Developing Monoclonal Antibodies for Immunohistochemistry

The experiences of a laboratory which pioneered the application of monoclonal antibodies to diagnostic histochemistry is described. This was achieved in four key steps: (1) Monoclonal antibodies were successfully produced to replace the difficult-to-produce and limited polyclonal antibodies available for immunohistochemistry. (2) Monoclonal antibodies were produced to improve the immunoenzymatic detection of bound antibodies, using immunoperoxidase or alkaline phosphatase, increasing sensitivity and allowing the use of two chromogens when applied together. The availability of a reliable alkaline phosphatase-based detection allowed the detection of antigens in tissues with high endogenous peroxidase. (3) Methodologies were developed to unmask antigens not detected in routinely processed paraffin-embedded tissue. (4) Synthetic peptides were used as immunising antigens for the direct production of specific molecules of diagnostic interest. This was expanded to include recombinant proteins. Many reacted with fixed tissue and recognised homologous molecules in other species. In addition to these developments, the laboratory promoted the collaboration and training of researchers to spread the expertise of monoclonal production for diagnosis.

Detection of Escherichia coli by Combining an Affinity-Based Method with Contactless Atmospheric Pressure Ionization Mass Spectrometry

Escherichia coli are common pathogens, whereas E. coli O157:H7 is the most notorious E. coli strain, owing to its high virulence that can cause serious adverse effects and death. E. coli contains abundant peroxidases. Thus, the presence of E. coli can be determined by mixing E. coli with its substrate such as 3,5,3′,5′ tetramethylbenzidines (TMB) for endogenous peroxidase reactions. Under the presence of a high concentration of E. coli, colorless TMB turned to bluish, owing to the generation of the complexity of TMB and its oxidized TMB. To further reduce the detectable cell concentration, we developed an affinity-based method combined with an endogenous peroxidase reaction and mass spectrometric detection to detect E. coli. Affinity probes (diameter: ~20 µm) modified with maltose were generated for the enrichment of E. coli from sample solutions. E. coli trapped by the affinity probes was reacted with TMB in the presence of hydrogen peroxide for endogenous peroxidase reactions. Contactless atmospheric pressure ionization mass spectrometry was used for the detection of the reaction product, oxidized TMB (TMB cationic radical), to indicate the presence of target bacteria. The results showed that the developed method can be used to rapidly determine the presence of E. coli from a sample solution based on the detection of the TMB cationic radicals. The lowest detectable concentration of our method against E. coli O157:H7 in buffers and in complex juice samples was as low as ~100 cfu mL−1.

Dual Enzyme-Like Performances of PLGA Grafted Maghemite Nanocrystals and Their Synergistic Chemo/Chemodynamic Treatment for Human Lung Adenocarcinoma A549 Cells

In recent years, the emergence of non-toxic but catalytically active inorganic nanoparticles has attracted great attention for cancer treatment, but the therapeutic effect has been affected by the limited reactive oxygen species in tumors. Therefore, the combination of chemotherapy and chemodynamic therapy is regarded as a promising therapeutic strategy. In this paper, we reported the preparation and bioactivity evaluation of poly(lactic acid-co-glycolic acid) (PLGA) grafted-γ-Fe2O3 nanoparticles with dual response of endogenous peroxidase and catalase like activities. Our hypothesis is that PLGAgrafted γ-Fe2O3 nanoparticles could be used as a drug delivery system for the anti-tumor drug doxorubicin to inhibit the growth of lung adenocarcinoma A549 cells; meanwhile, based on its mimic enzyme properties, this kind of nanoparticles could be combined with doxorubicin in the treatment of A549 cells. Our experimental results showed that the PLGAgrafted γ-Fe2O3 nanoparticles could simulate the activity of catalase and decompose hydrogen peroxide into H2O and oxygen in neutral tumor microenvironment, thus reducing the oxidative damage caused by hydrogenperoxide to lung adenocarcinoma A549 cells. In acidic microenvironment, PLGA grafted γ-Fe2O3 nanoparticles could simulate the activity of peroxidase and effectively catalyze the decomposition of hydrogen peroxide to generate highly toxic hydroxyl radicals, which could cause the death of A549 cells. Furthermore, the synergistic effect of peroxidase-like activity of PLGA-grafted γ-Fe2O3 nanoparticles and doxorubicin could accelerate the apoptosisand destruction of A549 cells, thus enhancing the antitumor effect of doxorubicin-loaded PLGA-grafted γ-Fe2O3 nanoparticles. Therefore, this study provides an effective nanoplatform based on dual inorganic biomimetic nanozymes for the treatment of lung cancer.

Dual Enzyme-like Performances of PLGA Grafted Maghemite Nanocrystals and Their Synergistic Chemo/Chemodynamic Therapy for Human Lung Adenocarcinoma A549 Cells

Advancing nanocatalytic therapies of tumors formed on non-toxic but catalytically active inorganic nanoparticles (NPs) have aroused great interest in tumor therapy recently, but the limited reactive oxygen species within tumors may limit treatment efficiency. Therefore, the combination of chemotherapy and chemodynamic therapy is a promising treatment strategy. Herein, poly(lactic acid-co-glycolic acid) (PLGA) grafted-γ-Fe2O3 nanoparticles (NPPLGA) with dual response of endogenous peroxidase (POD)- and catalase (CAT)-like activities has been reported. On the one hand, the NPPLGA could serve as a drug delivery system for doxorubicin (DOX), an anti-tumor drug used to treat lung adenocarcinoma A549 cells. On the other hand, based on the mimetic enzyme properties of NPPLGA, it can be combined with DOX to treat lung cancer. The results show that the NPPLGA could be simulated the CAT-like activity to decompose hydrogen peroxide (H2O2) into H2O and O2 under neutral tumor microenvironment, so as to reduce the oxidative damage caused by H2O2 to lung adenocarcinoma A549 cells. Under acidic microenvironment, NPPLGA could simulate POD-like activity to effectively catalyze the decomposition of H2O2 to produce highly toxic hydroxyl radicals (•OH) to induce the death of tumor-cell through regular catalytic reaction of Fenton. Furthermore, the POD-like activity of NPPLGA synergistic with DOX can promote the apoptosis and destruction of A549 cells and enhance the antitumor impact of DOX-NPPLGA. Therefore, this study provides an efficacious dual inorganic biomimetic nanozyme-based nanoplatform for lung tumor treatment.

Hydrocephalus-Induced Changes in the Endocrine Hypothalamus of the Hamster

Hydroceplanllus-induced changes in intracranial pressure (ICP), ventricular size, periventricular edema and erytlirocyte density(ED), median eminence thickness, and pituitary gland morphology were determined in adult female hamsters (Mesocricetus auratus). Hydrocephalus (HC) was induced by intracisternal injection of kaolin (0.03 ml, 50 mg/ml saline); controls did not receive kaolin. Animais were chronically cannulated and ICP measured periodically (Camino V 420). At selecied times control and treated animals were decapitated, brains removed with the pituitary gland attached and fixed by immersion in formalin. Coronal brain slices containing the paraventricular nuclei (PVN), supraoptic nuclei (SON), suprachiosmatic nuclei (SCN), subfornical organ (SFO) and median eminence (ME) were prepared using a rodent brain matrix slicer and ventricular arca was quantitated by morphometric analysis (Jandel Video Analysis System). Samples were collected from the cortex and periventricular white matter (containing the PVN, SON, and SCN) and the median eminence. The whole pituitary gland was also collected for determination of specific gravity by linear density column method. Erythrocyte Densiry was assessed by immersing hydrocephalic brain slices containing the select nuclei in Karnoviskys fixative on day 9 post HC induction, and in simerythrocytes stained for endogenous peroxidase by tretramethyl benzidine cylochemistry to facilitate visualization. Previous studies in our laboratory have establislied that HC can result in alterations in estrous cycle, water balance, food intake and growth. The result in part from decrease perfusion of the endocrine liypothalamus and pituitary gland, edema and pressure-induced changes in the median eminence structures.