Mechanisms of escape phenomenon of spinal cord and brainstem in human rabies

BACKGROUND: Rabies virus preferentially involves brainstem, thalamus and spinal cord in human furious and paralytic rabies beginning in the early stage of illness. Nevertheless, rabies patient remains alert until the pre-terminal phase. Weakness of extremities develops only when furious rabies patient becomes comatose; whereas peripheral nerve dysfunction is responsible for weakness in paralytic rabies. METHODS: Evidence of apoptosis and mitochondrial outer membrane permeabilization in brain and spinal cord of 10 rabies patients was examined and these findings were correlated with the presence of rabies virus antigen. RESULTS: Although apoptosis was evident in most of the regions, cytochrome c leakage was relatively absent in spinal cord of nearly all patients despite the abundant presence of rabies virus antigen. Such finding was also noted in brainstem of 5 patients. CONCLUSION: Cell death in human rabies may be delayed in spinal cord and the reticular activating system, such as brainstem, thus explaining absence of weakness due to spinal cord dysfunction and preservation of consciousness

Characterization of a Human Antibody Fragment Fab and Its Calcium Phosphate Nanoparticles that Inhibit Rabies Virus Infection with Vaccine

Recombinant antibody phage display technology has been used to mimic many aspects of the processes that govern the generation and selection of high-affinity natural human antibodies in the human immune system, especially for infectious disease prophylaxis. An anti-rabies virus immunized phage-display Fab library was constructed from peripheral blood lymphocytes from vaccinated volunteers. The immunized antibody library, with a diversity of 6.7×108, was used to select and produce antibodies that bound to rabies virus glycoprotein. After five rounds of immobilized fixed rabies virion panning, four unique DNA sequences were found in the higher binding clones, and only one, Fab094, showed neutralization activity. Fab094 components were analyzed by ELISA, immunoprecipitation and immunofluorescent staining. ELISA and immunofluorescence showed that Fab094 bound specifically to rabies virions. Immunoprecipitation and mass spectrometry showed that Fab094 reacted with rabies virus glycoprotein. To improve the penetration power of Fab094 antibodies, we developed Fab094 calcium phosphate nanoparticles (Fab094-CPNPs) and tested their efficacy. The rapid fluorescent focus inhibition test indicated that the neutralizing antibody titers of Fab094 and Fab094-CPNPs were reached at 200.17 IU/Kg and 246.12 IU/Kg, respectively. These findings were confirmed in vivo in a Kunming mouse challenge model. Our results demonstrate that human Fab094 and Fab094-CPNPs are efficacious candidate drugs to replace rabies immunoglobulin in post-exposure prophylaxis (PEP)

Distinct functional neutrophil phenotypes in sepsis patients correlate with disease severity

PurposeSepsis is a clinical syndrome defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Sepsis is a highly heterogeneous syndrome with distinct phenotypes that impact immune function and response to infection. To develop targeted therapeutics, immunophenotyping is needed to identify distinct functional phenotypes of immune cells. In this study, we utilized our Organ-on-Chip assay to categorize sepsis patients into distinct phenotypes using patient data, neutrophil functional analysis, and proteomics.MethodsFollowing informed consent, neutrophils and plasma were isolated from sepsis patients in the Temple University Hospital ICU (n=45) and healthy control donors (n=7). Human lung microvascular endothelial cells (HLMVEC) were cultured in the Organ-on-Chip and treated with buffer or cytomix ((TNF/IL-1β/IFNγ). Neutrophil adhesion and migration across HLMVEC in the Organ-on-Chip were used to categorize functional neutrophil phenotypes. Quantitative label-free global proteomics was performed on neutrophils to identify differentially expressed proteins. Plasma levels of sepsis biomarkers and neutrophil extracellular traps (NETs) were determined by ELISA.ResultsWe identified three functional phenotypes in critically ill ICU sepsis patients based on ex vivo neutrophil adhesion and migration patterns. The phenotypes were classified as: Hyperimmune characterized by enhanced neutrophil adhesion and migration, Hypoimmune that was unresponsive to stimulation, and Hybrid with increased adhesion but blunted migration. These functional phenotypes were associated with distinct proteomic signatures and differentiated sepsis patients by important clinical parameters related to disease severity. The Hyperimmune group demonstrated higher oxygen requirements, increased mechanical ventilation, and longer ICU length of stay compared to the Hypoimmune and Hybrid groups. Patients with the Hyperimmune neutrophil phenotype had significantly increased circulating neutrophils and elevated plasma levels NETs.ConclusionNeutrophils and NETs play a critical role in vascular barrier dysfunction in sepsis and elevated NETs may be a key biomarker identifying the Hyperimmune group. Our results establish significant associations between specific neutrophil functional phenotypes and disease severity and identify important functional parameters in sepsis pathophysiology that may provide a new approach to classify sepsis patients for specific therapeutic interventions

Transcriptomic profile of host response in Japanese encephalitis virus infection

<p>Abstract</p> <p>Background</p> <p>Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy with the highest mortality rate of 30-50%. The purpose of this study was to understand complex biological processes of host response during the progression of the disease. Virus was subcutaneously administered in mice and brain was used for whole genome expression profiling by cDNA microarray.</p> <p>Results</p> <p>The comparison between viral replication efficiency and disease progression confirms the active role of host response in immunopathology and disease severity. The histopathological analysis confirms the severe damage in the brain in a time dependent manner. Interestingly, the transcription profile reveals significant and differential expression of various pattern recognition receptors, chemotactic genes and the activation of inflammasome. The increased leukocyte infiltration and aggravated CNS inflammation may be the cause of disease severity.</p> <p>Conclusion</p> <p>This is the first report that provides a detailed picture of the host transcriptional response in a natural route of exposure and opens up new avenues for potential therapeutic and prophylactic strategies against Japanese encephalitis virus.</p

Monoclonal antibodies for prophylactic and therapeutic use against viral infections

Neutralizing antibodies play an essential part in antiviral immunity and are instrumental in preventing or modulating viral diseases. Polyclonal antibody preparations are increasingly being replaced by highly potent monoclonal antibodies (mAbs). Cocktails of mAbs and bispecific constructs can be used to simultaneously target multiple viral epitopes and to overcome issues of neutralization escape. Advances in antibody engineering have led to a large array of novel mAb formats, while deeper insight into the biology of several viruses and increasing knowledge of their neutralizing epitopes has extended the list of potential targets. In addition, progress in developing inexpensive production platforms will make antiviral mAbs more widely available and affordable

PrognoScan: a new database for meta-analysis of the prognostic value of genes

<p>Abstract</p> <p>Background</p> <p>In cancer research, the association between a gene and clinical outcome suggests the underlying etiology of the disease and consequently can motivate further studies. The recent availability of published cancer microarray datasets with clinical annotation provides the opportunity for linking gene expression to prognosis. However, the data are not easy to access and analyze without an effective analysis platform.</p> <p>Description</p> <p>To take advantage of public resources in full, a database named "PrognoScan" has been developed. This is 1) a large collection of publicly available cancer microarray datasets with clinical annotation, as well as 2) a tool for assessing the biological relationship between gene expression and prognosis. PrognoScan employs the minimum <it>P</it>-value approach for grouping patients for survival analysis that finds the optimal cutpoint in continuous gene expression measurement without prior biological knowledge or assumption and, as a result, enables systematic meta-analysis of multiple datasets.</p> <p>Conclusion</p> <p>PrognoScan provides a powerful platform for evaluating potential tumor markers and therapeutic targets and would accelerate cancer research. The database is publicly accessible at <url>http://gibk21.bse.kyutech.ac.jp/PrognoScan/index.html</url>.</p

Development of a Mouse Monoclonal Antibody Cocktail for Post-exposure Rabies Prophylaxis in Humans

As the demand for rabies post-exposure prophylaxis (PEP) treatments has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide the required passive immune component in PEP in countries where canine rabies is endemic. Replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a mouse monoclonal antibody (MoMAb) cocktail with the ultimate goal to develop a product at the lowest possible cost that can be used in developing countries as a replacement for RIG in PEP. Five MoMAbs, E559.9.14, 1112-1, 62-71-3, M727-5-1, and M777-16-3, were selected from available panels based on stringent criteria, such as biological activity, neutralizing potency, binding specificity, spectrum of neutralization of lyssaviruses, and history of each hybridoma. Four of these MoMAbs recognize epitopes in antigenic site II and one recognizes an epitope in antigenic site III on the rabies virus (RABV) glycoprotein, as determined by nucleotide sequence analysis of the glycoprotein gene of unique MoMAb neutralization-escape mutants. The MoMAbs were produced under Good Laboratory Practice (GLP) conditions. Unique combinations (cocktails) were prepared, using different concentrations of the MoMAbs that were capable of targeting non-overlapping epitopes of antigenic sites II and III. Blind in vitro efficacy studies showed the MoMab cocktails neutralized a broad spectrum of lyssaviruses except for lyssaviruses belonging to phylogroups II and III. In vivo, MoMAb cocktails resulted in protection as a component of PEP that was comparable to HRIG. In conclusion, all three novel combinations of MoMAbs were shown to have equal efficacy to HRIG and therefore could be considered a potentially less expensive alternative biological agent for use in PEP and prevention of rabies in humans

Identification of a biomarker panel for colorectal cancer diagnosis

<p>Abstract</p> <p>Background</p> <p>Malignancies arising in the large bowel cause the second largest number of deaths from cancer in the Western World. Despite progresses made during the last decades, colorectal cancer remains one of the most frequent and deadly neoplasias in the western countries.</p> <p>Methods</p> <p>A genomic study of human colorectal cancer has been carried out on a total of 31 tumoral samples, corresponding to different stages of the disease, and 33 non-tumoral samples. The study was carried out by hybridisation of the tumour samples against a reference pool of non-tumoral samples using Agilent Human 1A 60-mer oligo microarrays. The results obtained were validated by qRT-PCR. In the subsequent bioinformatics analysis, gene networks by means of Bayesian classifiers, variable selection and bootstrap resampling were built. The consensus among all the induced models produced a hierarchy of dependences and, thus, of variables.</p> <p>Results</p> <p>After an exhaustive process of pre-processing to ensure data quality--lost values imputation, probes quality, data smoothing and intraclass variability filtering--the final dataset comprised a total of 8, 104 probes. Next, a supervised classification approach and data analysis was carried out to obtain the most relevant genes. Two of them are directly involved in cancer progression and in particular in colorectal cancer. Finally, a supervised classifier was induced to classify new unseen samples.</p> <p>Conclusions</p> <p>We have developed a tentative model for the diagnosis of colorectal cancer based on a biomarker panel. Our results indicate that the gene profile described herein can discriminate between non-cancerous and cancerous samples with 94.45% accuracy using different supervised classifiers (AUC values in the range of 0.997 and 0.955).</p