Monoclonal antibodies specific against tumors and viral infections

Monoclonal antibodies are monospecific antibodies. They are generated also by hybridoma technology. They are useful in target specific therapy against dangerous diseases. This property of monoclonal antibody offers us a new hope to combat deadly diseases where chemotherapy fails to produce specific treatment without adverse effects. This review article deals with the use of monoclonal antibodies specific against tumors and viral infections.     

Enhanced yield and homogeneity of buffalo growth hormone by an improved chromatographic protocol

Loss of buffalo Growth Hormone (buGH) in the various side fractions of standard buGH purification protocol has been determined quantitatively by direct binding ELISA and qualitatively by SDS-PAGE and Western blot analysis. Accounting result indicated that there was a considerable loss of buGH in the side fractions. An alternative protocol to prevent loss and to obtain a high yield of buGH has been developed by introducing anion exchange chromatography, QAE-Sephadex. This has resulted in a simple, reproducible three-step protocol. In this protocol, an extract obtained at 250 mM (NH4)2 SO4, pH 5.5, was loaded onto the QAE-Sephadex column in 0.1 M NH4 HCO3. At this salt concentration, the bulk of the buGH came as QAE unbound fraction. Some amount of buGH, together with contaminating proteins, was bound to QAE-Sephadex and these could be eluted with 1 M KCl. The immunopotency of the enriched buGH preparation "QUB" (QAE unbound fraction) in a direct binding ELISA was similar to that of the semi-pure buGH (ECS/APECS) preparation obtained using the standard protocol, but the yield was 4 times higher. The SDS-PAGE data showed that the banding pattern of standard semi-pure buGH and QUB were quite similar and QUB can be loaded onto the Sephacryl S-200 gel filtration chromatography to yield a highly purified buGH. SDS-PAGE and Western blot analyses showed the major band of buGH in QUB at the same position as in the case of standard buGH. It has also been demonstrated here that it is possible to separate buffalo prolactin (buPRL) and buGH on QAE-Sephadex.     

IL-4 inhibits bone-resorbing activity of mature osteoclasts by affecting NF-kappa B and Ca2+ signaling

IL-4 is an important immune cytokine that regulates bone homeostasis. We investigated the molecular mechanism of IL-4 action on bone-resorbing mature osteoclasts. Using a highly purified population of mature osteoclasts, we show that IL-4 dose-dependently inhibits receptor activator of NF-kappaB ligand (RANKL)-induced bone resorption by mature osteoclasts. We detected the existence of IL-4R mRNA in mature osteoclasts. IL-4 decreases TRAP expression without affecting multinuclearity of osteoclasts, and inhibits actin ring formation and migration of osteoclasts. Interestingly, IL-4 inhibition of bone resorption occurs through prevention of RANKL-induced nuclear translocation of p65 NF-kappaB subunit, and intracellular Ca(2+) changes. Moreover, IL-4 rapidly decreases RANKL-stimulated ionized Ca(2+) levels in the blood, and mature osteoclasts in IL-4 knockout mice are sensitive to RANKL action to induce bone resorption and hypercalcemia. Furthermore, IL-4 inhibits bone resorption and actin ring formation by human mature osteoclasts. Thus, we reveal that IL-4 acts directly on mature osteoclasts and inhibits bone resorption by inhibiting NF-kappaB and Ca(2+) signaling.     

Bystander effects in radiation-induced genomic instability

Exposure of GM10115 hamster-human hybrid cells to X-rays can result in the induction of chromosomal instability in the progeny of surviving cells. This instability manifests as the dynamic production of novel sub-populations of cells with unique cytogenetic rearrangements involving the "marker" human chromosome. We have used the comet assay to investigate whether there was an elevated level of endogenous DNA breaks in chromosomally unstable clones that could provide a source for the chromosomal rearrangements and thus account for the persistent instability observed. Our results indicate no significant difference in comet tail measurement between non-irradiated and radiation-induced chromosomally unstable clones. Using two-color fluorescence in situ hybridization we also investigated whether recombinational events involving the interstitial telomere repeat-like sequences in GM10115 cells were involved at frequencies higher than random processes would otherwise predict. Nine of 11 clones demonstrated a significantly higher than expected involvement of these interstitial telomere repeat-like sequences at the recombination junction between the human and hamster chromosomes. Since elevated levels of endogenous breaks were not detected in unstable clones we propose that epigenetic or bystander effects (BSEs) lead to the activation of recombinational pathways that perpetuate the unstable phenotype. Specifically, we expand upon the hypothesis that radiation induces conditions and/or factors that stimulate the production of reactive oxygen species (ROS). These reactive intermediates then contribute to a chronic pro-oxidant environment that cycles over multiple generations, promoting chromosomal recombination and other phenotypes associated with genomic instability.     

Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene

Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis and forms stable lysogens in which the Bxb1 genome is integrated into the host chromosome. Bxb1 encodes an integrase of the large serine recombinase family that catalyses integration and excision of the Bxb1 genome. We show here that Bxb1 integrates into a chromosomal attB site located within the 3' end of the groEL1 gene such that integration results in alteration of the C-terminal 21 amino acid residues. An integration-proficient plasmid vector containing the Bxb1 integrase gene and flanking DNA sequences efficiently transforms M. smegmatis via integration at attB. Bxb1-integrated recombinants are stable and fully compatible with L5 integration vectors. Strand exchange occurs within an 8 bp common core sequence present in attB and within an attP site situated immediately upstream of the phage integrase gene. Establishment of a defined in vitro system for Bxb1 integration shows that recombination occurs efficiently without requirement for high-energy cofactors, divalent metals, DNA supercoiling or additional proteins.     

11,12-epoxyeicosatrienoic acid (11,12-EET): structural determinants for inhibition of TNF-alpha-induced VCAM-1 expression

A series of 11,12-EET analogues were synthesized and compared using a human endothelial cell based TNF-alpha-induced VCAM-1 expression assay. The resulting data were used to map a putative recognition/binding domain for 11,12-EET.     

Innate immune responses to Rhodococcus equi

We examined innate immune responses to the intracellular bacterium Rhodococcus equi and show that infection of macrophages with intact bacteria induced the rapid translocation of NF-kappa B and the production of a variety of proinflammatory mediators, including TNF, IL-12, and NO. Macrophages from mice deficient in MyD88 failed to translocate NF-kappa B and produced virtually no cytokines in response to R. equi infection, implicating a TLR pathway. TLR4 was not involved in this response, because C3H/HeJ macrophages were fully capable of responding to R. equi infection, and because RAW-264 cells transfected with a dominant negative form of TLR4 responded normally to infection by R. equi. A central role for TLR2 was identified. A TLR2 reporter cell was activated by R. equi, and RAW-264 cells transfected with a dominant negative TLR2 exhibited markedly reduced cytokine responses to R. equi. Moreover, macrophages from TLR2(-/-) mice exhibited diminished cytokine responses to R. equi. The role of the surface-localized R. equi lipoprotein VapA (virulence-associated protein A), in TLR2 activation was examined. Purified rVapA activated a TLR2-specific reporter cell, and it induced the maturation of dendritic cells and the production of cytokines from macrophages. Importantly, TLR2(-/-)-deficient but not TLR4(-/-)-deficient mice were found to be compromised in their ability to clear a challenge with virulent R. equi. We conclude that the efficient activation of innate immunity by R. equi may account for the relative lack of virulence of this organism in immunocompetent adults.