Tolerization as a tool for generating novel monoclonal antibodies

Standard hybridoma production involves the fusion of spleen cells from an immunized mouse with a non-secretory murine myeloma cell line. While this technology has provided numerous reagents that are highly valuable, demand is now increasing for monoclonal antibodies which can distinguish between closely related antigens. Induction of tolerance towards common antigens enables the recovery of high-specificity reagents that have previously proved elusive. This review details a number of strategies using either complex protein mixtures or purified proteins as tolerogens and subsequent immunization with a closely related immunogen.     

Expression of toll-like receptors on B lymphocytes

Toll-like receptors (TLRs) are a family of trans-membrane receptors that play an important role in the innate immune system. Most studies examining the cellular expression of TLRs on immune cells have focussed on neutrophils, monocytes and dendritic cells, but there is little evidence of TLRs being expressed on lymphocytes. Using 3-colour flow cytometry, expression of TLR-1, TLR-2, TLR-3, TLR-4, and TLR-9 on peripheral blood lymphocyte populations was determined. Further examination of TLRs on CD5- and CD5+ CD19+ B cell subsets was performed. The binding of TLR1 and TLR9 antibodies was detected on 15-90% of resting B cells, but not on resting T-cells. The higher expression of TLR1 and TLR9 on CD5+ B cells compared to CD5- B cells may reflect the role of B1 cells in more primitive, less specific antibody responses.     

Medical applications of leukocyte surface molecules--the CD molecules

Leukocytes are the cells of the immune system and are centrally involved in defense against infection, in autoimmune disease, allergy, inflammation, and in organ graft rejection. Lymphomas and leukemias are malignancies of leukocytes, and the immune system is almost certainly involved in most other cancers. Each leukocyte expresses a selection of cell surface glycoproteins and glycolipids which mediate its interaction with antigen, with other components of the immune system, and with other tissues. It is therefore not surprising that the leukocyte surface molecules (CD molecules) have provided targets for diagnosis and therapy. Among the "celebrities" are CD20, a target for lymphoma therapeutic antibodies which earns $2 billion annually (and makes a significant difference to lymphoma patients), and CD4, the molecule used by the human immunodeficiency virus (HIV) as an entry portal into cells of the immune system. This short review provides a background to the CD molecules and antibodies against them, and summarizes research, diagnostic, and therapeutic applications of antibodies against these molecules.     

Structure and Activity of the Peptidyl-Prolyl Isomerase Domain from the Histone Chaperone Fpr4 toward Histone H3 Proline Isomerization

The FK506-binding protein (FKBP) family of peptidyl-prolyl isomerases (PPIases) is characterized by a common catalytic domain that binds to the inhibitors FK506 and rapamycin. As one of four FKBPs within the yeast Saccharomyces cerevisiae, Fpr4 has been described as a histone chaperone, and is in addition implicated in epigenetic function in part due to its mediation of cis-trans conversion of proline residues within histone tails. To better understand the molecular details of this activity, we have determined the solution structure of the Fpr4 C-terminal PPIase domain by using NMR spectroscopy. This canonical FKBP domain actively increases the rate of isomerization of three decapeptides derived from the N terminus of yeast histone H3, whereas maintaining intrinsic cis and trans populations. Observation of the uncatalyzed and Fpr4-catalyzed isomerization rates at equilibrium demonstrate Pro¹⁶ and Pro³⁰ of histone H3 as the major proline targets of Fpr4, with little activity shown against Pro³⁸. This alternate ranking of the three target prolines, as compared with affinity determination or the classical chymotrypsin-based fluorescent assay, reveals the mechanistic importance of substrate residues C-terminal to the peptidyl-prolyl bond.     

A Knock-in Mouse Model of Human PHD2 Gene-associated Erythrocytosis Establishes a Haploinsufficiency Mechanism

The central pathway for controlling red cell mass is the PHD (prolyl hydroxylase domain protein):hypoxia-inducible factor (HIF) pathway. HIF, which is negatively regulated by PHD, activates numerous genes, including ones involved in erythropoiesis, such as the ERYTHROPOIETIN (EPO) gene. Recent studies have implicated PHD2 as the key PHD isoform regulating red cell mass. Studies of humans have identified erythrocytosis-associated, heterozygous point mutations in the PHD2 gene. A key question concerns the mechanism by which human mutations lead to phenotypes. In the present report, we generated and characterized a mouse line in which a P294R knock-in mutation has been introduced into the mouse Phd2 locus to model the first reported human PHD2 mutation (P317R). Phd2^P294R/+ mice display a degree of erythrocytosis equivalent to that seen in Phd2^+/− mice. The Phd2^P294R/+-associated erythrocytosis is reversed in a Hif2a^+/−, but not a Hif1a^+/− background. Additional studies using various conditional knock-outs of Phd2 reveal that erythrocytosis can be induced by homozygous and heterozygous knock-out of Phd2 in renal cortical interstitial cells using a Pax3-Cre transgene or by homozygous knock-out of Phd2 in hematopoietic progenitors driven by a Vav1-Cre transgene. These studies formally prove that a missense mutation in PHD2 is the cause of the erythrocytosis, show that this occurs through haploinsufficiency, and point to multifactorial control of red cell mass by PHD2.     

The human leucocyte differentiation antigens （HLDA） workshops： the evolving role of antibodies in research, diagnosis and therapy

The 8^th International Workshop on Human Leucocyte Differentiation Antigens （chaired by HZ and managed by BS） was run over a 4-year period and culminated in a conference in December 2004. Here we review the achievements of the HLDA Workshops and provide links to information on CD molecules and antibodies against them, including the 93 new CDs assigned in the 8^th Workshop. We consider what remains to be achieved （including an estimate of the number of leucocyte surface molecules still to be discovered）, and how the field can best move forward.     

Cross-reactivity of anti-human chemokine receptor and anti-TNF family antibodies with common marmoset (Callithrix jacchus) leukocytes

The common marmoset (Callithrix jacchus) is a New World primate species frequently employed for immunological models of human disease. We used flow cytometry to screen a panel of new anti-human antibodies from the HLDA8 workshop to establish cross-reactivity with marmoset peripheral blood mononuclear cells. Seventy-seven antibodies were screened of which nine antibodies showed binding. Cross-reactivity of anti-human monoclonal antibodies with CC and CXC chemokine receptors CCR3, CCR6, CCR7, and CCR8 was demonstrated on untreated marmoset mononuclear cells. Stimulation of marmoset mononuclear cells with ConA and/or PMA-ionomycin resulted in an up-regulated expression of CXCR1, CXCR3, and CXCR4. The expression of TNF-family related molecules TACI and APRIL on marmoset mononuclear cells was also identified. These studies extend the range of cross-reactive antibodies to now include anti-chemokine and anti-TNF family antibodies for this important pre-clinical model species and should provide useful tools for investigation of immunological processes in marmoset monkey models.     

An economical 20 litre bench-top fermenter

We describe an economical 20 litre bench-top fermenter suitable for production of recombinant antibody fragments in bacterial expression systems. The bacterial culture contained within a polycarbonate carboy is mixed (400-600 rpm) and aerated (1 vessel vol./min) by a high-shear radial flow impeller mounted on a hollow stainless steel shaft, through which pressurised air is pumped. Air is dispersed as fine bubbles into the culture medium by the turbine impeller, without the need for a porous sparger. A stainless steel baffle stabilised by a gliding counterweight increases mixing. The components can easily be disassembled for cleaning and sterilisation. Temperature (range 20-37 degrees C) and pH (range 7.0-7.5) are controlled manually. Using the apparatus, it proved possible to achieve Escherichia coli cell culture densities equivalent to an optical density at 600 nm (OD(600)) of 30-32, compared with OD(600) 4-6 in shake flasks. A yield of 40 mg/litre/day of a recombinant antibody fragment was obtained with the fermenter, which was 15-fold more than the yield of 2.5mg/litre/day achieved in shake flasks. The fermenter may be particularly suited for research purposes.     

Isolation of antigen-specific B cells

Cell separation techniques are important in immunology. Major cell populations can be separated successfully with high purity. However, isolation of cells which are specific for particular antigens is more challenging because of the relatively small numbers of antigen-specific cells, and the lack of independent markers available to determine the purity of the isolated population. In this review, the literature describing three principal techniques used to separate antigen-specific cells has been reviewed. Particular emphasis has been placed on yield and purity; the two most important parameters of any purification method. The most promising isolation methods have used immunomagnetic sorting and multiparametric flow cytometric analysis.     

Soursop leaves (Annona muricata L.) endophytic fungi anticancer activity against HeLa cells

Cervical cancer causes many deaths in females worldwide, including in Indonesia. Several studies have reported that soursop (Annona muricata L.) leaves can be used to treat cervical cancer. This study aims to determine the use of endophytic fungi of A. muricata leaves extract as an ingredient that inhibits cervical cancer. The isolated endophytic fungi from various soursop leave accessions were grown in culture media, then extracted using ethyl acetate. The extract was then tested against anti-yeast, cervical cancer cells, and on normal cells as control using the MTT method. Five isolated fungi were selected based on the greatest inhibition in one concentration, and the inhibitory concentration 50 (IC₅₀) value was determined. The soursop leaves endophytic fungi extracts showed cytotoxicity against cervical cancer cells by inhibiting the multiplication of HeLa cancer cells in vitro. The Sir-SM2 endophytic fungi crude ethyl acetate extract showed high cytotoxicity to cervical cancer cells (HeLa cells) but less harmful to the normal Chang cells; therefore can be a natural anticancer. Identification based on morphology shows that the isolated Sir-SM2 endophytic fungi belong to the Penicillium genus, and molecular identification based on Internal Transcribed Spacer shows high similarities with Penicillium crustosum.