Enhanced molecular volume of conservatively pegylated Hb: (SP-PEG5K)6-HbA is non-hypertensive

Recent studies have suggested that the "pressor effect" of acellular Hb is a consequence of perturbation of the macro-and microcirculatory system in multiple ways, and that PEGylation is an effective approach for controlling the same. In an attempt to confirm this concept, a new and simple thiolation mediated, maleimide chemistry-based conservative PEGylation protocol has been developed to conjugate multiple copies of PEG-chains to Hb. This approach combines the high reactivity of maleimides towards thiols with the propensity of iminothiolane to derivatize the epsilon-amino groups of proteins into reactive thiol groups, with conservation of their positive charge. One of the PEGylated products, namely (SP-PEG5K)6-HbA, that carries on an average six copies of PEG5000 chains per Hb, is non-hypertensive in hamster top load and in rat 50% exchange transfusion models. This hexa-PEGylated-Hb has (i) a hydrodynamic volume corresponding to that of an oligomerized Hb of 256kDa, (ii) a molecular radius of approximately 6.8 nm, (iii) high oxygen affinity, (iv) lowered Bohr effect, and (v) increased viscosity and colloidal osmotic pressure. These properties of (SP-PEG5K)6-HbA are consistent with the emerging new paradigms for the design of Hb based oxygen carriers and confirm the concept that the "pressor effect" of Hb is a multifactorial event. The thiolation mediated maleimide chemistry-based PEGylation protocol described here for the generation of (SP-PEG5K)6-Hb is simple, highly efficient, and is carried out under oxy conditions. The results demonstrate that a non-hypertensive PEG-Hb can be generated by conjugation of a lower number of PEG chains than previously reported.     

Differential modulation of Ca(v)2.1 channels by calmodulin and Ca2+-binding protein 1

Ca(v)2.1 channels, which mediate P/Q-type Ca2+ currents, undergo Ca2+/calmodulin (CaM)-dependent inactivation and facilitation that can significantly alter synaptic efficacy. Here we report that the neuronal Ca2+-binding protein 1 (CaBP1) modulates Ca(v)2.1 channels in a manner that is markedly different from modulation by CaM. CaBP1 enhances inactivation, causes a depolarizing shift in the voltage dependence of activation, and does not support Ca2+-dependent facilitation of Ca(v)2.1 channels. These inhibitory effects of CaBP1 do not require Ca2+, but depend on the CaM-binding domain in the alpha1 subunit of Ca(v)2.1 channels (alpha12.1). CaBP1 binds to the CaM-binding domain, co-immunoprecipitates with alpha12.1 from transfected cells and brain extracts, and colocalizes with alpha12.1 in discrete microdomains of neurons in the hippocampus and cerebellum. Our results identify an interaction between Ca2+ channels and CaBP1 that may regulate Ca2+-dependent forms of synaptic plasticity by inhibiting Ca2+ influx into neurons.     

Two-year evaluation of Borrelia burgdorferi culture and supplemental tests for definitive diagnosis of Lyme disease

Lyme disease is usually diagnosed and treated based on clinical manifestations. However, laboratory testing is useful for patients with confusing presentations and for validation of disease in clinical studies. Although cultivation of Borrelia burgdorferi is definitive, prior investigations have shown that no single test is optimal for Lyme disease diagnosis. We applied high-volume blood culture, skin biopsy culture, PCR, and serodiagnosis to a cohort of patients with suspected Lyme disease acquired in Maryland and southern Pennsylvania. The study was performed to confirm the relative utility of culture and to identify laboratory testing algorithms that will supplement clinical diagnosis. Overall, 30 of 86 patients (35%) were culture positive, whereas an additional 15 of 84 (18%) were seropositive only (51% total sero- and culture positive), and PCR on skin biopsy identified 4 additional patients who were neither culture nor seropositive. Among 49 laboratory test-positive patients, the highest sensitivity (100%) for diagnosis was obtained when culture, skin PCR, and serologic tests were used, although serologic testing with skin PCR was almost as sensitive (92%). Plasma PCR was infrequently positive and provided no additional diagnostic value. Although culture is definitive and has a relatively high sensitivity, the results required a mean of 3.5 weeks to recovery. The combination of acute-phase serology and skin PCR was 75% sensitive, offering a practical and relatively rapid alternative for confirming clinical impression. The full battery of tests could be useful for patients with confusing clinical signs or for providing strong laboratory support for clinical studies of Lyme disease.     

Characterization of BrkA Expression in Bordetella bronchiseptica

BrkA confers resistance to killing by complement in Bordetella pertussis. Complement resistance in Bordetella bronchiseptica was examined. Four B. bronchiseptica strains possessed the brkA gene; however, only three expressed the protein. Only the strain lacking BrkA was susceptible to complement. Introduction of the B. pertussis brkA gene restored BrkA expression to this strain but did not confer resistance. brkA was mutated in the strains that naturally expressed BrkA, and loss of BrkA did not confer sensitivity to complement. As a species, B. bronchiseptica is more resistant to complement than B. pertussis, and BrkA does not mediate resistance.     

Helicobacter-induced chronic active lymphoid aggregates have characteristics of tertiary lymphoid tissue

Susceptible strains of mice that are naturally or experimentally infected with murine intestinal helicobacter species develop hepatic inflammatory lesions that have previously been described as chronic active hepatitis. The inflammatory infiltrates in some models of chronic autoimmunity or inflammation resemble tertiary lymphoid organs hypothesized to arise by a process termed lymphoid organ neogenesis. To determine whether hepatic inflammation caused by infection with helicobacter could give rise to tertiary lymphoid organs, we used fluorescence-activated cell sorting, immunohistochemistry, and in situ hybridization techniques to identify specific components characteristic of lymphoid organs in liver tissue sections and liver cell suspensions from helicobacter-infected mice. Small venules (high endothelial venules [HEVs]) in inflammatory lesions in Helicobacter species-infected livers were positive for peripheral node addressin. Mucosal addressin cell adhesion molecule also stained HEVs and cells with a staining pattern consistent with scattered stromal cells. The chemokines SLC (CCL 21) and BLC (CXCL13) were present, as were B220-positive B cells and T cells. The latter included a na?ve (CD45lo-CD62Lhi) population. These findings suggest that helicobacter-induced chronic active hepatitis arises through the process of lymphoid organ neogenesis.     

Engineering porcine arteries: effects of scaffold modification

Techniques have been developed to culture bovine or porcine vascular cells on polyglycolic acid (PGA) scaffolds to form engineered vessels. Previously, it was shown that smooth muscle cells (SMCs) that were in close proximity to PGA remnants after 8 weeks of culture had lower expression of SMC markers of differentiation and were more mitotic compared with SMCs that were distant from polymer residuals. Modifications of PGA were explored as a means to minimize residual polymer fragments after culture. To hasten degradation, polymer was treated with heat, NaOH, or gamma-irradiation. Differential scanning calorimetry, mass and tensile strength degradation, and inherent viscosity were used to assess polymer characteristics. When polymer was maintained in aqueous conditions, tensile strength of treated PGA degraded to zero within 3 weeks for each treatment. Engineered vessel constructs cultured on NaOH and gamma-treated polymer displayed smooth muscle alpha-actin throughout the vessel wall. Scaffold treatment impacted graft morphology, cellular differentiation, and mechanical integrity.     

A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer

BACKGROUND: There is a continued need to develop more effective cancer immunotherapy strategies. Exosomes, cell-derived lipid vesicles that express high levels of a narrow spectrum of cell proteins represent a novel platform for delivering high levels of antigen in conjunction with costimulatory molecules. We performed this study to test the safety, feasibility and efficacy of autologous dendritic cell (DC)-derived exosomes (DEX) loaded with the MAGE tumor antigens in patients with non-small cell lung cancer (NSCLC). METHODS: This Phase I study enrolled HLA A2+ patients with pre-treated Stage IIIb (N = 4) and IV (N = 9) NSCLC with tumor expression of MAGE-A3 or A4. Patients underwent leukapheresis to generate DC from which DEX were produced and loaded with MAGE-A3, -A4, -A10, and MAGE-3DPO4 peptides. Patients received 4 doses of DEX at weekly intervals. RESULTS: Thirteen patients were enrolled and 9 completed therapy. Three formulations of DEX were evaluated; all were well tolerated with only grade 1-2 adverse events related to the use of DEX (injection site reactions (N = 8), flu like illness (N = 1), and peripheral arm pain (N = 1)). The time from the first dose of DEX until disease progression was 30 to 429+ days. Three patients had disease progression before the first DEX dose. Survival of patients after the first DEX dose was 52-665+ days. DTH reactivity against MAGE peptides was detected in 3/9 patients. Immune responses were detected in patients as follows: MAGE-specific T cell responses in 1/3, increased NK lytic activity in 2/4. CONCLUSION: Production of the DEX vaccine was feasible and DEX therapy was well tolerated in patients with advanced NSCLC. Some patients experienced long term stability of disease and activation of immune effectors.     

Use of isolated immature-stage B cells to understand negative selection and tolerance induction at the molecular level

Encounter with antigen by newly developing antigen receptor-positive B cells leads to negative selection. This process positions the B cell antigen receptor (BCR) in a central role for initiating the process of negative selection and suggests developmental regulation of its signaling. The observation that immature B cells are more susceptible to negative selection than are mature B cells has been demonstrated in a number of in vitro and in vivo model systems and support the idea of developmental regulation of BCR-initiated responses. Since identical antigen receptors are expressed on immature and mature B cells, the critical fate-determining distinction between these developmental stages must lie downstream of the receptor-ligand interaction itself, in the form of different BCR-linked signaling processes or with different secondary events occurring subsequent to BCR cross-linking. To address the first possibility, our laboratory and others have sought to define the differences in BCR-mediated signal transduction in immature and mature B lymphocytes. In this review article we will discuss current in vitro systems to study this question in primary, nontransformed murine B lymphocytes. In addition, we will discuss our previously published work in order to illustrate how these model systems have been useful in beginning to unravel the molecular basis for immune B cell negative selection and tolerance.     

Genetic basis for conversion of rough-to-smooth colony morphology in Actinobacillus actinomycetemcomitans

The basis of the rough-to-smooth conversion of Actinobacillus actinomycetemcomitans was examined. Smooth variants often contained mutations at the flp promoter region. Replacing the mutated flp promoter with the wild-type promoter restored the rough phenotype. The expression level of the flp promoter was approximately 100-fold lower in smooth than in rough strains. Mutations of the flp promoter are a cause of the rough-to-smooth conversion.