Targeting HER2: A report on the in vitro and in vivo pre-clinical data supporting trastuzumab as a radioimmunoconjugate for clinical trials

The potential of the HER2-targeting antibody trastuzumab as a radioimmunoconjugate useful for both imaging and therapy was investigated. Conjugation of trastuzumab with the acyclic bifunctional chelator CHX-A″-DTPA yielded a chelate:protein ratio of 3.4 ± 0.3; the immunoreactivity of the antibody unaffected. Radiolabeling was efficient, routinely yielding a product with high specific activity. Tumor targeting was evaluated in mice bearing subcutaneous (s.c.) xenografts of colorectal, pancreatic, ovarian and prostate carcinomas. High uptake of the radioimmunoconjugate, injected intravenously (i.v.), was observed in each of the models and the highest tumor %ID/g (51.18 ± 13.58) was obtained with the ovarian (SKOV-3) tumor xenograft. Specificity was demonstrated by the absence of uptake of ¹¹¹In-trastuzumab by melanoma (A375) s.c. xenografts and ¹¹¹In-HuIgG by s.c. LS-174T xenografts. Minimal uptake of i.v. injected ¹¹¹In-trastuzumab in normal organs was confirmed in non-tumor-bearing mice. The in vivo behavior of ¹¹¹In-trastuzumab in mice bearing intraperitoneal (i.p.) LS-174T tumors resulted in a tumor %ID/g of 130.85 ± 273.34 at 24 h. Visualization of tumor, s.c. and i.p. xenografts was achieved by γ-scintigraphy and PET imaging. Blood pool was evident as expected but cleared over time. The blood pharmacokinetics of i.v. and i.p. injected ¹¹¹In-trastuzumab was determined in mice with and without tumors. The data from these in vitro and in vivo studies supported advancement of radiolabeled trastuzumab into two clinical studies, a Phase 0 imaging study in the Molecular Imaging Program of the National Cancer Institute and a Phase 1 radioimmunotherapy study at the University of Alabama.Key words: monoclonal antibody, HER2, trastuzumab, radioimmunodiagnosis, radioimmunotherapy     

Phosphorus supply drives rapid turnover of membrane phospholipids in the diatom Thalassiosira pseudonana

In low-phosphorus (P) marine systems, phytoplankton replace membrane phospholipids with non-phosphorus lipids, but it is not known how rapidly this substitution occurs. Here, when cells of the model diatom Thalassiosira pseudonana were transferred from P-replete medium to P-free medium, the phospholipid content of the cells rapidly declined within 48 h from 45±0.9 to 21±4.5% of the total membrane lipids; the difference was made up by non-phosphorus lipids. Conversely, when P-limited T. pseudonana were resupplied with P, cells reduced the percentage of their total membrane lipids contributed by a non-phosphorus lipid from 43±1.5 to 7.3±0.9% within 24 h, whereas the contribution by phospholipids rose from 2.2±0.1 to 44±3%. This dynamic phospholipid reservoir contained sufficient P to synthesize multiple haploid genomes, suggesting that phospholipid turnover could be an important P source for cells. Field observations of phytoplankton lipid content may thus reflect short-term changes in P supply and cellular physiology, rather than simply long-term adjustment to the environment.     

ANTICANCER MEDICINES (DOXORUBICIN AND METHOTREXATE) CONJUGATED WITH MAGNETIC NANOPARTICLES FOR TARGETING DRUG DELIVERY THROUGH IRON

The uptake of iron is increased by cancer cells. Iron magnetic nanoparticles (MNP) can be used as a nanovehicle for immobilization of anticancer medicines and to integrate them at a target site. The anticancer medicines doxorubicin (DOX) and methotrexate (MTX) were immobilized separately and in combination onto MNP by a glutaraldehyde activation method and confirmed by magnetic nanoparticles linked immunosorbent assay (MagLISA) and Fourier-transform infrared (FTIR) spectroscopy. The phenol peaks of DOX and MTX at 2896.6 cm^?1 to 2912.5 cm^?1 in FTIR spectra of immobilized medicines indicated the conjugation. Affinity-purified anti-DOX and anti-MTX antibodies were used to evaluate the coupling of DOX and MTX onto MNP, and the binding was found 34.6% to 37.2% and 51.8% to 54.3% separately, respectively. The immobilization of DOX and MTX in combination onto MNP was 18% and 27%, respectively. HeLa and B cells were cultured with DOX-MNP, MTX-MNP, and DOX-MNP-MTX separately, and MagLISA indicated that the binding of DOX-MNP/MTX-MNP was 41.5% to 45% with HeLa cells and 20% to 26% with B cells. No significant difference was observed in binding of DOX-MNP-MTX with HeLa and B cells. Results also indicated that the release of medicines at pH 5.0 is more (39% to 44%) than at pH 7.4 (3.7% to 10.2%). Sixteen to 22% more killing effect was observed on HeLa cells than on B cells. In immunohistochemical staining, more deposition of brown color on HeLa cells than on B cells may be due to more expression of iron-binding sites on cancer cells. The dual property of MNP can be used for binding of medicines and for targeting drug delivery.     

Ameliorative role of aspirin in paraquat‐induced lung toxicity via mitochondrial mechanisms

Paraquat (PQ) has accounted for numerous suicide attempts in developing countries. Aspirin (ASA) as an adjuvant treatment in PQ poisoning has an ameliorative role. And, it's uncoupling of mitochondrial oxidative phosphorylation role has been well established. The current study aimed at examining the aspirin mechanism on lung mitochondria of rats exposed to PQ. Male rats were randomly allocated in five groups: Control group, PQ group (50 mg/kg; orally, only on the first day), and PQ + ASA (100, 200, and 400 mg/kg; i.p.) groups for 3 weeks. Mitochondrial indices and respiratory chain‐complex activities were determined. PQ induced lung interstitial fibrosis; however, ASA (400 mg/kg) led to decrease in this abnormal alteration. In comparison with PQ group, complex II and IV activity, and adenosine triphosphate content in ASA groups had significantly increased; however, reactive oxygen species production, mitochondrial membrane permeabilization, and mitochondrial swelling were significantly reduced. In conclusion, aspirin can alleviate lung injury induced by PQ poisoning by improving mitochondrial dynamics.     

Genetic islands of <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> strains NEM316 and 2603VR and their presence in other Group B Streptococcal strains

Background  

Streptococcus agalactiae (Group B Streptococcus; GBS) is a major contributor to obstetric and neonatal bacterial sepsis. Serotype III strains cause the majority of late-onset sepsis and meningitis in babies, and thus appear to have an enhanced invasive capacity compared with the other serotypes that cause disease predominantly in immunocompromised pregnant women. We compared the serotype III and V whole genome sequences, strains NEM316 and 2603VR respectively, in an attempt to identify genetic attributes of strain NEM316 that might explain the propensity of strain NEM316 to cause late-onset disease in babies. Fourteen putative pathogenicity islands were described in the strain NEM316 whole genome sequence. Using PCR- and targeted microarray- strategies, the presence of these islands were assessed in a diverse strain collection including 18 colonizing isolates from healthy pregnant women, and 13 and 8 invasive isolates from infants with early- and late-onset sepsis, respectively.     

Novel synthesis of mannosamine conjugated magnetic nanoparticles for purification and stabilization of human lysosomal β-mannosidase

Human β-mannosidase (MANB) was purified to homogeneity directly from lysosomes by using mannosamine conjugated magnetic (Fe₃O₄) nanoparticles, DE-52 cellulose, and sephadex G-200 chromatography. Fe₃O₄ nanoparticles were synthesized and utilized ammonia to attach the amino group on the nanoparticles. The particles were covalently attached with D-mannosamine by cross linker glutaraldehyde and confirmed by FTIR spectroscopy. In FTIR analysis, the peaks appeared at 2,356.6 cm⁻¹ for −N = CH linkage and at 3,378.4 cm⁻¹, 3,664.9 cm⁻¹ for −OH groups confirmed the conjugation of D-mannosamine with Fe₃O₄ nanoparticles. Results showed a single band of 97 kDa of purified MANB in SDS-PAGE. The isoelectric point was 4.5 and the K_m and Vmax values were 2.51 mM and 0.315 μM/min/mg, respectively. The purification fold was 329 with 68% yield. The optimal activity was at pH 5.0 and 75% activity was stable in 20% glycerol at 4°C. The enzyme activity was inhibited by Ni²⁺, Zn²⁺, Cd²⁺, Cu²⁺, Mo²⁺, Ag⁺¹, iodoacetate, SDS, DMF, DMSO, ethanol, and acetone; slightly reduced by Pb²⁺, Co²⁺, EDTA, DTT, and β-mercaptoethanol. The activity was not affected by Mg²⁺, Mn²⁺, Sn²⁺, Ca²⁺, Fe³⁺, PMSF, Triton X-100, D-mannosamine, D-mannose, D-mannitol, D-glucose, and D-fructose. The homogeneity of MANB enzyme was further confirmed by 2D-PAGE and immunoblot. This is the first novel report of conjugation of D-mannosamine with Fe₃O₄ nanoparticles for purification of human MANB enzyme.     

FREE CHOICE PROFILING OF CHILEAN GOAT CHEESE

Different goat cheeses from Chile were studied by Free-Choice Profile (FCP) analysis. Generalized Procrustes Analysis (GPA) applied to FCP data permitted differentiation between samples and informed on the attributes responsible for the observed differences. Appearance was a dominant factor in discriminating samples and to a lesser degree textural variables were also correlated with GPA dimensions. In acceptability the fresh cheeses were significantly preferred over the ripened ones.     

TGF-beta and IL-10 regulation of IFN-gamma produced in Th2-type schistosome granulomas requires IL-12

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) regulate CD4+ T cell interferon-gamma (IFN-gamma) secretion in schistosome granulomas. The role of IL-12 was determined using C57BL/6 and CBA mice. C57BL/6 IL-4-/- granuloma cells were stimulated to produce IFN-gamma when cultured with IL-10 or TGF-beta neutralizing monoclonal antibody. In comparison, C57BL/6 wild-type (WT) control granuloma cells produced less IFN-gamma. IL-12, IL-18, and soluble egg antigen stimulated IFN-gamma release from C57BL/6 IL-4-/- and WT mice. IFN-gamma production in C57 IL-4-/- and WT granulomas was IL-12 dependent, because IL-12 blockade partly abrogated IFN-gamma secretion after stimulation. All granuloma cells released IL-12 (p70 and p40), and IL-12 production remained constant after anti-TGF-beta, anti-IL-10, recombinant IL-18, or antigen stimulation. C57 WT and IL-4-/- mouse granuloma cells expressed IL-12 receptor (IL-12R) beta1-subunit mRNA but little beta2 mRNA. TGF-beta or IL-10 blockade did not influence beta1 or beta2 mRNA expression. CBA mouse dispersed granuloma cells released no measurable IFN-gamma, produced IL-12 p70 and little p40, and expressed IL-12R beta2 and little beta1 mRNA. In T helper 2 (Th2) granulomas of C57BL/6 WT and IL-4-/- mice, cells produce IL-12 (for IFN-gamma production) and IL-10 and TGF-beta modulate IFN-gamma secretion via mechanisms independent of IL-12 and IL-12R mRNA regulation. We found substantial differences in control of granuloma IFN-gamma production and IL-12 circuitry in C57BL/6 and CBA mice.