Modulators of cytoskeletal reorganization in CA1 hippocampal neurons show increased expression in patients at mid-stage Alzheimer's disease

During the progression of Alzheimer's disease (AD), hippocampal neurons undergo cytoskeletal reorganization, resulting in degenerative as well as regenerative changes. As neurofibrillary tangles form and dystrophic neurites appear, sprouting neuronal processes with growth cones emerge. Actin and tubulin are indispensable for normal neurite development and regenerative responses to injury and neurodegenerative stimuli. We have previously shown that actin capping protein beta2 subunit, Capzb2, binds tubulin and, in the presence of tau, affects microtubule polymerization necessary for neurite outgrowth and normal growth cone morphology. Accordingly, Capzb2 silencing in hippocampal neurons resulted in short, dystrophic neurites, seen in neurodegenerative diseases including AD. Here we demonstrate the statistically significant increase in the Capzb2 expression in the postmortem hippocampi in persons at mid-stage, Braak and Braak stage (BB) III-IV, non-familial AD in comparison to controls. The dynamics of Capzb2 expression in progressive AD stages cannot be attributed to reactive astrocytosis. Moreover, the increased expression of Capzb2 mRNA in CA1 pyramidal neurons in AD BB III-IV is accompanied by an increased mRNA expression of brain derived neurotrophic factor (BDNF) receptor tyrosine kinase B (TrkB), mediator of synaptic plasticity in hippocampal neurons. Thus, the up-regulation of Capzb2 and TrkB may reflect cytoskeletal reorganization and/or regenerative response occurring in hippocampal CA1 neurons at a specific stage of AD progression.     

Charge variants in IgG1: Isolation,characterization, in vitro binding properties and pharmacokinetics in rats

Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7–9.1 and was composed of about 20% acidic variants, 12% basic variants and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity or the PK properties in rats.Key words: mAb IgG1, charge heterogeneity, isoelectric point, neonatal Fc receptor (FcRn), pharmacokinetics, potency     

Calcium‐stimulated mitogen‐activated protein kinase activation elicits Bcl‐xL downregulation and Bak upregulation in notexin‐treated human neuroblastoma SK‐N‐SH cells

Notechis scutatus scutatus notexin induced apoptotic death of SK‐N‐SH cells accompanied with downregulation of Bcl‐xL, upregulation of Bak, mitochondrial depolarization, and ROS generation. Upon exposure to notexin, Ca²⁺‐mediated JNK and p38 MAPK activation were observed in SK‐N‐SH cells. Production of ROS was a downstream event followed by Ca²⁺‐mediated mitochondrial alteration. Notexin‐induced cell death, mitochondrial depolarization, and ROS generation were suppressed by SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor). Moreover, phospho‐p38 MAPK and phospho‐JNK were proved to be involved in Bcl‐xL degradation, and overexpression of Bcl‐xL attenuated the cytotoxic effect of notexin. Bak upregulation was elicited by p38 MAPK‐mediated ATF‐2 activation and JNK‐mediated c‐Jun activation. Suppression of Bak upregulation by ATF‐2 siRNA or c‐Jun siRNA attenuated notexin‐evoked mitochondrial depolarization and rescued viability of notexin‐treated cells. Taken together, our data indicate that notexin‐induced apoptotic death of SK‐N‐SH cells is mediated through mitochondrial alteration triggering by Ca²⁺‐evoked p38 MAPK/ATF‐2 and JNK/c‐Jun signaling pathways. J. Cell. Physiol. 222:177–186, 2010. © 2009 Wiley‐Liss, Inc.     

Invariant ankle moment patterns when walking with and without a robotic ankle exoskeleton

To guide development of robotic lower limb exoskeletons, it is necessary to understand how humans adapt to powered assistance. The purposes of this study were to quantify joint moments while healthy subjects adapted to a robotic ankle exoskeleton and to determine if the period of motor adaptation is dependent on the magnitude of robotic assistance. The pneumatically powered ankle exoskeleton provided plantar flexor torque controlled by the wearer's soleus electromyography (EMG). Eleven naïve individuals completed two 30-min sessions walking on a split-belt instrumented treadmill at 1.25 m/s while wearing the ankle exoskeleton. After two sessions of practice, subjects reduced their soleus EMG activation by ～36% and walked with total ankle moment patterns similar to their unassisted gait (r²=0.98±0.02, THSD, p>0.05). They had substantially different ankle kinematic patterns compared to their unassisted gait (r²=0.79±0.12, THSD, p<0.05). Not all of the subjects reached a steady-state gait pattern within the two sessions, in contrast to a previous study using a weaker robotic ankle exoskeleton (Gordon and Ferris, 2007). Our results strongly suggest that humans aim for similar joint moment patterns when walking with robotic assistance rather than similar kinematic patterns. In addition, greater robotic assistance provided during initial use results in a longer adaptation process than lesser robotic assistance.     

Epitope Mapping of Ibalizumab,a Humanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity in Infected Patients

Ibalizumab is a humanized monoclonal antibody that binds human CD4, the primary receptor for human immunodeficiency virus type 1 (HIV-1). With its unique specificity for domain 2 of CD4, this antibody potently and broadly blocks HIV-1 infection in vitro by inhibiting a postbinding step required for viral entry but without interfering with major histocompatibility complex class II (MHC-II)-mediated immune function. In clinical trials, ibalizumab has demonstrated anti-HIV-1 activity in patients without causing immunosuppression. Thus, a characterization of the ibalizumab epitope was conducted in an attempt to gain insight into the underlying mechanism of its antiviral activity as well as its safety profile. By studying mouse/human chimeric CD4 molecules and site-directed point mutants of CD4, amino acids L96, P121, P122, and Q163 in domain 2 were found to be important for ibalizumab binding, with E77 and S79 in domain 1 also contributing. All these residues appear to cluster on the interface between domains 1 and 2 of human CD4 on a surface opposite the site where gp120 and the MHC-II molecule bind on domain 1. Separately, the epitope of M-T441, a weakly neutralizing mouse monoclonal antibody that competes with ibalizumab, was localized entirely within domain 2 on residues 123 to 125 and 138 to 140. The results reported herein not only provide an appreciation for why ibalizumab has not had significant adverse immunological consequences in infected patients to date but also raise possible steric hindrance mechanisms by which this antibody blocks HIV-1 entry into a CD4-positive cell.The human immunodeficiency virus type 1 (HIV-1) epidemic continues to spread at the alarming rate of approximately 2.5 million new cases per year, despite intensive efforts from the scientific community. A safe and effective HIV-1 vaccine would be a key weapon to fight this epidemic; however, vaccine development has not yet proven successful. The extraordinary diversity of the virus, its capacity to evade adaptive immune responses, and the inability to induce broadly neutralizing antibodies against HIV-1 represent unprecedented challenges for vaccine development (3). Alternatively, the strategy of preexposure prophylaxis (PrEP) with antiretroviral drugs or even virus-specific immunoglobulins (Igs) (11) is gaining traction. Protection of rhesus macaques from challenge with simian immunodeficiency virus (SIV) has been observed after passive administration of anti-gp120 or anti-gp41 monoclonal antibodies, such as b12, 2G12, 2F5, and 4E10 (2, 20). However, the application of these antibodies as PrEP has been hindered due to their lack of potency or breadth or both. To this end, PrEP strategies could also consider antibodies to CCR5 (13) or CD4 (8, 12, 14), which have potent and broad inhibitory activities against HIV-1 without unwanted side effects.The CD4 molecule, a cell surface glycoprotein found primarily on T lymphocytes, is the primary receptor for the HIV-1 envelope gp160 glycoprotein (7, 18). A member of the immunoglobulin superfamily (19), CD4 consists of an extracellular segment composed of four tandem immunoglobulin-like domains (D1, D2, D3, and D4), a single transmembrane span, and a short C-terminal cytoplasmic tail (15, 24). It is worth noting that both human major histocompatibility complex (MHC) class II (26) and HIV-1 gp120 (16, 24) bind to the same surface on the first domain (D1) of the CD4 molecule.Ibalizumab (formerly known as TNX-355) is a humanized IgG4 monoclonal antibody that blocks HIV-1 entry by binding to human CD4 (8, 12, 14, 33). It was engineered from its mouse progenitor (5A8) by grafting the mouse complementary-determining region (CDR) onto a human IgG4 construct (4, 5). The IgG4 isotype was chosen to minimize the chances for CD4⁺ T-cell depletion by antibody- and complement-dependent cytotoxicity mediated by binding to Fc receptors. Ibalizumab or 5A8 blocks CD4-dependent virus entry and inhibits a broad spectrum of both laboratory-adapted and clinical HIV-1 isolates, including CCR5-tropic and CXCR4-tropic strains from multiple subtypes, with 50% inhibitory concentrations (IC₅₀s) of 0.0004 to 0.152 μg/ml (4, 5). In vivo, treatment with ibalizumab prominently reduced plasma viremia in rhesus macaques infected with SIV, because this monoclonal antibody has equal affinity for rhesus CD4 (22, 23). In HIV-1 patients, single as well as multiple doses of ibalizumab resulted in substantial reductions (∼10-fold) in viral loads and increases in CD4⁺ T-cell counts without evidence of serious adverse effects or immunologic impairments (12, 14).Efforts were made years ago to delineate the antibody binding site of 5A8 on human CD4 (hCD4) (5). Two stretches, amino acids (aa) 121 to 124 and aa 127 to 134, in domain 2 (D2) were found to be critical for binding. Since then, however, little work has been done to fine-map this epitope, and whether other parts of hCD4 are involved in the binding of this antibody has remained unexplored. The fact that an anti-hCD4-D2 antibody can noncompetitively, yet potently, block HIV-1 entry is intriguing, as viral gp120 binds to D1 of hCD4 (16, 24). Therefore, to gain a better understanding of the mechanism by which ibalizumab inhibits HIV-1 infection while avoiding undesired side effects, we sought to fine-map the epitope of this unique monoclonal antibody.     

Identification of an autoantibody against the proliferating cell nuclear antigen from a patient with systemic lupus erythematosus

Antibodies against the proliferating cell nuclear antigen (PCNA) was first discovered in the sera of systemic lupus erythematosus (SLE) patients. However, the reactivity and specificity of anti-PCNA autoantibodies are still unclear. To investigate the property of anti-PCNA autoantibodies, we conducted an ELISA screening of the anti-PCNA autoantibodies in sera of SLE patients. Eighteen out of 191 SLE sera were found to be positive for anti-PCNA antibodies giving a frequency of nearly 10%. Among the positive sera, a sample with the highest titer of anti-PCNA autoantibody preferentially recognizes the wild-type PCNA as compared to the Y114A mutation which contains a single amino acid substitution at 114 and fails to form the toroidal structure. Moreover, the autoantibody purified from this serum identifies only the free PCNA in crude mammalian cell extracts but not other associated cellular components. This finding raises a possibility that immunostaining with the human anti-PCNA autoantibodies in previous studies might have only partially PCNAs in tissues.     

Cold treatment for guava fruits infested with oriental fruit fly,Bactrocera dorsalis (Diptera: Tephritidae)

Guava (Psidium guajava Linnaeus) is an important horticulture crop in tropical and subtropical regions. However, guava transportation to the fruit market is regulated due to the risk of fruit fly infestations. The oriental fruit fly [Bactrocera dorsalis (Hendel), Tephritidae, Diptera], which damages numerous horticulture fruits, is the primary pest in guava. For fruit export, phytosanitary treatment is necessary to prevent the potential oriental fruit fly infestations through pest-infested fruits. In this study, cold treatment was administered to eliminate oriental fruit fly infestations in guava fruits. The optimal egg inoculation density was 200 eggs per fruit, and third instar larvae displayed the highest cold treatment tolerance. On assessing the efficacy of cold treatment, none of the third instar larvae in the fruits survived at a constant fruit core temperature of 0.5–1 °C for 11 days. A confirmatory test revealed that on maintaining a fruit core temperature of 0.5–1 °C for 12 days, 100% mortality was achieved among the fruit flies. In fruit quality assessment, guava fruits exposed to cold treatment still maintained their market value. Thus, cold treatment effectively disinfested guava fruits with possible fruit fly infestations, making it a viable quarantine treatment for guava fruits prior to their export.