Human monoclonal antibodies to West Nile virus identify epitopes on the prM protein

AbstractHybridoma cell lines (2E8, 8G8 and 5G12) producing fully human monoclonal antibodies (hMAbs) specific for the pre-membrane (prM) protein of West Nile virus (WNV) were prepared using a human fusion partner cell line, MFP-2, and human peripheral blood lymphocytes from a blood donor diagnosed with WNV fever in 2004. Using site-directed mutagenesis of a WNV-like particle (VLP) we identified 4 amino acid residues in the prM protein unique to WNV and important in the binding of these hMAbs to the VLP. Residues V19 and L33 are important epitopes for the binding of all three hMAbs. Mutations at residue, T20 and T24 affected the binding of hMAbs, 8G8 and 5G12 only. These hMAbs did not significantly protect AG129 interferon-deficient mice or Swiss Webster outbred mice from WNV infection

A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen

<p>Abstract</p> <p>Background</p> <p>We have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients.</p> <p>Methods</p> <p>The current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification.</p> <p>Results</p> <p>By application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein.</p> <p>Conclusion</p> <p>We have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.</p

Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13

The emergence of artemisinin (ART) resistance in Plasmodium falciparum intra-erythrocytic parasites has led to increasing treatment failure rates with first-line ART-based combination therapies in Southeast Asia. Decreased parasite susceptibility is caused by K13 mutations, which are associated clinically with delayed parasite clearance in patients and in vitro with an enhanced ability of ring-stage parasites to survive brief exposure to the active ART metabolite dihydroartemisinin. Herein, we describe a panel of K13-specific monoclonal antibodies and gene-edited parasite lines co-expressing epitope-tagged versions of K13 in trans. By applying an analytical quantitative imaging pipeline, we localize K13 to the parasite endoplasmic reticulum, Rab-positive vesicles, and sites adjacent to cytostomes. These latter structures form at the parasite plasma membrane and traffic hemoglobin to the digestive vacuole wherein artemisinin-activating heme moieties are released. We also provide evidence of K13 partially localizing near the parasite mitochondria upon treatment with dihydroartemisinin. Immunoprecipitation data generated with K13-specific monoclonal antibodies identify multiple putative K13-associated proteins, including endoplasmic reticulum-resident molecules, mitochondrial proteins, and Rab GTPases, in both K13 mutant and wild-type isogenic lines. We also find that mutant K13-mediated resistance is reversed upon co-expression of wild-type or mutant K13. These data help define the biological properties of K13 and its role in mediating P. falciparum resistance to ART treatment

Native human autoantibodies targeting GIPC1 identify differential expression in malignant tumors of the breast and ovary

<p>Abstract</p> <p>Background</p> <p>We have been studying the native humoral immune response to cancer and have isolated a library of fully human autoantibodies to a variety of malignancies. We previously described the isolation and characterization of two fully human monoclonal antibodies, 27.F7 and 27.B1, from breast cancer patients that target the protein known as GIPC1, an accessory PDZ-domain binding protein involved in regulation of G-protein signaling. Human monoclonal antibodies, 27.F7 and 27.B1, to GIPC1 demonstrate specific binding to malignant breast cancer tissue with no reactivity with normal breast tissue.</p> <p>Methods</p> <p>The current study employs cELISA, flow cytometry, Western blot analysis as well as immunocytochemistry, and immunohistochemistry. Data is analyzed statistically with the Fisher one-tail and two-tail tests for two independent samples.</p> <p>Results</p> <p>By screening several other cancer cell lines with 27.F7 and 27.B1 we found consistently strong staining of other human cancer cell lines including SKOV-3 (an ovarian cancer cell line). To further clarify the association of GIPC1 with breast and ovarian cancer we carefully studied 27.F7 and 27.B1 using immunocytochemical and immunohistochemical techniques. An immunohistochemical study of normal ovarian tissue, benign, borderline and malignant ovarian serous tumors, and different types of breast cancer revealed high expression of GIPC1 protein in neoplastic cells. Interestingly, antibodies 27.F7 and 27.B1 demonstrate differential staining of borderline ovarian tumors. Examination of different types of breast cancer demonstrates that the level of GIPC1 expression depends on tumor invasiveness and displays a higher expression than in benign tumors.</p> <p>Conclusion</p> <p>The present pilot study demonstrates that the GIPC1 protein is overexpressed in ovarian and breast cancer, which may provide an important diagnostic and prognostic marker and will constitute the basis for further study of the role that this protein plays in malignant diseases. In addition, this study suggests that human monoclonal antibodies 27.F7 and 27.B1 should be further evaluated as potential diagnostic tools.</p

Tissue ACE phenotyping in lung cancer.

BACKGROUND:Pulmonary vascular endothelium is the main metabolic site for Angiotensin I-Converting Enzyme (ACE)-mediated degradation of several biologically-active peptides (angiotensin I, bradykinin, hemo-regulatory peptide Ac-SDKP). Primary lung cancer growth and lung cancer metastases decrease lung vascularity reflected by dramatic decreases in both lung and serum ACE activity. We performed precise ACE phenotyping in tissues from subjects with lung cancer. METHODOLOGY:ACE phenotyping included: 1) ACE immunohistochemistry with specific and well-characterized monoclonal antibodies (mAbs) to ACE; 2) ACE activity measurement with two ACE substrates (HHL, ZPHL); 3) calculation of ACE substrates hydrolysis ratio (ZPHL/HHL ratio); 4) the pattern of mAbs binding to 17 different ACE epitopes to detect changes in ACE conformation induced by tumor growth (conformational ACE fingerprint). RESULTS:ACE immunostaining was dramatically decreased in lung cancer tissues confirmed by a 3-fold decrease in ACE activity. The conformational fingerprint of ACE from tumor lung tissues differed from normal lung (6/17 mAbs) and reflected primarily higher ACE sialylation. The increase in ZPHL/HHL ratio in lung cancer tissues was consistent with greater conformational changes of ACE. Limited analysis of the conformational ACE fingerprint in normal lung tissue and lung cancer tissue form the same patient suggested a remote effect of tumor tissue on ACE conformation and/or on "field cancerization" in a morphologically-normal lung tissues. CONCLUSIONS/SIGNIFICANCE:Local conformation of ACE is significantly altered in tumor lung tissues and may be detected by conformational fingerprinting of human ACE

Tissue ACE phenotyping in lung cancer

Background Pulmonary vascular endothelium is the main metabolic site for Angiotensin I-Converting Enzyme (ACE)-mediated degradation of several biologically-active peptides (angiotensin I, bradykinin, hemo-regulatory peptide Ac-SDKP). Primary lung cancer growth and lung cancer metastases decrease lung vascularity reflected by dramatic decreases in both lung and serum ACE activity. We performed precise ACE phenotyping in tissues from subjects with lung cancer. Methodology ACE phenotyping included: 1) ACE immunohistochemistry with specific and well-characterized monoclonal antibodies (mAbs) to ACE; 2) ACE activity measurement with two ACE substrates (HHL, ZPHL); 3) calculation of ACE substrates hydrolysis ratio (ZPHL/HHL ratio); 4) the pattern of mAbs binding to 17 different ACE epitopes to detect changes in ACE conformation induced by tumor growth (conformational ACE fingerprint). Results ACE immunostaining was dramatically decreased in lung cancer tissues confirmed by a 3-fold decrease in ACE activity. The conformational fingerprint of ACE from tumor lung tissues differed from normal lung (6/17 mAbs) and reflected primarily higher ACE sialylation. The increase in ZPHL/HHL ratio in lung cancer tissues was consistent with greater conformational changes of ACE. Limited analysis of the conformational ACE fingerprint in normal lung tissue and lung cancer tissue form the same patient suggested a remote effect of tumor tissue on ACE conformation and/or on "field cancerization" in a morphologically-normal lung tissues. Conclusions/Significance Local conformation of ACE is significantly altered in tumor lung tissues and may be detected by conformational fingerprinting of human ACE.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Human monoclonal antibodies to West Nile virus identify epitopes on the prM protein

AbstractHybridoma cell lines (2E8, 8G8 and 5G12) producing fully human monoclonal antibodies (hMAbs) specific for the pre-membrane (prM) protein of West Nile virus (WNV) were prepared using a human fusion partner cell line, MFP-2, and human peripheral blood lymphocytes from a blood donor diagnosed with WNV fever in 2004. Using site-directed mutagenesis of a WNV-like particle (VLP) we identified 4 amino acid residues in the prM protein unique to WNV and important in the binding of these hMAbs to the VLP. Residues V19 and L33 are important epitopes for the binding of all three hMAbs. Mutations at residue, T20 and T24 affected the binding of hMAbs, 8G8 and 5G12 only. These hMAbs did not significantly protect AG129 interferon-deficient mice or Swiss Webster outbred mice from WNV infection

Potential use of melatonergic drugs in analgesia : mechanisms of action

Melatonin is a remarkable molecule with diverse physiological functions. Some of its effects are mediated by receptors while other, like cytoprotection, seem to depend on direct and indirect scavenging of free radicals not involving receptors. Among melatonin´s many effects, its antinociceptive actions have attracted attention. When given orally, intraperitoneally, locally, intrathecally or through intracerebroventricular routes, melatonin exerts antinociceptive and antiallodynic actions in a variety of animal models. These effects have been demonstrated in animal models of acute pain like the tail-flick test, formalin test or endotoxin-induced hyperalgesia as well as in models of neuropathic pain like nerve ligation. Glutamate, gamma-aminobutyric acid, and particularly, opioid neurotransmission have been demonstrated to be involved in melatonin´s analgesia. Results using melatonin receptor antagonists support the participation of melatonin receptors in melatonin´s analgesia. However, discrepancies between the affinity of the receptors and the very high doses of melatonin needed to cause effects in vivo raise doubts about the uniqueness of that physiopathological interpretation. Indeed, melatonin could play a role in pain through several alternative mechanisms including free radicals scavenging or nitric oxide synthase inhibition. The use of melatonin analogs like the MT1/MT2 agonist ramelteon, which lacks free radical scavenging activity, could be useful to unravel the mechanism of action of melatonin in analgesia. Melatonin has a promising role as an analgesic drug that could be used for alleviating pain associated with cancer, headache or surgical procedures

Malaria : therapeutic implications of melatonin

Abstract: Malaria, which infects more than 300 million people annually, is a serious disease. Epidemiological surveys indicate that of those who are affected, malaria will claim the lives of more than one million individuals, mostly children. There is evidence that the synchronous maturation of Plasmodium falciparum, the parasite that causes a severe form of malaria in humans and P. chabaudi, responsible for rodent malaria, could be linked to circadian changes in melatonin concentration. In vitro melatonin stimulates the growth and development of P. falciparum through the activation of specific melatonin receptors coupled to phospholipase-C activation and the concomitant increase of intracellular Ca2+. The Ca2+ signaling pathway is important to stimulate parasite transition from the trophozoite to the schizont stage, the final stage of intraerythrocytic cycle, thus promoting the rise of parasitemia. Either pinealectomy or the administration of the melatonin receptor blocking agent luzindole desynchronizes the parasitic cell cycle. Therefore the use of melatonin antagonists could be a novel therapeutic approach for controlling the disease. On the other hand, the complexity of melatonin’s action in malaria is underscored by the demonstration that treatment with high doses of melatonin is actually beneficial for inhibiting apoptosis and liver damage resulting from the oxidative stress in malaria. The possibility that the coordinated administration of melatonin antagonists (to impair the melatonin signal that synchronizes P. falciparum) and of melatonin in doses high enough to decrease oxidative damage could be a novel approach in malaria treatment is discussed

Malaria : therapeutic implications of melatonin

Malaria, which infects more than 300 million people annually, is a serious disease. Epidemiological surveys indicate that of those who are affected, malaria will claim the lives of more than one million individuals, mostly children. There is evidence that the synchronous maturation of Plasmodium falciparum, the parasite that causes a severe form of malaria in humans and P. chabaudi, responsible for rodent malaria, could be linked to circadian changes in melatonin concentration. In vitro melatonin stimulates the growth and development of P. falciparum through the activation of specific melatonin receptors coupled to phospholipase-C activation and the concomitant increase of intracellular Ca2+. The Ca2+ signaling pathway is important to stimulate parasite transition from the trophozoite to the schizont stage, the final stage of intraerythrocytic cycle, thus promoting the rise of parasitemia. Either pinealectomy or the administration of the melatonin receptor blocking agent luzindole desynchronizes the parasitic cell cycle. Therefore the use of melatonin antagonists could be a novel therapeutic approach for controlling the disease. On the other hand, the complexity of melatonin’s action in malaria is underscored by the demonstration that treatment with high doses of melatonin is actually beneficial for inhibiting apoptosis and liver damage resulting from the oxidative stress in malaria. The possibility that the coordinated administration of melatonin antagonists (to impair the melatonin signal that synchronizes P. falciparum) and of melatonin in doses high enough to decrease oxidative damage could be a novel approach in malaria treatment is discussed