Development of a Mouse Model of Abdominal Cutaneous Flaps for Breast Reconstruction

Autologous tissue transfer, in addition to replacing tissue that was lost during injury or surgery, offers women an excellent option to improve cosmetic appearance and self-confidence following mastectomy due to breast cancer. However, flap necrosis is a complication in obese patients undergoing this procedure. We created a mouse model to study the flap-related complications that leads to decreased flap survival in autologous breast reconstruction.

Methods

Left superficial inferior epigastric (SIE) pedicle abdominal-cutaneous flaps were elevated in 8 week-old, obese ob/ob male mice and their lean littermates. Flaps were followed by serial photography. Area of flap necrosis was measured at 7 days. Statistical analysis was performed.

Results

Necrosis was observed at the distal margin of the flaps, in both lean and obese groups. Lean left SIE flaps (n = 8) had a total area flap necrosis of 9.1% at 7 days whereas obese left SIE flaps (n = 8) had a total area flap necrosis of 45.5% at 7 days. Obese flaps had a statistically significant increase in necrosis compared to the lean flaps, p = 0.001.

Conclusions

There was a significant difference between flap survival in lean and obese SIE pedicle flaps in our mouse model. We have developed the first flap model of obesity utilizing the superficial epigastric pedicle in the mouse. This model is optimal for future studies to dissect out mechanisms that lead to the complications related to flap survival for breast reconstruction, especially in obese subjects.     

Monoclonal antibody therapeutics with up to five specificities: Functional enhancement through fusion of target-specific peptides

The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvβ3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model.     

A murrel interferon regulatory factor-1: molecular characterization,gene expression and cell protection activity

In this study, we have reported a first murrel interferon regulatory factor-1 (designated as Murrel IRF-1) which is identified from a constructed cDNA library of striped murrel Channa striatus. The identified sequence was obtained by internal sequencing method from the library. The Murrel IRF-1 varies in size of the polypeptide from the earlier reported fish IRF-1. It contains a DNA binding domain along with a tryptophan pentad repeats, a nuclear localization signal and a transactivation domain. The homologous analysis showed that the Murrel IRF-1 had a significant sequence similarity with other known fish IRF-1 groups. The phylogenetic analysis exhibited that the Murrel IRF-1 clustered together with IRF-1 members, but the other members including IRF-2, 3, 4, 5, 6, 7, 8, 9 and 10 were clustered individually. The secondary structure of Murrel IRF-1 contains 27 % α-helices (85 aa residues), 5.7 % β-sheets (19 aa residues) and 67.19 % random coils (210 aa residues). Furthermore, we predicted a tertiary structure of Murrel IRF-1 using I-Tasser program and analyzed the structure on PyMol surface view. The RNA structure of the Murrel IRF-1 along with its minimum free energy (?284.43 kcal/mol) was also predicted. The highest gene expression was observed in spleen and its expression was inducted with pathogenic microbes which cause epizootic ulcerative syndrome in murrels such as fungus, Aphanomyces invadans and bacteria, Aeromonas hydrophila, and poly I:C, a viral RNA analog. The results of cell protection assay suggested that the Murrel IRF-1 regulates the early defense response in C. striatus. Moreover, it showed Murrel IRF-1 as a potential candidate which can be developed as a therapeutic agent to control microbial infections in striped murrel. Overall, these results indicate the immune importance of IRF-1, however, the interferon signaling mechanism in murrels upon infection is yet to be studied at proteomic level.     

Intereukin-10 and Kupffer cells protect steatotic mice livers from ischemia-reperfusion injury

Steatotic livers are more sensitive to ischemia/reperfusion (I/R) and are thus routinely rejected for transplantation because of their increased rate of primary nonfunction (PNF). Lean livers have less I/R-induced damage and inflammation due toKupffer cells (KC), which are protective after total, warm, hepatic I/R with associated bowel congestion. This protection has been linked to KC-dependent expression of the potent anti-inflammatory cytokine interleukin-10 (IL-10).We hypothesized that pretreatment with exogenous IL-10would protect the steatotic livers of genetically obese (ob/ob) mice from inflammation and injury induced by I/R. Lean and ob/ob mice were pretreated with either IL-10 or liposomally-encapsulated bisphosphonate clodronate (shown to deplete KC) prior to total, warm, hepatic I/R. IL-10 pretreatment increased survival of ob/ob animals at 24 hrs post-I/R from 30% to 100%, and significantly decreased serum ALT levels. At six hrs post-I/R, IL-10 pretreatment increased IL-10 mRNA expression, but suppressed up-regulation of the pro-inflammatory cytokine IL-1β mRNA. However, ALT levels were elevated at six hrs post-I/R in KC-depleted animals. These data reveal that pretreatment with IL-10 protects steatotic livers undergoing I/R, and that phagocytically active KC retain a hepatoprotective role in the steatotic environment.     

Influenza A Virus Encoding Secreted Gaussia Luciferase as Useful Tool to Analyze Viral Replication and Its Inhibition by Antiviral Compounds and Cellular Proteins

Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1–3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels.     

Spatial distribution of nematode genera in relation to host plants of Western Ghats,Tamil Nadu,India

A survey of plant parasitic nematodes (PPN) abundance and diversity was conducted in foothills of Western Ghats at Coimbatore and Nilgiris districts in Tamil Nadu, India. Our study pointed to evaluate the PPN association with weeds, shrubs, and herbs including some agricultural crop cover both agro and forest ecosystems. Soil samples were collected around the rhizosphere of the plants and nematodes were isolated, counted under a microscope and identified by morphological characters. We collected 415 soil samples around the rhizosphere of 84 major plant families. Among 84 plant families, we found 13 PPN genera those generally responsible for cash crop yield loss. The genus Helicotylenchus spp., and Meloidogyne spp., were found frequent (226,136 samples) and rarely Hemicycliophora spp.,. The PPN diversity was measured by Shannon diversity index which showed that the diversity of PPN found higher in plant species those from the families Rosaceae (2.20) and Rubiaceae (2.13). We also recorded the PPN richness was dominated in four plant families. This result reveals that the PPN diversity and richness are positively correlated with host plant species diversity and altitude. Further, our study concludes that a wild plant in the forest ecosystem behaves like a reservoir of PPN.     

The hypervirulent Mycobacterium tuberculosis strain HN878 induces a potent TH1 response followed by rapid down-regulation

The HN878 strain of Mycobacterium tuberculosis is regarded as "hypervirulent" due to its rapid growth and reduced survival of infected mice when compared with other clinical isolates. This property has been ascribed due to an early increase in type I IFNs and a failure to generate TH1-mediated immunity, induced by a response to an unusual cell wall phenolic glycolipid expressed by the HN878 isolate. We show, however, that although type I IFN does play an inhibitory role, this response was most apparent during the chronic disease stage and was common to all M. tuberculosis strains tested. In addition, we further demonstrate that the HN878 infection was associated with a potent TH1 response, characterized by the emergence of both CD4 and CD8 T cell subsets secreting IFN-gamma. However, where HN878 differed to the other strains tested was a subsequent reduction in TH1 immunity, which was temporally associated with the rapid emergence of a CD4+CD25+FoxP3+CD223+IL-10+ regulatory T cell population. This association may explain the paradoxical initial emergence of a TH1 response in these mice but their relatively short time of survival.     

The cellular immune response to Mycobacterium tuberculosis infection in the guinea pig

Pulmonary tuberculosis in guinea pigs is an extremely useful model for drug and vaccine testing due to the fact that its pathological disease process is similar to that present in humans. Progress in this field has been hindered because the tools necessary to undertake a complete immunological analysis of the guinea pig cellular immune response against Mycobacterium tuberculosis have been lacking. In this study, we combined a new flow cytometric gating strategy with immunohistochemistry to track T cells, B cells, and the MIL4 Ab, which detects both guinea pig heterophils (neutrophils) and eosinophils, to provide the first documentation of the kinetics of influx and positioning of these cell populations. The results show that the responding T cells are mostly CD4 cells and that after day 30 of the infection numbers of these cells in the lungs drops dramatically. These appear to be replaced by a steady increase in B cells and granulocytes which was associated with worsening lung pathology. These data reveal new information about the cellular phenotypes which mediate protective immunity or host immunopathogenesis during M. tuberculosis infection in this key animal model.     

Can Currently Available Advanced Combustion Biomass Cook-Stoves Provide Health Relevant Exposure Reductions? Results from Initial Assessment of Select Commercial Models in India

EcoHealth - Household air pollution from use of solid fuels is a major contributor to the national burden of disease in India. Currently available models of advanced combustion biomass cook-stoves...