Analysis of sFlt Isoforms as Biomarkers for the Development of Preeclampsia

Preeclampsia is a multi-system disorder characterized by hypertension, edema and proteinuria affecting between 5-10% of pregnancies. A subset of cases progress to severe preeclampsia with exacerbated hypertension/proteinuria and evidence of nervous system, liver and/or kidney dysfunction, in addition to fetal growth restriction. Soluble fms-like tyrosine kinase-1 (sFlt) is minimally expressed in many tissues, including the placenta, and is a circulating antagonist to vascular endothelial growth factor. With progression of pregnancy, sFlt levels significantly rise, especially in women who develop preeclampsia. Diagnostic tests to predict preeclampsia in pregnant women are limited and current tests measure total sFlt in relationship to placental growth factor with varying sensitivity and specificity. We hypothesized that a pregnancy-specific splice variant of sFlt (sFlt1-14), almost exclusively expressed by the placenta, would serve as an improved serum biomarker for the development of preeclampsia. Monoclonal antibodies (mAbs) were developed that specifically bind the two predominant isoforms of sFlt (sFlt1 and sFlt1-14) by hybridoma generation from wild type mice immunized with c-terminal peptides of the two isoforms. Western blot, ELISA and affinity analysis indicated the mAbs were specific for sFlt1 or sFlt1-14 splice variants and recognized these proteins in biological fluids (amniotic fluid or serum). A quantitative capture ELISA was developed whereby total sFlt in biological fluid is captured by a unique human mAb and specific levels of sFlt1 or sFlt1-14 are detected by their respective mouse mAb, followed by anti-murine secondary antibody development. Using recombinant sFlt1 or sFlt1-14 as standards, these endogenous proteins were quantified in commercially available third trimester human pregnant sera. Future studies will measure these isoforms in sera prospectively collected from women with known outcomes of a healthy pregnancy or preeclampsia and the ability of absolute quantitation of the isoform(s) or a ratio of the two to predict the likely onset and severity of preeclampsia will be evaluated

Pre-exposure prophylaxis with OspA-specific human monoclonal antibodies protects mice against tick transmission of Lyme disease spirochetes

Background. Tick transmission of Borrelia spirochetes to humans results in significant morbidity from Lyme disease worldwide. Serum concentrations of antibodies against outer surface protein A (OspA) were shown to correlate with protection from infection with Borrelia burgdorferi, the primary cause of Lyme disease in the United States. Methods. Mice transgenic for human immunoglobulin genes were immunized with OspA protein of B. burgdorferi to generate human monoclonal antibodies (HuMabs) against OspA. HuMabs were generated and tested in in vitro borreliacidal assays and animal protection assays. Results. Nearly 100 unique OspA specific HuMabs were generated and four HuMabs (221-7, 857-2, 319-44, and 212-55) were selected as lead candidates based on borreliacidal activity. HuMab 319-44, 857-2 and 212-55 were borreliacidal against one or two Borrelia genospecies, whereas 221-7 was borreliacidal (IC50 \u3c 1nM) against B. burgdorferi, B. afzelii and B. garinii, the three main genospecies endemic in the US, Europe and Asia. All four HuMabs completely protected mice from infection at 10 mg/kg in a murine model of tick-mediated transmission of B. burgdorferi. Conclusions. Our study indicates that OspA-specific HuMabs can prevent the transmission of Borrelia and administration of these antibodies could be employed as pre-exposure prophylaxis for Lyme disease

Human monoclonal antibodies directed against toxins A and B prevent Clostridium difficile-induced mortality in hamsters

Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea, and recent outbreaks of strains with increased virulence underscore the importance of identifying novel approaches to treat and prevent relapse of Clostridium difficile-associated diarrhea (CDAD). CDAD pathology is induced by two exotoxins, toxin A and toxin B, which have been shown to be cytotoxic and, in the case of toxin A, enterotoxic. In this report we describe fully human monoclonal antibodies (HuMAbs) that neutralize these toxins and prevent disease in hamsters. Transgenic mice carrying human immunoglobulin genes were used to isolate HuMAbs that neutralize the cytotoxic effects of either toxin A or toxin B in cell-based in vitro neutralization assays. Three anti-toxin A HuMAbs (3H2, CDA1, and 1B11) could all inhibit the enterotoxicity of toxin A in mouse intestinal loops and the in vivo toxicity in a systemic mouse model. Four anti-toxin B HuMAbs (MDX-1388, 103-174, 1G10, and 2A11) could neutralize cytotoxicity in vitro, although systemic toxicity in the mouse could not be neutralized. Anti-toxin A HuMAb CDA1 and anti-toxin B HuMAb MDX-1388 were tested in the well-established hamster model of C. difficile disease. CDA1 alone resulted in a statistically significant reduction of mortality in hamsters; however, the combination treatment offered enhanced protection. Compared to controls, combination therapy reduced mortality from 100% to 45% (P\u3c0.0001) in the primary disease hamster model and from 78% to 32% (P\u3c0.0001) in the less stringent relapse model

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Effect of progesterone in the development of gestational hypertension in the SHHF/Mcc-fa(cp) rat

This study, sponsored by the University of Massachusetts Medical School, examined the relationship between progesterone and gestational hypertension in spontaneously hypertensive heart failure (SHHF) rats. Blood pressure, water consumption, and blood and urine analyses displayed differences between SHHF and normotensive Wistar-Kyoto (WKY) rats. SHHF rats exhibited higher levels of progesterone receptor A in the kidney and lower levels of serum progesterone compared to WKY rats. These differences were associated with the onset of hypertension in pregnant SHHF rats, possibly playing a role in the disease

Waste disposal in the tambon of Sang Khom, Thailand

Our project team evaluated the current waste disposal system in the villages of Sang Khom, province of Udon Thani, Thailand, by obtaining a physical description of the system and assessing its potential health and environmental problems. We assessed the feasibility of alternative systems by considering the technical feasibility and social and environmental implications. We considered Sang Khon's available budget to create recommendations for improving their wasted disposal system. We also recommended education programs to increase community awareness concerning waste disposal

Pre-exposure Prophylaxis With OspA-Specific Human Monoclonal Antibodies Protects Mice Against Tick Transmission of Lyme Disease Spirochetes

BACKGROUND: Tick transmission of Borrelia spirochetes to humans results in significant morbidity from Lyme disease worldwide. Serum concentrations of antibodies against outer surface protein A (OspA) were shown to correlate with protection from infection with Borrelia burgdorferi, the primary cause of Lyme disease in the United States. METHODS: Mice transgenic for human immunoglobulin genes were immunized with OspA from B. burgdorferi to generate human monoclonal antibodies (HuMabs) against OspA. HuMabs were generated and tested in in vitro borreliacidal assays and animal protection assays. RESULTS: Nearly 100 unique OspA-specific HuMabs were generated, and 4 HuMabs (221-7, 857-2, 319-44, and 212-55) were selected as lead candidates on the basis of borreliacidal activity. HuMabs 319-44, 857-2, and 212-55 were borreliacidal against 1 or 2 Borrelia genospecies, whereas 221-7 was borreliacidal (half maximal inhibitory concentration, \u3c 1 nM) against B. burgdorferi, Borrelia afzelii, and Borrelia garinii, the 3 main genospecies endemic in the United States, Europe, and Asia. All 4 HuMabs completely protected mice from infection at 10 mg/kg in a murine model of tick-mediated transmission of B. burgdorferi CONCLUSIONS: Our study indicates that OspA-specific HuMabs can prevent the transmission of Borrelia and that administration of these antibodies could be employed as preexposure prophylaxis for Lyme disease

Circulating Levels of sFlt1 Splice Variants as Predictive Markers for the Development of Preeclampsia

Angiogenic biomarkers, including soluble fms-like tyrosine kinase 1 (sFlt1), are thought to be predictors of preeclampsia onset; however, improvement is needed before a widespread diagnostic test can be utilized. Here we describe the development and use of diagnostic monoclonal antibodies specific to the two main splice variants of sFlt1, sFlt1-1 and sFlt1-14. These antibodies were selected for their sensitivity and specificity to their respective sFlt1 isoform in a capture ELISA format. Data from this pilot study suggest that sFlt1-1 may be more predictive of preeclampsia than total sFlt1. It may be possible to improve current diagnostic platforms if more specific antibodies are utilized

Identification and Characterization of Broadly Neutralizing Human Monoclonal Antibodies Directed against the E2 Envelope Glycoprotein of Hepatitis C Virus▿

Nearly all livers transplanted into hepatitis C virus (HCV)-positive patients become infected with HCV, and 10 to 25% of reinfected livers develop cirrhosis within 5 years. Neutralizing monoclonal antibody could be an effective therapy for the prevention of infection in a transplant setting. To pursue this treatment modality, we developed human monoclonal antibodies (HuMAbs) directed against the HCV E2 envelope glycoprotein and assessed the capacity of these HuMAbs to neutralize a broad panel of HCV genotypes. HuMAb antibodies were generated by immunizing transgenic mice containing human antibody genes (HuMAb mice; Medarex Inc.) with soluble E2 envelope glycoprotein derived from a genotype 1a virus (H77). Two HuMAbs, HCV1 and 95-2, were selected for further study based on initial cross-reactivity with soluble E2 glycoproteins derived from genotypes 1a and 1b, as well as neutralization of lentivirus pseudotyped with HCV 1a and 1b envelope glycoproteins. Additionally, HuMAbs HCV1 and 95-2 potently neutralized pseudoviruses from all genotypes tested (1a, 1b, 2b, 3a, and 4a). Epitope mapping with mammalian and bacterially expressed proteins, as well as synthetic peptides, revealed that HuMAbs HCV1 and 95-2 recognize a highly conserved linear epitope spanning amino acids 412 to 423 of the E2 glycoprotein. The capacity to recognize and neutralize a broad range of genotypes, the highly conserved E2 epitope, and the fully human nature of the antibodies make HuMAbs HCV1 and 95-2 excellent candidates for treatment of HCV-positive individuals undergoing liver transplantation

Identification of human monoclonal antibodies specific for human SOD1 recognizing distinct epitopes and forms of SOD1.

Mutations in the gene encoding human SOD1 (hSOD1) can cause amyotrophic lateral sclerosis (ALS) yet the mechanism by which mutant SOD1 can induce ALS is not fully understood. There is currently no cure for ALS or treatment that significantly reduces symptoms or progression. To develop tools to understand the protein conformations present in mutant SOD1-induced ALS and as possible immunotherapy, we isolated and characterized eleven unique human monoclonal antibodies specific for hSOD1. Among these, five recognized distinct linear epitopes on hSOD1 that were not available in the properly-folded protein but were available on forms of protein with some degree of misfolding. The other six antibodies recognized conformation-dependent epitopes that were present in the properly-folded protein with two different recognition profiles: three could bind hSOD1 dimer or monomer and the other three were specific for hSOD1 dimer only. Antibodies with the capacity to bind hSOD1 monomer were able to prevent increased hydrophobicity when mutant hSOD1 was exposed to increased temperature and EDTA, suggesting that the antibodies stabilized the native structure of hSOD1. Two antibodies were tested in a G93A mutant hSOD1 transgenic mouse model of ALS but did not yield a statistically significant increase in overall survival. It may be that the two antibodies selected for testing in the mouse model were not effective for therapy or that the model and/or route of administration were not optimal to produce a therapeutic effect. Therefore, additional testing will be required to determine therapeutic potential for SOD1 mutant ALS and potentially some subset of sporadic ALS