Therapeutic administration of anti-PcrV F(ab')(2) in sepsis associated with Pseudomonas aeruginosa.

The effects of rabbit-derived polyclonal Ab against PcrV, a protein involved in the translocation of type III secreted toxins of Pseudomonas aeruginosa, was investigated in two animal models of P. aeruginosa sepsis. In a mouse survival study, the i.v. administration of anti-PcrV IgG after the airspace instillation of a lethal dose of P. aeruginosa resulted in the complete survival of the animals. In a rabbit model of septic shock associated with Pseudomonas-induced lung injury, animals treated with anti-PcrV IgG intratracheally or i.v. had significant decreases in lung injury, bacteremia, and plasma TNF-alpha and significant improvement in the hemodynamic parameters associated with shock compared with animals treated in a similar manner with nonspecific control IgG. The administration of anti-PcrV F(ab')(2) showed protective effects comparable to those of whole anti-PcrV IgG. These results document that the therapeutic administration of anti-PcrV IgG blocks the type III secretion system-mediated virulence of P. aeruginosa and prevents septic shock and death, and that these protective effects are largely Fc independent. We conclude that Ab therapy neutralizing the type III secretion system has significant potential against lethal P. aeruginosa infections.     

T cell epitopes of human myelin oligodendrocyte glycoprotein identified in HLA-DR4 (DRB1*0401) transgenic mice are encephalitogenic and are presented by human B cells.

Myelin oligodendrocyte glycoprotein (MOG) is an Ag present in the myelin sheath of the CNS thought to be targeted by the autoimmune T cell response in multiple sclerosis (MS). In this study, we have for the first time characterized the T cell epitopes of human MOG restricted by HLA-DR4 (DRB1*0401), an MHC class II allele associated with MS in a subpopulation of patients. Using MHC binding algorithms, we have predicted MOG peptide binding to HLA-DR4 (DRB1*0401) and subsequently defined the in vivo T cell reactivity to overlapping MOG peptides by testing HLA-DR4 (DRB1*0401) transgenic mice immunized with recombinant human (rh)MOG. The data indicated that MOG peptide 97-108 (core 99-107, FFRDHSYQE) was the immunodominant HLA-DR4-restricted T cell epitope in vivo. This peptide has a high in vitro binding affinity for HLA-DR4 (DRB1*0401) and upon immunization induced severe experimental autoimmune encephalomyelitis in the HLA-DR4 transgenic mice. Interestingly, the same peptide was presented by human B cells expressing HLA-DR4 (DRB1*0401), suggesting a role for the identified MOG epitopes in the pathogenesis of human MS.     

Acetylcholine receptor peptide recognition in HLA DR3-transgenic mice: in vivo responses correlate with MHC-peptide binding.

HLA DR3 is an MHC molecule that reportedly predisposes humans to myasthenia gravis (MG). Though MG is an Ab-mediated autoimmune disease, CD4+ T cells are essential for the generation of high-affinity Abs; hence the specificities of autoreactive CD4+ T cells are important. In this study we report the HLA DR3-restricted T cell determinants on the extracellular region sequence of human acetylcholine receptor alpha subunit. We find two promiscuous determinants on this region 141-160 and 171-190 as defined by their immunogenicity in HLA DR3-, HLA DQ8-, and HLA DQ6-transgenic mice in the absence of endogenous mouse class II molecules. We also studied the minimal determinants of these two regions by truncation analysis, and the MHC binding affinity of a set of overlapping peptides spanning the complete sequence region of human acetylcholine receptor alpha subunit. One of the peptide sequences strongly immunogenic in HLA DR3-transgenic mice also had the highest binding affinity to HLA DR3. Identification of T cell determinants restricted to an MHC molecule known to predispose to MG may be an important step toward the development of peptide-based immunomodulation strategies for this autoimmune disease.     

The basics of preclinical drug development for neurodegenerative disease indications

Background

Because of the emerging intersections of HIV infection and Alzheimer's disease, we examined cerebrospinal fluid (CSF) biomarkers related of amyloid and tau metabolism in HIV-infected patients.

Methods

In this cross-sectional study we measured soluble amyloid precursor proteins alpha and beta (sAPPα and sAPPβ), amyloid beta fragment 1-42 (Aβ_1-42), and total and hyperphosphorylated tau (t-tau and p-tau) in CSF of 86 HIV-infected (HIV+) subjects, including 21 with AIDS dementia complex (ADC), 25 with central nervous system (CNS) opportunistic infections and 40 without neurological symptoms and signs. We also measured these CSF biomarkers in 64 uninfected (HIV-) subjects, including 21 with Alzheimer's disease, and both younger and older controls without neurological disease.

Results

CSF sAPPα and sAPPβ concentrations were highly correlated and reduced in patients with ADC and opportunistic infections compared to the other groups. The opportunistic infection group but not the ADC patients had lower CSF Aβ_1-42 in comparison to the other HIV+ subjects. CSF t-tau levels were high in some ADC patients, but did not differ significantly from the HIV+ neuroasymptomatic group, while CSF p-tau was not increased in any of the HIV+ groups. Together, CSF amyloid and tau markers segregated the ADC patients from both HIV+ and HIV- neuroasymptomatics and from Alzheimer's disease patients, but not from those with opportunistic infections.

Conclusions

Parallel reductions of CSF sAPPα and sAPPβ in ADC and CNS opportunistic infections suggest an effect of CNS immune activation or inflammation on neuronal amyloid synthesis or processing. Elevation of CSF t-tau in some ADC and CNS infection patients without concomitant increase in p-tau indicates neural injury without preferential accumulation of hyperphosphorylated tau as found in Alzheimer's disease. These biomarker changes define pathogenetic pathways to brain injury in ADC that differ from those of Alzheimer's disease.     

Scanning a DRB3*0101 (DR52a)-restricted epitope cross-presented by DR3: overlapping natural and artificial determinants in the human acetylcholine receptor

A recurring epitope in the human acetylcholine receptor (AChR) alpha subunit (alpha146-160) is presented to specific T cells from myasthenia gravis patients by HLA-DRB3*0101-"DR52a"-or by DR4. Here we first map residues critical for DR52a in this epitope by serial Ala substitution. For two somewhat similar T cells, this confirms the recently deduced importance of hydrophobic "anchor" residues at peptide p1 and p9; also of Asp at p4, which complements this allele's distinctive Arg74 in DRbeta. Surprisingly, despite the 9 sequence differences in DRbeta between DR52a and DR3, merely reducing the bulk of the peptide's p1 anchor residue (Trp149-->Phe) allowed maximal cross-presentation to both T cells by DR3 (which has Val86 instead of Gly). The shared K71G73R74N77 motif in the alpha helices of DR52a and DR3 thus outweighs the five differences in the floor of the peptide-binding groove. A second issue is that T cells selected in vitro with synthetic AChR peptides rarely respond to longer Ag preparations, whereas those raised with recombinant subunits consistently recognize epitopes processed naturally even from whole AChR. Here we compared one T cell of each kind, which both respond to many overlapping alpha140-160 region peptides (in proliferation assays). Even though both use Vbeta2 to recognize peptides bound to the same HLA-DR52a in the same register, the peptide-selected line nevertheless proved to depend on a recurring synthetic artifact-a widely underestimated problem. Unlike these contaminant-responsive T cells, those that are truly specific for natural AChR epitopes appear less heterogeneous and therefore more suitable targets for selective immunotherapy.     

The basics of preclinical drug development for neurodegenerative disease indications

Preclinical development encompasses the activities that link drug discovery in the laboratory to initiation of human clinical trials. Preclinical studies can be designed to identify a lead candidate from several hits; develop the best procedure for new drug scale-up; select the best formulation; determine the route, frequency, and duration of exposure; and ultimately support the intended clinical trial design. The details of each preclinical development package can vary, but all have some common features. Rodent and nonrodent mammalian models are used to delineate the pharmacokinetic profile and general safety, as well as to identify toxicity patterns. One or more species may be used to determine the drug's mean residence time in the body, which depends on inherent absorption, distribution, metabolism, and excretion properties. For drugs intended to treat Alzheimer's disease or other brain-targeted diseases, the ability of a drug to cross the blood brain barrier may be a key issue. Toxicology and safety studies identify potential target organs for adverse effects and define the Therapeutic Index to set the initial starting doses in clinical trials. Pivotal preclinical safety studies generally require regulatory oversight as defined by US Food and Drug Administration (FDA) Good Laboratory Practices and international guidelines, including the International Conference on Harmonisation. Concurrent preclinical development activities include developing the Clinical Plan and preparing the new drug product, including the associated documentation to meet stringent FDA Good Manufacturing Practices regulatory guidelines. A wide range of commercial and government contract options are available for investigators seeking to advance their candidate(s). Government programs such as the Small Business Innovative Research and Small Business Technology Transfer grants and the National Institutes of Health Rapid Access to Interventional Development Pilot Program provide funding and services to assist applicants in preparing the preclinical programs and documentation for their drugs. Increasingly, private foundations are also funding preclinical work. Close interaction with the FDA, including a meeting to prepare for submission of an Investigational New Drug application, is critical to ensure that the preclinical development package properly supports the planned phase I clinical trial.     

Computational study of biomechanical drivers of renal cystogenesis

Biomechanics and Modeling in Mechanobiology - Renal cystogenesis is the pathological hallmark of autosomal dominant polycystic kidney disease, caused by PKD1 and PKD2 mutations. The formation of...     

Evidence That the Mechanism of Prenatal Germ Cell Death in the Mouse Is Apoptosis

Using fluorescence-activated cell sorting combined with fluorescence microscopy the mechanism of embryonic germ cell death in the mouse has been shown to be apoptosis. Primordial germ cells (PGCs) from embryos at specific developmental stages have been analyzed, and cells with apoptotic morphology have been isolated by cell sorting. In the female, apoptotic oogonia at Day 13 and apoptotic oocytes at Days 15 and 17 were found. In the male, apoptotic cells were seen on Day 13 through Day 17. Apoptotic germ cells were not detected at Day 12 (combined male and female PGCs). Examination of sorted cells by fluorescence microscopy and by light microscopic analysis after alkaline phosphatase staining confirmed that the cells are apoptotic germ cells. Electron microscopy further confirmed that cells showing the morphological characteristics of apoptosis are present.