in a dose-dependent manner by addition of the MTOR inhibitor pp242. Our findings indicate that the dominant genetic drivers of GCTs regardless of the site of origin are activation of the MAPK and/or PI3K pathways by somatic point mutations. Mutated MTOR represents a potential target for novel targeted therapies for refractory GCTs.

Calcium signaling via Orai1 is essential for induction of the nuclear orphan receptor pathway to drive Th17 differentiation.

Orai1 is the pore subunit of Ca(2+) release-activated Ca(2+) (CRAC) channels that stimulate downstream signaling pathways crucial for T cell activation. CRAC channels are an attractive therapeutic target for alleviation of autoimmune diseases. Using high-throughput chemical library screening targeting Orai1, we identified a novel class of small molecules that inhibit CRAC channel activity. One of these molecules, compound 5D, inhibited CRAC channel activity by blocking ion permeation. When included during differentiation, Th17 cells showed higher sensitivity to compound 5D than Th1 and Th2 cells. The selectivity was attributable to high dependence of promoters of retinoic-acid-receptor-related orphan receptors on the Ca(2+)-NFAT pathway. Blocking of CRAC channels drastically decreased recruitment of NFAT and histone modifications within key gene loci involved in Th17 differentiation. The impairment in Th17 differentiation by treatment with CRAC channel blocker was recapitulated in Orai1-deficient T cells, which could be rescued by exogenous expression of retinoic-acid-receptor-related orphan receptors or a constitutive active mutant of NFAT. In vivo administration of CRAC channel blockers effectively reduced the severity of experimental autoimmune encephalomyelitis by suppression of differentiation of inflammatory T cells. These results suggest that CRAC channel blockers can be considered as chemical templates for the development of therapeutic agents to suppress inflammatory responses.

Interaction between anti-Alzheimer and antipsychotic drugs in modulating extrapyramidal motor disorders in mice.

Antipsychotics are often used in conjunction with anti-Alzheimer drugs to treat the behavioral and psychological symptoms of dementia (BPSD). Here, we examined the effects of cholinesterase inhibitors (ChEIs), donepezil and galantamine, on antipsychotic-induced extrapyramidal side effects (EPS) in mice. The effects of serotonergic agents on the EPS drug interaction were also evaluated. Donepezil (0.3-3 mg/kg) did not induce EPS signs by itself; however, it significantly potentiated bradykinesia induction with a low dose of haloperidol (0.5 mg/kg) in dose-dependent and synergistic manners. Galantamine (0.3-3 mg/kg) elicited mild bradykinesia at a high dose and dose-dependently augmented haloperidol-induced bradykinesia. The EPS potentiation by galantamine was blocked by trihexyphenidyl (a muscarinic antagonist), but not by mecamylamine (a nicotinic antagonist). In addition, the bradykinesia potentiation by galantamine was significantly reduced by (±)-8-hydroxy-2-(di-n-propylamino)-tetralin (a 5-HT1A agonist), ritanserin (a 5-HT2 antagonist), and SB-258585 (a 5-HT6 antagonist). The present results give us a caution for the antipsychotics and ChEIs interaction in inducing EPS in the treatment of BPSD. In addition, second generation antipsychotics, which can stimulate 5-HT1A receptors or antagonize 5-HT2 and 5-HT6 receptors, seem to be favorable as an adjunctive therapy for BPSD.

Inhibitory effect of HIV-specific neutralizing IgA on mucosal transmission of HIV in humanized mice.

HIV-1 infections are generally initiated at mucosal sites. Thus, IgA antibody, which plays pivotal roles in mucosal immunity, might efficiently prevent HIV infection. However, mounting a highly effective HIV-specific mucosal IgA response by conventional immunization has been challenging and the potency of HIV-specific IgA against infection needs to be addressed in vivo. Here we show that the polymeric IgA form of anti-HIV antibody inhibits HIV mucosal transmission more effectively than the monomeric IgA or IgG1 form in a comparable range of concentrations in humanized mice. To deliver anti-HIV IgA in a continual manner, we devised a hematopoietic stem/progenitor cell (HSPC)-based genetic approach using an IgA gene. We transplanted human HSPCs transduced with a lentiviral construct encoding a class-switched anti-HIV IgA (b12-IgA) into the humanized bone marrow-liver-thymus (BLT) mice. The transgene was expressed specifically in B cells and plasma cells in lymphoid organs and mucosal sites. After vaginal HIV-1 challenge, mucosal CD4(+) T cells in the b12-IgA-producing mice were protected from virus-mediated depletion. Similar results were also obtained in a second humanized model, "human immune system mice." Our study demonstrates the potential of anti-HIV IgA in immunoprophylaxis in vivo, emphasizing the importance of the mucosal IgA response in defense against HIV/AIDS.

Predictive significance of mean apparent diffusion coefficient value for responsiveness of temozolomide-refractory malignant glioma to bevacizumab: preliminary report.

BACKGROUND: Recurrent glioblastoma after initial radiotherapy plus concomitant and adjuvant temozolomide is problematic. Here, patients with temozolomide-refractory high-grade gliomas were treated with bevacizumab (BV) and evaluated using apparent diffusion coefficient (ADC) for response. METHODS: Nine post-temozolomide recurrent or progressive high-grade glioma patients (seven with glioblastoma and two with anaplastic astrocytoma) were treated with BV monotherapy. Average age was 57 years (range, 22-78), median Karnofsky Performance Scale (KPS) was 70 (30-80) and median BV line number was 2 (2-5). Two had additional stereotactic radiotherapy within 6 months prior to BV. Magnetic resonance (MR) imaging after BV therapy was performed within 2 weeks with calculation of mean ADC (mADC) values of enhancing tumor contours. RESULTS: Post-BV treatment MR imaging showed decreased tumor volumes in eight of nine cases (88.9 %). Partial response was obtained in four cases (44.4 %), four cases had stable disease, and one had progressive disease. Of 15 evaluable enhancing lesions, 11 shrank and four did not. Pretreatment mADC values were above 1100 (10(-6) mm(2)/s) in all responding tumors, while all non-responding lesions scored below 1100 (p = 0.001). mADC decreased after the first BV treatment in all lesions except one. KPS improved in four cases (44.4 %). Median progression-free survival and overall survival for those having all lesions with high mADC (>1100) were significantly longer than those with a low mADC (<1100) lesion (p = 0.018 and 0.046, respectively). CONCLUSIONS: Bevacizumab monotherapy is effective in patients with temozolomide-refractory recurrent gliomas and tumor mean ADC value can be a useful marker for prediction of BV response and survival.

HIV latency in the humanized BLT mouse.

Even after extended treatment with powerful antiretroviral drugs, HIV is not completely eliminated from infected individuals. Latently infected CD4(+) T cells constitute one reservoir of replication-competent HIV that needs to be eliminated to completely purge virus from antiretroviral drug-treated patients. However, a major limitation in the development of therapies to eliminate this latent reservoir is the lack of relevant in vivo models that can be used to test purging strategies. Here, we show that the humanized BLT (bone marrow-liver-thymus) mouse can be used as both an abundant source of primary latently infected cells for ex vivo latency analysis and also as an in vivo system for the study of latency. We demonstrate that over 2% of human cells recovered from the spleens of HIV-infected BLT mice can be latently infected and that this virus is integrated, activation inducible, and replication competent. The non-tumor-inducing phorbol esters prostratin and 12-deoxyphorbol-13-phenylacetate can each induce HIV ex vivo from these latently infected cells, indicating that this model can be used as a source of primary cells for testing latency activators. Finally, we show activation-inducible virus is still present following suppression of plasma viral loads to undetectable levels by using the antiretroviral drugs zidovudine, indinavir sulfate, and didanosine, demonstrating that this model can also be used to assess the in vivo efficacy of latency-purging strategies. Therefore, the HIV-infected BLT mouse should provide a useful model for assessment of HIV latency activators and approaches to eliminate persistent in vivo HIV reservoirs.

Pathophysiological roles of serotonergic system in regulating extrapyramidal motor functions.

The serotonergic nervous system plays crucial roles in regulating psycho-emotional, cognitive, sensori-motor and autonomic functions. It is now known that multiple serotonin (5-hydroxytryptamine; 5-HT) receptors regulate extrapyramidal motor functions, which are implicated in pathogenesis and/or treatment of various neurological disorders (e.g., Parkinson's disease and drug-induced extrapyramidal motor deficits). Specifically, antagonism of 5-HT2A/2C receptors alleviates antipsychotic-induced extrapyramidal side effects (EPS) by relieving the 5-HT2A/2C receptor-mediated inhibition of nigral dopaminergic neuron activity and striatal dopamine release. Indeed, many of the second generation antipsychotics (e.g., risperidone, perospirone and olanzapine) commonly possess potent 5-HT2A/2C blocking actions which contribute to their atypical antipsychotic property. In addition, activation of 5-HT1A receptors also improves antipsychotic-induced EPS and motor disabilities in animal models of Parkinson's disease. Microinjection studies revealed that stimulation of postsynaptic 5-HT1A receptors in the striatum or motor cortex plays an important role in the antiparkinsonian actions. Furthermore, recent studies demonstrated that antagonism of 5-HT3 and 5-HT6 receptors alleviates extrapyramidal motor disorders while 5-HT4, 5-HT5, and 5-HT7 receptors are mostly inactive. These results encourage drug discovery research into new 5-HT receptor ligands that could improve current therapies for extrapyramidal motor disorders.