The dendritic cell (DC) possesses the ability to stimulate both T helper 1 (Th1) and Th2 responses depending on activation stimuli. Although it is known that chemically or genetically modified DC can be used therapeutically to steer immune responses towards either Th1 or Th2, cellular therapy with ex vivo manipulated DC is clinically difficult. Here we demonstrate a novel method of switching immune responses from Th1 to Th2 through in vivo immune modulation by administration of siRNA. We demonstrate that siRNA targeting of the IL-12p35 gene leads to a Th2 bias in vitro through an IL-10 dependent mechanism. In vivo administration of siRNA admixed with the oil-based contrast agent lipiodol in the presence of antigen and adjuvant induced a deviation in recall response to reduced production of IFN-γ and augmented IL-4 response using either KLH or ovalbumin. This simple method of in vivo modification of immune response possesses therapeutic potential in Th1-mediated diseases such as multiple sclerosis and autoimmune diabetes. It is known that the immune response can be polarized into two broad subsets based on cytokine secretion of the T helper cells. T helper-1 cells (Th1) responses are associated with anti-viral and anti-cancer immune reactions and are characterized by high secretion of the cytokine IFN-γ and IL-2 , multipl+ T helper cells (Th0) are programmed by the antigen presenting cell in order to become polarized into either the Th1 or Th2 phenotype thymidine (Amersham) was added to each well for 16 h. Using an automated cell harvester, the cells were collected onto glass microfiber filter, and the radioactive labeling incorporation was measured by a Wallac Betaplate liquid scintillation counter.Proliferative recall responses to KLH and ovalbumin in immunized mice were assessed by sacrificing C57/BL6 mice 14 days after immunization with antigen-loaded DC. T cells were purified from suspensions of lymph nodes using CD4+ T cell column (R&D Systems) after washing in PBS. Purified T cells were cultured in 96 well plates with irradiated syngeneic splenocytes in triplicate and mixed with serial dilutions of KLH or OVA at concentrations ranging from 0–10 ug/ml. Following a 72-h incubation, 1 μCi of [The supernatants from recall response T cell cultures or MLR were harvested and assessed for DC cytokines  and T cell cytokines  by ELISA. Cytokine-specific ELISA  was used for detecting cytokine concentrations in culture supernatants according to the manufacturer's instructions using a Benchmark Microplate Reader .We have previously demonstrated that transfection of DC with siRNA specific for the p35 component of IL-12 induces potent gene specific silencing at the mRNA transcript level as demonstrated by RT-PCR and subsequently reduced expression of the IL-12 p70 heterodimer as witnessed by ELISA protein . FurtherIn our laboratory, the combination of 10 ng/ml of LPS and TNF-α, respectively, (LPS/TNF) is used as a standard method of inducing activation of bone marrow derived DC for production of IL-12, as well as upregulation of costimulatory molecules such as CD40, CD80 and CD86 . Using tWe have previously demonstrated that DC silenced for the IL-12p35 subunit possess an increased production of IL-10 and are poor stimulators of MLR . Using t+ CD25+ T regulatory cells [In our previous studies we have observed that silencing IL-12 is associated with an upregulation in IL-10 production . While Iry cells  and TR1 ry cells . We wantry cells  and thatry cells . With thWhile we previously demonstrated that IL-12 silenced DC possessed similar levels of costimulatory molecules as wild-type DC , we did Having demonstrated above that lipiodol can serve as a transfection reagent for uptake of siRNA in DC, combined with the fact that lipiodol is commonly used for a variety of clinical applications ,41, we aLipiodol (trade name Ultra-Fluide™) is an iodized poppy seed oil fatty acid ethylester commonly used as contrast media for radiography . CommonlWe therefore continued to seek whether lipiodol transfer of siRNA can still mediate the immune modulatory effects previously reported by us in terms of switching Th1 to Th2 responses . AlthougWhile we were able to induce in vivo immune modulation through administration of the lipiodol/siRNA mixture, we did not demonstrate that in vivo the DC were indeed the culprits for immune modulation. As previously stated, lipiodol in vivo is pinocytosed by endothelial cells , and mosRegardless of the cellular mechanisms involved, the ability to immune modulate through administration of a "vaccine composition" with siRNA opens a whole world of novel possibilities both therapeutic and experiments. Through alleviating the need for ex vivo cellular processing, simple siRNA based vaccines can be developed for a variety of disease indications where a Th switch is desired. In terms of clinical applicability, the addition of siRNA for embolization of tumors would allow for induction of immune stimulation at the site of tissue necrosis. This would permit induction of immune responses to antigens released during the embolization procedure. Indeed similar approach used by Adema et al has demonstrated successful induction of immunity through tissue damage combined with immune stimulation .In conclusion, we demonstrate that siRNA can be administered through a clinically useful method, thus opening the possibilities of genetic immune modulation without the use of viruses or ex vivo cellular manipulation.


Wolbachia has spread to pandemic levels. Little is known about the microbial basis of cytoplasmic incompatibility (CI) except that bacterial densities and percentages of infected sperm cysts associate with incompatibility strength. The recent discovery of a temperate bacteriophage (WO-B) of Wolbachia containing ankyrin-encoding genes and virulence factors has led to intensifying debate that bacteriophage WO-B induces CI. However, current hypotheses have not considered the separate roles that lytic and lysogenic phage might have on bacterial fitness and phenotype. Here we describe a set of quantitative approaches to characterize phage densities and its associations with bacterial densities and CI. We enumerated genome copy number of phage WO-B and Wolbachia and CI penetrance in supergroup A- and B-infected males of the parasitoid wasp Nasonia vitripennis. We report several findings: (1) variability in CI strength for A-infected males is positively associated with bacterial densities, as expected under the bacterial density model of CI, (2) phage and bacterial densities have a significant inverse association, as expected for an active lytic infection, and (3) CI strength and phage densities are inversely related in A-infected males; similarly, males expressing incomplete CI have significantly higher phage densities than males expressing complete CI. Ultrastructural analyses indicate that approximately 12% of the A Wolbachia have phage particles, and aggregations of these particles can putatively occur outside the Wolbachia cell. Physical interactions were observed between approximately 16% of the Wolbachia cells and spermatid tails. The results support a low to moderate frequency of lytic development in Wolbachia and an overall negative density relationship between bacteriophage and Wolbachia. The findings motivate a novel phage density model of CI in which lytic phage repress Wolbachia densities and therefore reproductive parasitism. We conclude that phage, Wolbachia, and arthropods form a tripartite symbiotic association in which all three are integral to understanding the biology of this widespread endosymbiosis. Clarifying the roles of lytic and lysogenic phage development in Wolbachia biology will effectively structure inquiries into this research topic.By manipulating arthropod reproduction worldwide, the heritable endosymbiont  (Wolbachia) and a parasitic wasp host are affected by a mobile element—a temperate bacteriophage of Wolbachia. In contrast to recent reports that suggest bacteriophage WO-B may induce reproductive parasitism, the authors' quantitative and ultrastructural analyses indicate that lytic phage WO-B are lethal and therefore associate with a reduction in both Wolbachia densities and reproductive parasitism. Based on these data, the authors propose a phage density model in which lytic phage development specifically leads to a reduction, rather than induction, of reproductive parisitism. The study is among the first investigations to show that lytic bacteriophage inversely associate with the densities and phenotype of an obligate intracellular bacterium.Symbiotic bacteria that are maternally inherited are widespread in terrestrial invertebrates. Such bacteria infect the cells of reproductive tissues and can have important evolutionary and developmental effects on the host. Often these inherited symbionts develop beneficial relationships with their hosts, but some species can also selfishly alter invertebrate reproduction to increase the numbers of infected females (the transmitting sex of the bacteria) in the population. Bacterial-mediated distortions such as male-killing, feminization, parthenogenesis induction, and cytoplasmic incompatibility are collectively known as “reproductive parasitism.” In this article, the investigators show that the associations between the most common reproductive parasite in the biosphere  Wolbachia are α–proteobacterial endosymbionts that are recognized for their widespread distribution and inductions of reproductive parasitism, including feminization, male-killing, parthenogenesis, and cytoplasmic incompatibility (CI) in arthropods  + [(four lysogenic phages per Wolbachia) × (the observed frequency of virion-free Wolbachia)]. This equation [(24.66 + 4.0)(0.1176)] + [(4.0)(0.8829)] yields an estimate of 6.9 phage copies per Wolbachia, a number that parallels the real-time quantitative PCR estimate of an average density of 5.8 phages per bacterium. Despite many assumptions requiring proper caution, it can be concluded that the consistency of the RT-qPCR and TEM studies, and relatively low frequency of observed virion-containing Wolbachia (11.78%), produces an overall, moderate intrahost ratio of phage to bacteria.We employed transmission electron microscopy (TEM) to directly confirm the lytic phage development evident from the quantitative PCR studies. l testes B–5E. Thel stages . As a reermatids B, which d testes B–5E, 11. pipiens . The meaolbachia , we can Wolbachia ranged in diameter from 35 to 45 nm, some of which had an observable tail-like structure that ranged in length from 10 to 20 nm. Bacteriophage tails are typically required for host membrane attachment and infection. Particles were distributed along the inner membrane and in the middle of the cell, which was often irregularly shaped, degenerative, and lysed. We observed virion-containing Wolbachia with dense masses in the middle of the cell and detached membrane structures analogous to phage-infected Chlamydia [Wolbachia widely varied from four to 65. In at least three of six cases, virions were observed leaving lysed cells into the extracellular matrix , potential modulation of intracellular Wolbachia densities , and, by association, penetrance of Wolbachia-mediated reproductive parasitism such as CI.In this study, we examined the tripartite associations and ultrastructure of a phage, bacterial endosymbiont, and host arthropod that collectively represent a widespread and evolutionary important symbiotic system. The effects that lytic phage growth and virion production can have on this tripartite symbiosis span important evolutionary and ecological processes such as lateral phage transfer between Wolbachia, as expected for an active lytic phage. Ultrastructural analyses confirm the ability of Wolbachia phage to form virion particles and lyse the host cell. Furthermore, the negative association between CI levels and phage densities suggests that the genes encoding CI “toxins” apparently are not expressed in replicating or mature phage. Otherwise, we would expect a positive correlation between phage densities and CI levels. Genes in phage WO-B may still encode CI “toxins” while the genome is integrated as a prophage during lysogeny, but a complete transcriptome characterization of the phage during lysogeny is necessary to address this issue. Sex-specific expression of phage-related genes is consistent with a possible role of lysogenic phage in reproductive parasitism and has been observed in infected Culex mosquitoes [Wolbachia and do not impart a benefit to the bacterium.Our quantitative and ultrastructural analyses comprise the first studies to illustrate a negative density relationship between phage WO-B and squitoes ,47. HoweWolbachia, benefits from the presence of a lytic phage since host level and phage level selection could both favor an active bacteriophage infection that lyses Wolbachia cells and thus reduces the bacterial load in the arthropod host. This host level selection could also explain its absence from mutualistic Wolbachia in filarial nematodes [Wolbachia that is required for host oogenesis and larval development. The results presented here also raise the interesting possibility that common environmental  or host genetic factors known to modulate Wolbachia densities do so by inducing prophage excision, virion production, and cell lysis. Future investigations should simultaneously examine the effects of these extrinsic factors on phage and insect endosymbiont densities. Finally, an active and inducible temperate phage in Wolbachia is a promising genetic tool to engineer Wolbachia for biocontrol strategies or as a biotherapy tool to combat the devastating pathologies of Wolbachia-assisted filariasis in humans. It is clear that the Wolbachia symbiosis has been shaped by at least three genetic entities and experimental and theoretical studies on their interactions will collectively assemble a general model of the host-reproductive parasite interactions.One hypothesis for the widespread distribution of phage WO-B  based onematodes  in whichN. vitripennis are gregarious parasitoid wasps of fly pupae. They are raised on Sarcophaga bullata (flesh fly) pupae in the laboratory, with constant light and temperature (21 °C). Three strains were used in these studies: a single A-infected strain (12.1), a single B-infected lab strain (4.9), and an uninfected strain (13.2). These strains were derived in 1996 from a double AB-infected isofemale strain by spontaneous loss of Wolbachia following prolonged diapause [diapause .All crosses were set up as single pair matings between uninfected virgin females and virgin males of each of the three strains. Only those pairings where copulation occurred within 10 min of observation were used. Males were immediately frozen at −80 °C, and each female was provided with four hosts for feeding and egg laying. After 48 h, the females were transferred to new vials and given a single host for 6 h. Females were then discarded from each vial and the parasitized hosts were left undisturbed until adult emergence in approximately 2 wk. Adults were scored upon death for sex ratios and total family size. Two replicate experiments were performed 8 mo apart.Wolbachia-specific primers. RT-qPCR was performed in an iCycler system . Reaction volumes of 25 μl contained 12.5 μl of BioRad SYBR Green Supermix, 10.5 μl of sterile water, 0.5 μl each of 10 μM forward and reverse primer, and 1 μl of target DNA in single wells of a 96-well plate (Bio-Rad). For the selective amplification of a small portion of the Nasonia S6 Kinase gene (133 bp), Wolbachia groEL (97 bp), and WO-B ORF7 (125 bp), the following primers were designed and employed: NvS6KQTF2 (5′-GGCATTATCTACAGAGATTTG-3′), NvS6KQTR2 (5′-GCTATATGACCTTCTGTATCAAG-3′), NvWGroQTF1 (5′-CAACCTTTACTTCCTATTCTTG-3′), NvWGroQTR1 (5′-CTAAAGTGCTTAATGCTTCACCTTC-3′), NvWOF1 (5′-GTCTGGAAAGCTTACAAAAAG-3′), and NvWOR1 (5′-CTCGCCAAAATATAGCCCTGC-3′). PCR conditions comprised an initial melting at 95 °C for 3 min, followed by 40 cycles with melting at 95 °C (15 s) and primer annealing at 55.6 °C (1 min). A melting curve analysis was performed at the end of the PCRs to check for primer-dimers and nonspecific amplification. Standard curves for each gene were constructed with a log10 dilution series of known amounts of PCR products cloned into plasmid vectors using the TOPO TA cloning kit . RT-qPCR assays were performed in triplicate on all DNAs and two replicate experiments were performed in total.Genomic DNA was isolated using the DNeasy Tissue Kit  from single adult males and suspended in 70 μl of double-distilled sterile water. Infection status on a subset of these adults was confirmed by PCR with p = 0.15 prior to removal of four outliers. We expect more variability in this correlation analysis since the phage densities and CI strength are only indirectly related. Linear or logarithmic trend lines were added to the charts to illustrate patterns using Microsoft Excel.Correlation coefficients were calculated using the nonparametric Spearman's rho methods in JMP version 5.0.1a  Multivariate outliers were identified by the jackknifed Mahalanobis Distance in JMP and removed from the datasets . Outliers can artificially increase or decrease the value of a correlation coefficient and their removal can have recommended benefits that are not typically considered in practice ,64. Remoz*-transformation of data for small datasets. Normalization was then performed by reconstructing the RT-qPCR standard curve with the average threshold cycles of the enumerated dilution series across separate plates. Ct values for experimental DNAs for each gene were compared against the “average” standard curve to specify the normalized starting quantities of template DNA. No plate effects were observed after data normalization.Small plate effects are common in RT-qPCR experiments and were apparent by a trend of slightly elevated or reduced threshold cycle (Ct) values for the same template DNA used for the standard curve across two different plates. Before normalization of the plate effects, individual correlation coefficients (Spearman's rho) on the data from each plate were determined to specify the same association trends (positive or negative) by a homogeneity test  were isolated using buffers and centrifugation steps previously described [Whole-insect homogenates from infected escribed , with th


Nephila clavipes to their biochemical composition and physicochemical properties. Five layers of different make-ups could be distinguished. Of these only the two core layers contained the known silk proteins, but all can vitally contribute to the mechanical performance or properties of the silk fibre. Understanding the composite nature of silk and its supra-molecular organisation will open avenues in the production of high performance fibres based on artificially spun silk material.Albeit silks are fairly well understood on a molecular level, their hierarchical organisation and the full complexity of constituents in the spun fibre remain poorly defined. Here we link morphological defined structural elements in dragline silk of  Spider silks display extraordinary mechanical features 1-helical and type II β-turn structures The properties of dragline silk are believed to depend equally on both the molecular design of its two main protein components termed major ampullate spidroins 1 (MaSp1) and 2 (MaSp2) and the hierarchical organisation of structural elements Based on morphological evidence a hierarchical organisation of the fibre into distinct structural elements has been reported, in particular an inner core was discriminated from an outer skin layer and fibrils running parallel to the fibre axis have been observed Due to silk's biomimetic potential attempts are ongoing to mass produce parts of the known repetitive sequences of spidroins and to spin the material into fibres Nephila clavipes confirmed a core-skin structure, but revealed in addition two thin layers on top of the skin (Transmission Electron (TEM) and Atomic Force Microscopy (AFM) micrographs of cross-sectioned dragline silk fibres from the spider the skin . The extthe skin , inset, the skin .The more interior of the outer two layers exhibited a thickness of approximately 40–100 nm and was distinguished in TEM images by its strong osmiophilic nature that is a strong staining with osmium tetroxide . The layBelow the inward of the outer two tiers a skin layer of 50–100 nm was discernible in TEM, which was richer in contrast than the subjacent core material . This laThe core layer comprises most of the fibre. Speckles of higher contrast are visible throughout the core's extension in TEM images . In AFM A lipid character of the outermost layer was indicated by the weak staining of silk with the lipophilic dye oil red . This stGlycosylation that has been previously demonstrated for dragline silk The material of the next inward skin layer is composed of proteins that mostly displayed molecular weights similar to the known MaSps, but that were immunological distinct to them . They reThe inner material is represented by the core and is made up by the known spidroins since it reacted with all silk specific sera . The corThe restriction of the two known MaSps to the core was proved by staining laser ablated fibres and fibre cross-sections with silk specific antibodies. Antibodies specific for MaSp 1 (S1Rx) and MaSp 2 (S2Rx) reacted exclusively with the inner material at lesions, but not the surface of the fibre . In contGlycosylation was evident in the glyco and skin layers as well a throughout the core. Reactivity of Concavalin A per area was strongest for the glyco layer, followed by the skin and the core in accordance to their reactivity in western blots . This inEvidence for a robust nature of the skin was found in experiments where filaments were treated with chaotropic agents or acids. In both cases the skin showed increased resistance to these chemicals compared to the core . In acidIf treated with hexafluorisopropanol (Hfip) grooves on the surface became obvious that first ran parallel and at later time points of treatment also perpendicular to the fibre axis . At restUrea treatment induced swelling of the fibre resulting in diameters of ∼8 µm without dissolving the material . ObviousParticularly by video contrast microscopy (VCM) of urea treated and Coomassie stained samples fibrils were evident, which were oriented parallel to the fibre axis . These fIf dragline was incubated in urea or chloride salts of lithium (LiCl) and guanidinium (GdmCl) only a swelling of the fibre was noticed .Nephila clavipes looked unstructured in transmission electron microscopy (TEM), the dragline of N. edulis showed the existence of a multilayer assembly N. clavipes. First, the skin could be mechanically separated from the core by freeze-thaw cycles  have been identified MaSp 1, however, seemed to be missing in the draglines produced by spiders of the genera The protein feedstock for dragline silk is produced and stored in the major ampullate gland As a consequence dragline silk represents a composite material of high complexity. By altering its composition the spider is able to achieve better silk functionality and to facilitate spinning in adaptation to changing environmental conditions Nephila clavipes were done as described Livestock breeding and silking of the golden orb-weaving spider Technical details for light microscopy (LM) For manipulations silk fibres were either fixed prior to or after different treatments to glass slides  as well as glass squares (5×5 mm), glimmer or aluminium blocks (SEM) by conductive silver adhesive or nail varnish. Bulk fibres were treated with different solvents in tubes, whereas solvents were overlaid onto the fixed fibres. Reagents used were the chaotropic agents lithium thiocyanate (LiSCN) at different concentrations, 8 M urea with and without Coomassie Brilliant Blue G250 staining (0.1% in PBS) and hexafluorisopropanol  or a mixture of acetic acid (HAc) and hydrochloric acid (HCl) at different ratios. At various times samples were analysed by phase contrast LM using a CCD camera, by VCM or SEM.Solubilisation experiments were carried out by incubation of approximately 1 mg of dragline silk in 1ml solution. Used solvents were 8M urea, 99% Hexafluorisopropanol (Hfip), saturated lithium chloride (LiCl), saturated lithium bromide (LiBr), saturated lithium isothiocyanate (LiSCN), 6 M guanidinium hydrochloride (GdmHCl) and 6 M guanidinium isothocyanate (GdmSCN). After 24 hrs incubation in the respective solvent the sample was centrifuged at 14 000 g to remove any particulate matter and the supernatant was dialysed against 8 M urea. After a second centrifugation step 10 µl of the supernatant were analysed by sodium dodecyl sulphate-polyacrylamide gelelectrophoresis (SDS-PAGE) in a 5–20% pre-cast gel (BioRad). The gel was either directly Coomassie stained or blotted onto nitrocellulose filters. Filters were stained with specific antibodies using the enhanced chemiluminescence (ECL) for detection.The setup for laser ablation experiments was as detailed elsewhere Water and ether treated fibres were stained for 20 min. in a 0.2% oil red solution in 60% 2-propanol . After a brief wash in 60% 2-propanol and A. bidest., fibres were analysed in transmitted light by LM.40) for 1 hour; 3 brief washes in PBS; mounting and analysis by fluorescent LM or SEM.Silk fibres were decorated with antibodies or lectins using the following work-flow: brief wash in phosphate buffered saline (PBS); saturation of unspecific binding sites for 20 minutes in PBS/2% bovine serum albumin (BSA); 3 brief washes with PBS; incubation with the primary antibody (1∶10 up to 1∶1000 dilution) or biotinylated Concavalin A  (1 mg/ml in a 1∶100 dilution) for 2 hours; 3 brief washes in PBS; incubation with a fluorescein labelled secondary antibody  or streptavidin (1 mg/ml in a 1∶100 dilution ) coupled to gold particles . Next fibres were 10 times vigorously washed by vortexing in 0.1% Triton-X-100 . In the next step, fibres were 10 times flash frozen in liquid nitrogen. After each freezing cycle, fibres were washed in water . Cross-sections of fibres were prepared for each step and analysed by TEM.For gel electrophoresis fibres were extracted in bulk twice with ether. The extracts were combined, the ether evaporated and the dried material suspended in Hfip (lipid fraction). Then the fibres were washed twice gently in water and mechanically sheared using a Polytron device. After that, fibres were vigorously washed 10 times by vortexing in 0.1% Triton X-100. The washes were combined and concentrated in Microcon ultracentrifugal filter devices . After washing twice with water, the retained material was suspended in Hfip (glycoprotein fraction). Next, fibres were 10 times flash frozen in liquid nitrogen, followed by quick thawing. The fibres were then incubated two times for 10 minutes in 25% lithiumbromide (LiBr) to solubilise the inner core. Particulate matter was sedimented by ultracentrifugation and suspended in Hfip (skin fraction). The solute of the supernatant, handled with filter pipet tips to avoid contamination with any floating particulate matter from the centrifugation step, was changed to Hfip by two successive centrifugation-wash cycles using Microcon ultracentrifugal devices (core fraction). All samples were then dialyzed against 8M urea. Fractions were separated on 5–20% gradient gels (BioRad), and blotted to nitrocellulose membranes. Proteins were either revealed by Ponceau S staining (Sigma-Aldrich) or reacted with biotinylated Concavalin A or silk specific antibodies. Concavalin A was detected with streptavidin and antibodies by secondary antibodies, both coupled to peroxidase. Detection was with enhanced chemiluminescence .


The limited fossil fuel prompts the prospecting of various unconventional energy sources to take over the traditional fossil fuel energy source. In this respect the use of hydrogen gas is an attractive alternate source. Attributed by its numerous advantages including those of environmentally clean, efficiency and renew ability, hydrogen gas is considered to be one of the most desired alternate. Cyanobacteria are highly promising microorganism for hydrogen production. In comparison to the traditional ways of hydrogen production , Cyanobacterial hydrogen production is commercially viable. This review highlights the basic biology of cynobacterial hydrogen production, strains involved, large-scale hydrogen production and its future prospects. While integrating the existing knowledge and technology, much future improvement and progress is to be done before hydrogen is accepted as a commercial primary energy source. Its advantages as fuel are numerous: it is eco-friendly, efficient, renewable, and during its production and utilization no COenerated . By the Gloeocapsa alpicola under sulphur starvation shows increased hydrogen production [Arthrospira (Spirulina platensis) can produce hydrogen (1 μmole H2/12 hr/mg cell dry weight) in complete anaerobic and dark condition [Anabaena cylindrica, produces hydrogen and oxygen gas simultaneously in an argon atmosphere for 30 days in light limited condition [Cycas revoluta  and Zamia furfuracea showed a significant in vivo hydrogen uptake [Anabaena sp. is able to produce significant amount of hydrogen. Among them nitrogen-starved cells of Anabaena cylindrica produces highest amount of hydrogen (30 ml of H2/lit culture/hour). Hydrogenase-deficient cyanobacteria Nostoc punctiforme NHM5 when incubated under high light for a long time, until the culture was depleted of CO2 shows increase in hydrogen production [Cyanobacteria form a large and diverse group of oxygenic photoautotrophic prokaryotes, many of which have the ability to produce hydrogen Table . Hydrogeoduction . Arthrosondition . Anotherondition . Symbiotn uptake . Anabaenoduction .Nitrogenase and it is found in the heterocysts of filamentous cyanobacteria when they grow under nitrogen limiting conditions. Hydrogen is produced as a byproduct of fixation of nitrogen into ammonia. The reaction consumes ATP and has the general form:Cyanobacteria are photoautotrophic microorganisms -23 that nifD and nifK, α and β respectively) and the other is dinitrogenase reductase . Dinitrogenase is a α2β2 heterotetramer, having molecular weight of about 220 to 240 kDa respectively, breaks apart the atoms of nitrogen. Dinitrogenase reductase is a homodimer of about 60 to 70 kDa and plays the specific role of mediating the transfer of electrons from the external electron donor (a ferredoxin or a flavodoxin) to the dinitrogenase [A Nitrogenase enzyme consists of two parts:one is dinitrogenase  [hoxFUYH) and it can either take up or produce hydrogen. Uptake hydrogenase enzymes are found in the thylakoid membrane of heterocysts from filamentous cyanobacteria, where it transfers the electrons from hydrogen for the reduction of oxygen via the respiratory chain in a reaction known as oxyhydrogenation or Knallgas reaction. The enzyme consists of two subunits. The hupL-coded protein is responsible for the up taking hydrogen and the smaller subunit that is coded by hupS looks after the reduction affair. The hydrogen formed is usually re oxidized by an uptake hydrogenase via a Knallgas reaction and hence there is no net H2 production in strains with uptake hydrogenases under ambient conditions. So it is counterproductive when the goal is to produce hydrogen on a commercial scale. The reaction catalyzed by the uptake hydrogenase takes the following form:The other hydrogen-metabolizing/producing enzymes in cyanobacteria are y hupSL) , has thebidirectional or reversible hydrogenase, is poorly understood and thought to control ion levels in the organism. Reversible hydrogenase is associated with the cytoplasmic membrane and likely functions as an electron acceptor from both NADH and H2 [+ reducing type consisting of a hydrogenase dimmer coded by hoxYH gene. Maturation of reversible hydrogenases requires the action of several auxillary proteins collectively termed as hyp  [The biological role of H and H2 . The revH and H2 ,35. Molend hypB) . Unlike Hydrogen photo evolution catalyzed by nitrogenases or hydrogenases can only function under anaerobic conditions due to their extreme sensitivity to oxygen. Since oxygen is a byproduct of photosynthesis, organisms have developed the following spatial and temporal strategies to protect the enzyme from inactivation by oxygen, these are:(a) Heterocyst-containing cyanobacteria physically separate oxygen evolution from nitrogenase activity by segregating oxygenic photosynthetic activity in vegetative cells and nitrogenase activity in heterocystis with reduced oxygen-permeability  and(b) Non-heterocystous cyanobacteria separate oxygen-evolution from nitrogenase activity by performing these functions during light and dark periods respectively .Important parameters for hydrogen production: Efficiency of hydrogen production depends on several factors or parameters that are more critical when hydrogen production is aimed at a large scale.Various parameters influence hydrogen production in different ways, a few selected parameters are presented with examples:A. Environmental conditions: Several environmental conditions such as light, temperature, salinity, nutrient availability, gaseous atmosphere play a role in hydrogen production. Requirement of different cyanobacterial species are different for optimum hydrogen production.Arthrospira platensis) produces hydrogen under anaerobic conditions, both in the dark and in the light [Synechococcus PCC7942 occurs under in the dark under anaerobic condition [Spirulina platensis can produce hydrogen optimally at 32°C in complete anaerobic and dark condition [Anabaena variabilis ATCC 29413 and in its mutant PK84 [Anabaena variabilis PK84 with air containing 2% CO2 was stimulated by light [Nostoc muscorum is catalyzed by nitrogenase more hydrogen is produced in this strain in the light than in the dark [Anabaena cylindrica, produces hydrogen under an argon atmosphere for 30 days in light limited (luminous intensity 6.0 W/m-2) and 18 days under elevated light (luminous intensity 32 W/m-2) [Anabaena cylindrica for a prolonged period under light limited condition occurs in the absence of exogenous nitrogen [2 to 60 W/m2 [I) Light: Although most cyanobacterial species preferentially absorb red light near 680 nm , light rhe light  but sevehe light . Hydrogeondition . Spiruliondition . The higthe dark . Anabaen2 W/m-2) . Continunitrogen . The effnitrogen . Nitroge 60 W/m2 .Nostoc cultured at 22°C showed higher rates of hydrogen production than at 32°C [Nostoc muscorum SPU004 showed optimum hydrogen production at 40°C [Anabaena variabilis SPU 003 on the other hand show optimum hydrogen production at 30°C [II) Temperature: The optimum temperature for hydrogen production for most cyanobacterial species is between 30–40°C and varies from species to species of cyanobacteria. For example,  at 32°C , while N at 40°C . Anabaen at 30°C .+ ions from within the cells or prevention of Na+ influx [III) Salinity: Salinity does effect hydrogen production by cyanobacteria . In gene+ influx .Anabaena variabilis SPU003 is highly sensitive to Co, Cu, Mn, Zn, Ni, Fe ions and shows no hydrogen production at concentrations below 10 mM for these ions [Anabaena cylindrica grown with 5.0 mg of Ferric ions per liter produce hydrogen at a rate about twice that of culture with 0.5 mg of Ferric ions per liter [IV) Micronutrients: Trace elements such as cobalt (Co), copper (Cu), molyblednum (Mo), zinc (Zn), and nickel (Ni) effects hydrogen production . Many ofese ions . A cultuer liter .V) Carbon source: Carbon sources are also known to influence the hydrogen production considerably by influencing nitrogenase activity . PresencAnabaena variabilis SPU003 and Anabaena cylindrical [Anabaena cylindrical ammonium addition (0.2 mM NH4+) at a given time point eventually suppresses hydrogen production, but periodic addition of lower amounts (0.1 mM ammonium chloride) do not inhibit hydrogen evolution [Anabaena cylindrica culture, oxygen production gets dominated with the incremental addition of ammonium chloride (from 0.1 mM to 0.5 mM) [VI) Nitrogen source: Several inorganic nitrogenous compound influence hydrogen production rates in many ways. Nitrite, nitrate and ammonia have been reported to inhibit nitrogenase in indrical ,46. Geneindrical . Althougvolution  while otvolution . In Anab 0.5 mM) . The hyd 0.5 mM) .VII) Molecular nitrogen: Molecular nitrogen is a competitive inhibitor for hydrogen production and removal of molecular nitrogen if often very necessary for hydrogen production. Hydrogen production may be considerably inhibited in presence of molecular nitrogen .VIII) Effect of oxygen on hydrogen production: Due to their extreme sensitivity to oxygen, hydrogen photo evolution catalyzed by nitrogenases or hydrogenases can function only under anaerobic conditions  As oxygeGloeocapsa alpicola and Synechocystis PCC 6803). It is possible to inhibit the oxygenic photosynthesis and enhance hydrogen production by incubating the cynobacteria in nutrients that lack sulfur [IX) Effect of sulfur on hydrogen production: Sulfur starvation enhances the rate of hydrogen production in several cyanobacterial species  Effect of Methane: Increased hydrogen production (up to four times) is observed in lination .B. Intrinsic factors affecting hydrogen production: There are several intrinsic factors such as genetic components or sensitive proteins in cyanobacteria that may affect hydrogen production.I) Presence of uptake hydrogenase and decreased hydrogen yield: The net hydrogen yield is affected in strains containing uptake hydrogenase. Much of the produced hydrogen is lost due to the activity of the uptake hydrogenase . KnockinII) Sensitivity of hydrogenase and nitrogenase to molecular oxygen: The molecular oxygen acts as an inhibitor for hydrogenase and nitrogenase. However, innovative technical interdisciplinary solutions, as described below, are now available to reduce or eliminate presence of molecular oxygen and increase yield of hydrogen .a) With the advances in nanotechnology it has become possible to build semi-permeable membranes around the organisms. An example is a membrane that discriminates by size with an active transport system . NanotechydS and hydL from Thiocapsa roseopersicina into sensitive organisms may help reducing the oxygen sensitivity [b) Problem to oxygen sensitivity can also be addressed by using sulfur stress phenomenon to down-regulate photosynthesis as described before. This creates the anaerobic conditions required for hydrogen production. This problem may also be addressed by engineering the native hydrogenase. Engineering oxygen-tolerant hydrogenase genes, for example, sitivity . An exprsitivity .2 fixation [hetR . Recombinant strains can be created with HetR protein harboring vectors that may allow with variable and controllable heterocyst frequency [III) Heterocystous cyanobacteria are more efficient to produce hydrogen than cyanobacteria with vegetative cells ,51. Thesrequency .Bioreactors are essential for large-scale production of hydrogen. Since light is an essential parameter for cyanobacterial growth so all such bioreactors must be transparent and hence are called photobioreactors ,55. All Several types of bioreactors have been used for hydrogen production. These can be mainly divided into three types of photobioreactors (PBRs): vertical column reactor, tubular type and flat panel photobioreactor. A reactor for photobiological hydrogen production must meet several conditions:1) Photobioreactor should be an enclosed system so that the produced hydrogen may be collected without any loss.2) The reactor design must allow sterilization with convenience and ease.3) To maximize the area of incident light  photobioreactor design should provide high surface to volume ratio.Rhodobactor sp. [(a) Vertical column reactor (air-lift loop reactor and bubble column): Such PBRs consists of a transparent column usually made up of high quality glass and surrounded by a water jacket that while allowing maintenance of the temperature with circulating water allows adequate entry of light. Reactor top has provision for medium inlet and outlets for the gases such as argon and for the hydrogen. Fresh medium is added from a reservoir from above the PBR ,58. Micrctor sp. ,57,58. T2. Alternately red light emitting diode (LED peaking at 665 nm) is used as the light source on one side [(b) Flat panel Photobioreactor: A typical flat-panel PBR consists of a stainless-steel frame and three polycarbonate panels . The reaone side . A membrone side . The pro(c) Tubular photobioreactors: Tubular PBRs consists of long transparent tubes with diameters ranging from 3 to 6 cm, and lengths ranging from 10 to 100 m . The culCyanobacterial hydrogen production is poised to be a very useful commodity provided various effective utilization of the produced hydrogen is devised. There are various applications where the process of biological hydrogen production by cyanobacteria can be well utilized. The examples can be included from food and chemical industries, which employ the process of hydrogenation to produce derivatives that are used as food additives, commodities, and fine chemicals:5 and C4 polyols can be obtained from cheap C6 carbohydrates by oxidative decarbonylation followed by hydrogenation. These polyols blends are useful for the manufacture of polyesters or alkyl resins employed in the manufacture of paints. Sorbitol is another important polyol produced from hydrogenation of glucose, which is used industrially in a variety of physical and chemical processes, . Derivatives are also used in protecting coatings, plasticizers, emulsifiers and detergents.(a) Hydrogenation of cheap carbohydrates into high valued derivatives: High value-added C(b) Hydrogenation of fatty acids: Hydrogenation of fatty acids is used to manufacture margarines, shortenings, and shortening oils. The hydrogenation of oils converts liquid oils into hard fats by adding hydrogen to the fat molecule. Oils can be hydrogenated to varying degrees, depending on the hardness. These fats are desirable for its melting point, allowing for high temperature cooking and frying. Hydrogenation involves the artificial saturation of unsaturated bond(s) present in fatty acids. In the process fatty acids are put under pressure, using hydrogen gas at temperatures of 120–210°C (248–410°F) in the presence of a metal catalyst   for six All these process requires the rigorous hydrogenation. If hydrogen produced by cyanobacteria is very economical in comparison to the traditional large-scale hydrogen production. Cyanobacterial hydrogen produced in a photobioreactor can easily be directed to separate compartments containing the substrate for hydrogenation and specific catalysts. Provisions of external heating as well as purifying the products from different contaminants  may be easily provided as well. Hydrogen generated in near vicinity in PBRs will reduce requirement for transport and hazards associated with transport. Apart from hydrogenation, hydrogen is combustible so it can be well applied as a substitute for conventional fuel or may be used in fuel cells to generate electricity.2 in combustion, liberates large amounts of energy per unit weight in combustion, easily converted to electricity, and is an inexhaustible resource. Biological hydrogen production has several advantages over other conventional hydrogen production processes. It requires the use of a simple photobioreactor akin to a transparent closed box, with low energy requirements and it is very cost effective. Electrochemical hydrogen production via solar battery-based water splitting requires the use of solar batteries with high-energy requirements. Low conversion efficiencies of biological systems can be compensated for, by low energy requirements and reduced initial investment costs. The most appealing aspect of the biological hydrogen production is the source of hydrogen, which is nothing but water. With the existing knowledge of bioengineering it possible to obtain sufficient amount of hydrogen that on combustion will liberate energy and therefore could act as a substitute of coal in several operations [3) is much lower compared to that produced by photovoltaic splitting of water ($170/m3) [Hydrogen gas is seen, as a future energy carrier by virtue of the fact that it does not evolve the "greenhouse gas" COerations . Reporte$170/m3) . Though 2 Carbon dioxide; ATP Adenosine triphosphate; NOx Nitrogen oxides; Mo Molybdenum; Fe Iron; V Vanadium; PBR Photo bioreactor; H2 Hydrogen; O2 Oxygen; N2 Nitrogen; NADP Nicotinamide Adenine di nucleotide Phosphate; Ni Nickel; Co Cobalt; Cu Copper; Zn Zinc; Mn Manganese; Na+ Sodium ion; CH4 Methane; C4 Four carbon compounds; C5 Five carbon compounds; C6 Six-carbon compounds.ADP Adenosine di-phosphate; CODD and DD collected and read the papers and prepared a rough skeleton of manuscript. SC co-directed and supervised the work. SKB directed and prepared the final manuscript.


Understanding the predictors of appropriate implantable cardioverter defibrillator (ICD) therapy could help to better identify candidates for ICD implantation.One hundred and sixty two patients with ICD (111 with coronary artery disease [CAD] and 51 with dilated cardiomyopathy [DCM]) were included in the study. Clinical, electrocardiographic, and ICD stored data and electrograms were collected.During mean follow up of 15±11 months 54 patients (33%) received ≥ 1 appropriate ICD therapy (AICDT). We used binary logistic regression analysis with forward selection method to find the potential predictors of appropriate ICD therapy after device implantation. Male gender , DCM as underlying heart disease , and QRS width > 100 ms  were correlated with increased likelihood of AICDT during the follow up period. In subgroup analysis of the patients with CAD and DCM, QRS duration > 100 ms was correlated with the probability of ≥ 1 AICDT. In our patients indication of ICD implantation (primary versus secondary prevention) did not influence probability of ≥ 1 AICDT QRS width could be used as an additional simple risk stratifier beyond EF to identify potential candidates who would benefit more from ICD implantation. This may have practical implications for patient selection especially in developing countries. Indication of ICD implantation (primary versus secondary prevention) did not affect the probability of ≥ 1 AICDT during the follow up period. Development of implantable cardioverter-defibrillators (ICD) has been a dramatic advancement in the management of patients with ventricular arrhythmias. A major issue in patients with ICD is the high incidence of ICD therapies-7, whichBetween January 2001 and January 2005, 162 patients with coronary artery disease (CAD) or dilated cardiomyopathy (DCM) underwent ICD implantation at our centre. Among these 94 (58%) received single-chamber and 68 (42%) received dual-chamber ICD. The left ventricular ejection fraction (EF) was measured by transthoracic echocardiography. All the patients gave written informed consent for the procedure of ICD implantation. The mean age was 58.2 ± 13.5 years. The implanted devices were manufactured by Medtronic  and St. Jude . In implanted devices all the detection and discrimination criteria were activated with the nominal values. In all the devices we defined ventricular fibrillation zone (300 ms) plus one VT zone (400 ms). If the patient had an episode of spontaneous or induced sustained monomorphic VT slower than 370 ms we extended the VT zone to VT cycle length plus 40ms. In the VT detection zone the first therapy was three antitachycardia bursts pacing. We used the nominal values of the ICDs for the duration and tachyarrhythmia detection criteria.After ICD implantation the patients were followed on a regular basis (3 months) and upon receiving high voltage therapy in our outpatient ICD clinic. The devices were interrogated at each session and the complete set of data (including intracardiac electrograms) was recorded on floppy diskettes. The summary of the episodes were also recorded in the patient’s file. The floppy diskettes were used in this study to retrieve all spontaneous sustained arrhythmia episodes resulted in ICD therapy. These episodes resulted in ICD therapies, studied by two independent electrophysiologists (AA and MRD) to define the diagnosis. In case of discrepancy in diagnosis the final analysis of the arrhythmia episode was made by a consensus of three electrophysiologists . Beside from diagnosis, the time of arrhythmia after implantation and the mode of therapy were recorded.Appropriate ICD therapy was defined as an antitachycardia pacing or shock therapy for ventricular tachycardia or fibrillation. Indication of ICD implantation was defined as secondary prevention (n=117) in patients who had experienced aborted sudden cardiac death, sustained ventricular arrhythmia, or syncope . The indication of ICD implantation in all the other patients  was categorized as primary prevention [Variables are expressed as mean ± SD, and percentage. Differences in frequency of characteristics were assessed by independent sample student’s t-test for continuous variables. Chi-square statistics (or fisher’s exact test if applicable) used for discrete variables. We used binary logistic regression analysis with forward selection method to find the potential predictors of appropriate ICD therapy after device implantation. A two-tailed p-value < 0.05 was considered statistically significant. We used SPSS® 13.0  for data storage and analysis.One hundred sixty two patients 123 men) with ICD were followed for a mean of 15±11 months. 3 men witDuring the follow up period, 54 patients (33%) received appropriate ICD therapy . There wDuring binary logistic regression analysis QRS width > 100 ms (P=0.003), male sex (P = 0.021), and DCM as underlying heart disease (P = 0.001) were correlated with ≥ 1 appropriate ICD therapy in all patients . We chosnumber of appropriate ICD therapies.During our follow up period the above mentioned patients received mean number of 17±29 (range 1 – 132) appropriate ICD therapy. Among these the number of appropriate ATP was 11.9±28 (range 0 – 131) and the number of appropriate shock therapy was 5.1±9.9 (range 1 – 56). The success rate of ATP therapy among the episodes in the VT detection zone was 88%. However, as our study is retrospective it is possible that after first appropriate ICD therapy, electrical and/or medical treatment was adjusted or optimized to prevent new VT recurrences. Therefore, to minimize this effect we just assessed the predictors of ≥ 1 appropriate ICD therapy rather than total During subgroup analysis in patients with CAD and DCM, QRS duration > 100 ms was correlated with the probability of ≥ 1 appropriate ICD therapy during binary logistic regression .The main findings of this retrospective study are 1) QRS width might be a useful risk stratifier (beyond ejection fraction) in patients with CAD and DCM; 2) adjunctive amiodarone therapy in our patients failed to decrease the incidence of ≥ 1 appropriate ICD therapy; 3) indication of ICD implantation did not influenced the probability of ≥ 1 appropriate ICD therapy during the follow up period. Several points merit consideration. The rate of appropriate ICD therapy was different in our patients compared to the other studies ,7. This currently indicated in patients who received an ICD. These patients frequently receive ICD shocks, which severely impair quality of life. Intravenous amiodarone followed by oral dose, in patients with electrical storm results in successful management of ventricular arrhythmias and possibly a long-term prognosis similar to patients who do not have electrical storm [No empiric antiarrhythmic therapy (including amiodarone) is al storm ,10. The al storm . Amiodaral storm .Patients with ICD who received it for primary prevention of sudden cardiac death received frequent appropriate ICD therapy. Among our patients the probability of ≥ 1 appropriate ICD therapy among those who received ICD for primary and secondary prevention was comparable. Although this may be partially explained by our restricted criteria of ICD implantation for primary prevention (see definition) it also highlights the importance of implementing guidelines of primary prevention of sudden cardiac death in daily clinical practice. However, it should be mentioned that practically for the majority of the countries  and health care providers it is simply economically impossible to follow all the indications derived from the recent primary prevention ICD-trials in which the major risk stratifier is EF . TherefoSeveral epidemiological studies showed that QRS duration  predicts sudden and all-cause mortality -16. ThesFinally, only 57% of our patients received beta-blocker therapy. There is substantial evidence that beta blocker therapy has positive effects on morbidity, and mortality, in patients who have been diagnosed with heart failure and/or CAD. Beta blockers should be considered a cornerstone of therapy for these patients -20. TherAlthough we showed that (1) the adjunctive amiodarone therapy do not reduce the incidence of ≥ 1 appropriate ICD therapy and (2) beta-blockers failed also to reduce it, our study was retrospective and non-randomized. Before making any conclusion from our data we have to wait for results of randomized studies such as OPTIC.


Historically cancer vaccines have yielded suboptimal clinical results.  We have developed a novel strategy for eliciting antitumor immunity based upon homology between neoplastic tissue and the developing placenta. Placenta formation shares several key processes with neoplasia, namely: angiogenesis, activation of matrix metalloproteases, and active suppression of immune function. Immune responses against xenoantigens are well known to break self-tolerance. Utilizing xenogeneic placental protein extracts as a vaccine, we have successfully induced anti-tumor immunity against B16 melanoma in C57/BL6 mice, whereas control xenogeneic extracts and B16 tumor extracts where ineffective, or actually promoted tumor growth, respectively. Furthermore, dendritic cells were able to prime tumor immunity when pulsed with the placental xenoantigens. While vaccination-induced tumor regression was abolished in mice depleted of CD4 T cells, both CD4 and CD8 cells were needed to adoptively transfer immunity to naïve mice. Supporting the role of CD8 cells in controlling tumor growth are findings that only freshly isolated CD8 cells from immunized mice were capable of inducing tumor cell caspases-3 activation ex vivo. These data suggest feasibility of using xenogeneic placental preparations as a multivalent vaccine potently targeting not just tumor antigens, but processes that are essential for tumor maintenance of malignant potential. For successful pregnancy to occur, establishment of hemochorial placentation is essential. The process of placenta formation involves tissue morphogenesis , activatInduction of immune responses towards self-proteins associated with tumor progression is a possible therapeutic approach to cancer. While administration of syngeneic or allogeneic proteins induces poor immune response, it is reported that administration of xenogeneic homologous proteins are capable of eliciting immunity against the endogenous self-protein. Vaccination with xenogeneic FGF-R , VEGF 2, PAP 2121, and MBased on the above rationale, we hypothesized that immunization with xenogeneic placental extracts would result in a potent immune response to the inhibitory cytokines, MMPs, and immune suppressive factors produced by the cancer. The success of this approach would pave the way for cancer vaccines effective against a wide variety of tumors since it does not involve targeting tissue-specific markers, but molecules essential for the existence and propagation of the tumor. Accordingly, in this study we sought to demonstrate feasibility of such an approach using the B16 murine model of melanoma, an accepted model of immunotherapy. We have demonstrated the ability of a single immunization to inhibit tumor growth in a CD4+ T cell dependent manner and the need for both CD4 and CD8 T cells to adoptively transfer immunity. Furthermore immunization using syngeneic dendritic cells pulsed with xenogenic placental extracts induced immunity at the time of tumor inoculation in a semi-therapeutic model. These data demonstrate feasibility of using this novel antigenic source as a starting point for therapeutic vaccine development.Female C57/BL6 mice , 5 wk of age, were kept in filter-top cages at the Animal Care and Veterinary Services Facility, the University of Western Ontario according to the Canadian Council for Animal Care Guidelines. Mice were fed by food and water ad libitum and allowed to settle for 2 wk before initiation of experimentationsPorcine placental tissue was obtained from delivering sows and washed in sterile phosphate buffered saline (PBS) containing 5% penicillin streptomycin mixture and placed on ice for transportation. Vascularized placental tissue was homogenized with a tissue grinder and exposed to 4 freeze-thaw cycles alternating from liquid nitrogen to 42 Celsius water bath. Cell debris was pelleted by centrifugation at 1500 g for 45 minutes. Supernatant was collected and sterilized with 0.2 micron Millipore filters. Total protein concentration was determined using the Bradford Assay (BioRad). For control tissue, porcine liver and B16 melanoma cell line proteins were isolated using identical protocol. Experiments utilizing allogeneic placental extracts were performed using term-BALB/c placental extracts prepared in a manner identical to the xenogeneic placental extracts. The whole protein preparations were dissolved into sterile, injection-grade PBS at a concentration of 2 mg/ml, and injections of 50 uL  were performed subcutaneously into C57/BL6 mice 7 days before tumor challenge.6 cells/well in 4 ml of complete medium  supplemented with recombinant GM-CSF  and recombinant mouse IL-4 . All cultures were incubated at 37°C in 5% humidified CO2. Non-adherent cells were removed after 48 h of culture (Day 2) and fresh medium was added. After 7 days of culture, >90% of the cells expressed the characteristic DC-specific marker CD11c asdetermined by FACS. DC were washed and plated in 24-well platesat a concentration of 2 × 105 cells/well in 400 μl ofserum-free RPMI 1640. Pulsing with XPE and/or OVA was performed by addition of said antigens at a concentration of 10 ug/ml for 24 hours.At Day 0, bone marrow cells were flushed from the femurs and tibias of C57/BL6 mice, washed and cultured in 6-well plates  at 4 × 103H]thymidine (Amersham) was added to each well for 16 h. Using an automated cell harvester, the cells were collected onto glass microfiber filter, and the radioactive labeling incorporation was measured by a Wallac Betaplate liquid scintillation counter.Proliferative recall responses to XPE and ovalbumin in immunized mice were assessed by sacrificing C57/BL6 mice, 14 days after immunization with antigen-loaded DC. T cells were purified from suspensions of lymph nodes using CD4+ T cell column (R&D Systems) after washing in PBS. Purified T cells were cultured in 96 well plates with irradiated syngeneic splenocytes in triplicate and mixed with serial dilutions of OVA at concentrations ranging from 0–10 ug/ml. Following a 72-h incubation, 1 μCi of 8], CD55 The knowledge that placental extracts may be stimulatory of anticancer immune responses has been postulated to account for the lower incidence of certain tumors in multi-parous women . Indeed We have demonstrated that immunization with a preparation of xenogenic placental extract induced a CD4-dependent immune response capable of inhibiting growth of B16 melanoma cells in the C57/BL6 mouse. Demonstration of CD8 activity in the induction of tumor regression was performed in experiments showing that freshly isolated CD8 cells from immunized mice were capable of inducing both tumor cell, as well as xenogeneic trophoblast apoptosis cascades. Immune-mediated clearance seemed to be specific to the XPE since immunization with control porcine liver extracts did not mediate protection. Additionally, the xenogeneic component of XPE seems to be critical since apoptosis-inducing cells were not detected in mice immunized with allogeneic placental extracts. Although we did not elucidate specific antigenic targets of xenoplacental immunization, preliminary data seems to suggest that inhibition of neoangiogenesis may be one of the potent mechanisms. Studies using purified immunoglobulin from XPE immunized mice demonstrate the ability to inhibit in vitro proliferation of endothelial cells. This work is still in progress and will be reported in a subsequent publication.The ability of XPE to inhibit tumor growth may not be surprising given the high concentration of endothelial cells in placental tissue, and previous reports that immunization with xeno-endothelial cells causes anticancer responses ,49. HoweAlthough numerous tumor antigens are currently under intense investigation, the ability for a multi-epitopic composition such as XPE to induce immunity that is transferable to naïve recipients is somewhat surprising given the lack of immunological adjuvants or multi-injection regimens used in other studies. In conclusion, XPE appears to be a potent stimulator of anticancer immune responses in a CD4 and CD8 T cell dependent manner. The characterization of both cross-reactive tumor antigens, as well as the apparent ability to act as an immunological adjuvant, will provide interesting new avenues of research, with the possibility of developing novel, clinically applicable therapeutics from a relatively benign antigenic source.


EHP 114:1083–1091; Goth et al.][.Thimerosal, an ethylmercury-based compound used for decades as a vaccinepreservative, has previously been linked to neurotoxic effects. Newresearch reveals that it may also affect the immune system by alteringhow dendritic cells respond to biochemical signals Dendritic cells are influential primary actors in the immune system’sresponse to infectious invasion of the body. Once activated, asingle dendritic cell can direct hundreds of T cells against an infectiousagent. This ability, however, depends on the dendritic cell respondingappropriately to signals.3R and RyR, respectively), which are known thimerosal targets. These gatekeepersof intracellular calcium stores are essential for signaling activitiesaffecting dendritic cell function and maturation.Previous studies by other researchers have indicated that thimerosal isan immunotoxicant, but its specific targets were unknown. Hypothesizingthat dendritic cells might be sensitive targets, the researchers culturedbone marrow–derived dendritic cells from mice and assayedhow both mature and immature cells responded to activation followingtreatment with thimerosal. They especially focused on the responsesof inositol 1,4,5-trisphosphate and ryanodine receptors (IP3R1 and RyR1. Upon activation with the cellular energy source adenosinetriphosphate, immature control cells responded with a measurable riseand fall in intracellular calcium concentration that involved RyR1 buildingupon the initial IP3R1-controlled calcium release and afterward working with IP3R1 to bring calcium down to resting levels.The team showed for the first time that both mature and immature dendriticcells express isoforms of these receptors, IPExposure to thimerosal at concentrations as low as 20 ppb altered the timecourse of these responses, however, and prolonged the length of timethat intracellular calcium levels remained elevated. One possible consequenceof these sustained calcium levels is a change in the rate andtiming of dendritic cells’ secretion of interleukin-6, a chemicalthat triggers further immune system action. Exposure to thimerosalat concentrations above 200 ppb caused immature dendritic cells todie.RyR1 gene is highly polymorphic, an observation that raises several questionsabout the role of RyR1 in the immune system’s genetic vulnerabilityto mercury.The continuing use of thimerosal in some vaccines and other products warrantsfurther investigation of possible immunotoxic effects of this compoundand its constituent ethylmercury. The researchers also note thatthe human


Polo-like kinases control multiple events during cell division, including mitotic entry, centrosome organization, spindle formation, chromosome segregation and cytokinesis. Their roles during cytokinesis, however, are not well understood because the requirement of these kinases during early stages of mitosis complicates the study of their functions after anaphase onset.Drosophila tissue culture cells during mitosis. After anaphase onset, Polo::GFP concentrated at the spindle midzone, but also diffused along the entire length of the central spindle. Using RNA interference we demonstrate that the microtubule-associated proteins Feo and Klp3A are required for Polo recruitment to the spindle midzone, but not the kinesin Pavarotti as previously thought. Moreover, we show that Feo and Klp3A form a complex and that Polo co-localizes with both proteins during cytokinesis.We used time-lapse microscopy to analyze the dynamics of Polo::GFP in feo RNAi, our data imply that Polo recruitment to the central spindle is not required for furrowing, but some other aspect of cytokinesis.Our results reveal that the Feo/Klp3A complex is necessary for Polo recruitment to the spindle midzone. A similar finding has also been recently reported in mammalian cells  Spindle microtubules dictate the position of the cleavage plane at the metaphase/anaphase transition and then reorganize into bundles of antiparallel and interdigitating microtubules known as the central spindle during cytokinesis Drosophila and ZEN-4 in C. elegansanaphase-promoting complex (APC) can centralspindlin translocate to the plus ends of microtubules where it promotes central spindle assembly Drosophila and SPD-1 in C. elegans) prevents its interaction with the kinesin KIF-4 and consequent localization to the spindle midzone  In mammals, the cyclin-dependent kinase 1 (CDK1) prevents central spindle assembly prior to anaphase onset by inhibiting the activity of at least two MAP proteins: MKLP1 and PRC1 Drosophila, Pav has been described to interact with Polo kinase and they appear mutually dependent for localization on the central spindle Conversely, Polo and Aurora B kinases are necessary for successful cytokinesis and, consistent with this, they both translocate to the spindle midzone after anaphase onset Drosophila. We show that GFP-tagged Polo undergoes a bi-phasic accumulation at the central spindle after anaphase onset, first localizing to the spindle midzone and then spreading over the entire length of the central spindle microtubules. We also demonstrate that Polo recruitment to the spindle midzone does not require Pav, but a complex formed by Fascetto (Feo) and Klp3A, the Drosophila homologue of KIF-4 Here we investigate the dynamics of Polo kinase during cytokinesis in To investigate Polo dynamics during cell division we generated a cell line stably expressing a C-terminal GFP tagged version of this kinase. Polo::GFP initially accumulated on centrosomes and astral microtubules during prophase , but as pav RNAi cells examined (n = 10) . This is in contrast with previous observations that Pav is required for proper localization of Polo to the central spindle We used the cell line described above to analyze Polo dynamics after depleting the kinesin Pav by RNAi. As previously described for other cell lines, Pav RNAi knockdown caused cytokinesis failure after 48 hours of treatment and no cleavage furrow formation or ingression was observed  [24]. Unfeo RNAi; time points –01:00, 00:00 and 02:30), but no fluorescence was observed at the spindle midzone after anaphase onset , or after furrow formation and ingression . Interestingly, Polo::GFP was still able to spread along the central spindle microtubules in late telophase feo RNAi cells  and, consistent with this finding, we have recently identified another MAP necessary for this localization . As previously reported for other cell lines, RNAi depletion of Feo in Polo:GFP cells caused late cytokinesis failure and the central spindle appeared much less robust than in control cells These results prompted us to analyze Polo::GFP localization to the central spindle after RNAi knock-down of several kinesins and MAPs known to be required for cytokinesis. In this way, we found that Polo::GFP consistently failed to localize to the spindle midzone in cells (n = 12) treated for 72 hours with dsRNA against the MAP Feo . Two disDrosophila homologue of KIF-4, Klp3A, in Polo::GFP cells klp3A RNAi cells klp3A RNAi cells 72 hours after RNAi treatment, we were able to avoid this prometaphase block and found that, as for feo RNAi cells, little or no accumulation of Polo::GFP on the spindle midzone could be observed after anaphase onset in all examined cells (n = 7)  appeared normal in late anaphase but were then diffuse in late telophase. To confirm and extend these findings, we filmed cells expressing Pav::GFP during cytokinesis after depletion of Feo. Pav::GFP accumulated at the spindle midzone two minutes after anaphase onset in both control and feo RNAi cells  and compacted as the furrow ingressed .The effects on Polo::GFP behavior observed after depletion of Feo could be caused by the general defect in the assembly and/or organization of the central spindle observed in these cells  [13]. Thngressed  and S7. feo RNAi cells whereas the Polo::GFP signal was totally absent , suggesting that the mechanism in flies could be slightly different. Consistent with this, human Plk1 does not localize to the central spindle when heterologously expressed in Drosophila cells and embryos Our experiments reveal that Feo and Klp3A form a complex during cytokinesis and their interaction appears to be regulated by dephosphorylation , similarfeo RNAi cells  was used in all the experiments. These cells have been adapted to grow in Serum Free Medium (SFM). Generation of dsRNAs and RNAi treatments were performed as described Plasmids expressing Polo::GFP, Feo::GFP and Feo::PtA under the control of the Actin5C promoter were constructed used Gateway® technology (Invitrogen) as described Immunocytochemistry was carried out as previously described 8 cells were resuspended in lysis buffer with or without a cocktail of phosphatase inhibitors (Sigma). The proteins were then separated on an 8–16% acrylamide gel and the bands excised and analyzed by LC-MS/MS. Reverse phase liquid chromatographic separation of peptides was carried out using a PepMap C18 reverse phase, 75 mm i.d., 15-cm column  on a Eksigent nano LC system attached to a LTQ-Orbitrap (Thermo Finnigan) mass spectrometer. The MS/MS fragmentation data achieved was used to search the National Center for Biotechnology Information and Flybase databases using the MASCOT search engine (http://www.matrixscience.com). Probability-based MASCOT scores were used to evaluate identifications. Only matches with P < 0.05 for random occurrence were considered significant.Protein-A affinity purification was performed as previously described with minor modifications 7 cells were collected and processed as above and the proteins fractioned on an 8–16% SDS-PAGE gel and transferred onto a PVDF membrane as previously described For pull-down assays, 5x10Figure S1Depletion of Feo causes similar cytokinesis defects in cells expressing different transgenes.(A) Western blot analysis of protein levels after RNAi treatment. Polo::GFP cells were incubated with dsRNAs directed against Feo (feo RNAi), Klp3A (klp3A RNAi) or no dsRNA as a control. After 72 or 96 hours the proteins were extracted and separated on a 10% SDS gel, transferred onto a PVDF membrane and probed with antibodies against Feo, Klp3A and α-tubulin. (B) Increase of multinucleate cells after feo RNAi in cells expressing Polo::GFP, Pav::GFP or no transgene (control). Results are means and s.d. from 3 distinct experiments using two different dsRNAs. More than 1000 cells were counted in each experiment.(6.05 MB TIF)Click here for additional data file.Figure S2Localization of Feo::GFP during mitosis. Cells were fixed and stained to detect Feo::GFP (green in the merged panels), DNA (blue in the merged panels) and Tubulin (red in the merged panels). Bar, 10 µm(3.13 MB TIF)Click here for additional data file.Movie S1Polo::GFP behavior during cell division. This movie shows Polo::GFP dynamics during mitosis, from prophase to cytokinesis. See (11.40 MB AVI)Click here for additional data file.Movie S2Polo::GFP dynamics during cytokinesis in control cells. This movie shows Polo::GFP dynamics from prometaphase in a control cells. See (9.90 MB AVI)Click here for additional data file.Movie S3Pav is not necessary for Polo recruitment to the spindle midzone. Polo::GFP cells were treated with dsRNA directed against Pav for 48 hours and then filmed from prometaphase. See (5.99 MB AVI)Click here for additional data file.Movie S4Polo is not recruited to the spindle midzone after feo RNAi. Polo::GFP cells were treated with dsRNA directed against Feo for 72 hours and then filmed from prometaphase. See (6.10 MB AVI)Click here for additional data file.Movie S5Polo is not recruited to the spindle midzone after klp3A RNAi. Polo::GFP cells were treated with dsRNA directed against Klp3A for 72 hours and then filmed from prometaphase. See (5.50 MB AVI)Click here for additional data file.Movie S6Pav::GFP behavior during cytokinesis. This movie shows Pav::GFP dynamics from prometaphase in a control cells. See (5.91 MB AVI)Click here for additional data file.Movie S7Pav is recruited to the spindle midzone after feo RNAi. Pav::GFP cells were treated with dsRNA directed against Feo for 72 hours and then filmed from prometaphase. See (2.94 MB AVI)Click here for additional data file.


The aim of the current study was to present a comprehensive evaluation of the 8-gauge vacuum-assisted Mammotome® procedures that were performed under ultrasound guidance by a single surgeon from March 2004 to December 2006. Multiple variables, including patient demographics, characteristics of the breast lesion (based on ultrasound and mammography), procedural and histopathology variables, and interval follow-up variables (based on ultrasound and mammography), were evaluated.A retrospective analysis was conducted of a series of 304 consecutive 8-gauge Mammotome® core acquisition, while 69 (23%) were performed with only partial excision of the ultrasound lesion during Mammotome® core acquisition (diagnostic tissue sampling only). 100% of all ultrasound lesions were accurately diagnosed, demonstrating no apparent false-negative results among the 256 patients that were compliant with follow-up at a median interval follow-up duration of 11 months (range 1 to 37). Likewise, 89% of all appropriately selected ultrasound lesions were completely excised, as demonstrated on interval follow-up ultrasound at a median time of 6 months . There were no independent predictors of successful complete excision of any given appropriately selected ultrasound lesion by the ultrasound-guided 8-gauge Mammotome® biopsy technique.Among 304 procedures, 235 (77%) were performed with the presumption of complete excision of the ultrasound lesion during Mammotome® system is highly accurate for ultrasound-guided diagnostic biopsy of suspicious breast lesions and is highly successful for complete excision of appropriately selected presumed benign breast lesions. This particular technology should be routinely offered to all appropriately selected patients that are evaluated by physicians involved in breast-specific health care.The 8-gauge vacuum-assisted Mammotome The thrThe software program SPSS 14.0 for Windows  was used for all statistical analyses. For univariate comparisons of categorical variables, either Pearson chi-square test or Fisher exact test was utilized. Continuous variables were expressed as median (range). For univariate comparisons of continuous variables, one-way analysis of variance (ANOVA) was utilized. All reported univariate P-values were two-sided. All univariate P-values determined to be 0.05 or less were considered to be significant. Multivariate logistic regression analysis was performed in appropriately selected situations on all variables with a univariate P-value of 0.10 or less, in order to assess for the determination of possible independent predictors.® procedure ultrasound are shown in Table ® biopsy procedures performed, 235 (77%) were performed with the presumption that the ultrasound lesion had been completely excised during Mammotome® core acquisition, while 69 (23%) were performed with only partial excision of the ultrasound lesion during Mammotome® core acquisition (diagnostic tissue sampling only). Patients in which the ultrasound lesion appeared to be completely excised during Mammotome® core acquisition were generally younger, had a predilection toward BI-RADS category 3 and 4 classification of the breast lesion, and had a significantly smaller original breast lesion size  based on the pre-Mammotome® procedure ultrasound findings. Whereas, patients in which only diagnostic tissue sampling was undertaken during Mammotome® core acquisition were generally older, had a predilection toward BI-RADS category 4 and 5 classification of the breast lesion, and had a significantly larger original breast lesion size based on the pre-Mammotome® procedure ultrasound findings.Patient demographics and characteristics of the original breast lesions seen on the pre-Mammotome® biopsy procedures are shown in Table ® core acquisition, significantly more tissue was removed  and the final histopathology more frequently rendered a benign diagnosis (predominately fibroadenomas). For those cases in which only diagnostic tissue sampling was undertaken during Mammotome® core acquisition, significantly less tissue was removed and the final histopathology more frequently rendered a malignant diagnosis (predominately invasive breast carcinomas). The overall number of post-procedural complications  and the severity of such complications seen were not significantly different between the two groups. No intraoperative surgical management of any post-procedural complications was ever required. No post-procedural infectious complications were encountered.Procedural and histopathology variables for the ultrasound-guided 8-gauge Mammotome® biopsy procedure, 256 (84%) patients were compliant and returned for interval follow-up, while 48 (16%) were noncompliant and did not returned for interval follow-up. The frequency of such interval follow-up was identical both for those patients in which the ultrasound lesion appeared to be completely excised during Mammotome® core acquisition and for those patients in which only diagnostic tissue sampling was undertaken during Mammotome® core acquisition. Interval follow-up breast imaging was performed in 189 (62%) patients at a median time of 6 months . This included 188 (62%) patients who underwent interval follow-up ultrasound and 179 (59%) patients who underwent interval follow-up mammogram. Instead of interval follow-up breast imaging, 64 (21%) of the patients underwent some sort of subsequent excisional breast procedure at a time shortly after their initial ultrasound-guided 8-gauge Mammotome® biopsy procedure. This included 46 invasive breast carcinomas, 4 cases of ductal carcinoma in situ, 2 intracystic papillary carcinomas, 1 subcutaneous basal cell carcinoma of the breast, 2 cases of epithelial atypia, 3 benign breast tumors requiring complete excision, 4 benign breast findings that were incidentally excised within the same excisional specimen as a known breast carcinoma, and 2 cases in which a benign finding was incidentally excised as part a planned prophylactic mastectomy. Far fewer patients in the group in which only diagnostic tissue sampling was undertaken during Mammotome® core acquisition had interval follow-up breast imaging since far more of those same patients had a malignant breast lesion that required some sort of subsequent excisional breast procedure. For those patients with a breast malignancy, 39 of 53 (74%) underwent subsequent successful breast conservation therapy, while 14 of 53 (26%) ultimately required a mastectomy .Interval follow-up variables are shown in Table ® core acquisition had no residual ultrasound lesion visible on the interval follow-up ultrasound , as compared to patients in which the procedure was performed for the purpose of only diagnostic tissue sampling during Mammotome® core acquisition . Thus, complete ultrasound lesions removal was confirmed on interval follow-up ultrasound in 89% of patients in which the ultrasound lesion appeared to be completely excised during Mammotome® core acquisition. Overall, 34 (18%) patients who underwent an ultrasound-guided 8-gauge Mammotome® biopsy procedure and who had interval follow-up ultrasound demonstrated a residual ultrasound lesion on the interval follow-up ultrasound, consisting of 19 (11%) of those in which an attempt was made to completely excise the original ultrasound breast lesion and 15 (83%) of those in which the procedure was performed for the purpose of only diagnostic tissue sampling.As is shown in Table Residual ultrasound lesion characteristics seen on interval follow-up ultrasound imaging are shown in Table ® core acquisition had a residual ultrasound lesion on interval follow-up, comparatively only 4 of 8 (50%) of the same such patients had a residual mammographic lesion on interval follow-up.In Table ® biopsy procedure did not significantly influence whether a residual ultrasound lesion could be seen on interval follow-up ultrasound imaging. However, and as would be intuitively deducted, the median size of the original ultrasound breast lesion  significantly correlated with whether a residual ultrasound lesion could be seen on interval follow-up ultrasound imaging, with smaller such original ultrasound breast lesions being seen in the group having no residual ultrasound lesion seen on interval follow-up ultrasound imaging and with larger such original ultrasound breast lesions being seen in the group having a residual ultrasound lesion seen on interval follow-up ultrasound imaging. Despite this apparent significant size correlation that was demonstrated on ANOVA, multivariate analysis using logistic regression failed to show that any single original ultrasound breast lesion size parameter could independently predict ones ability to know whether a residual ultrasound lesion would be seen on interval follow-up ultrasound imaging.As is shown in Table ® biopsy was performed that has subsequently required an additional breast biopsy procedure. These have included three repeat ultrasound-guided 8-gauge Mammotome® biopsy procedures  and one excisional biopsy . All four such repeat breast biopsy procedures simply showed benign findings that were consistent with the results of their previous original Mammotome® biopsy findings. Therefore, to date, there have been no instances within the group of 256 patients that were compliant with interval follow-up in which the histopathology results of the original ultrasound-guided 8-gauge Mammotome® biopsy have changed .To date, the median duration of interval follow-up for those 256 patients who were compliant and who returned for interval follow-up has been 11 months  . In this regard, since over 50% of the invasive breast cancers diagnosed in the present series were less than one centimeter in size, it is very apparent that the ultrasound-guided 8-gauge Mammotome® biopsy technique is highly advantageous for allowing generous and representative tissue sampling and for high accuracy of correctly diagnosing suspicious small subcentimeter breast lesions. In a similar regard, the ultrasound-guided 8-gauge Mammotome® biopsy device may potentially minimize the risk of histopathologic underestimation and false negative results [In the current study, 100% of all ultrasound lesions were accurately diagnosed by the ultrasound-guided 8-gauge Mammotome® biopsy technique, as demonstrated on interval follow-up ultrasound at a median time of 6 months . This figure of a nearly 90% success of complete excision on ultrasound-guided 8-gauge Mammotome® biopsy exceeds the 73% success of complete excision previously reported by Fine et al [® device in the current series versus the use of both 8-gauge and 11-gauge Mammotome® devices in the Fine et al series [® biopsy technique allows for on-going, real-time, ultrasound-guided "tailoring" of the excision cavity of any given presumed benign lesion that the operator is attempting to completely excise at the time of the Mammotome® procedure. This is itself a potential advantage over that of any percutaneous en bloc excision technique, since such percutaneous en bloc excision techniques generally allow for only a one-time attempt at tissue acquisition and can theoretically require an additional diagnostic procedure for complete excision any given presumed benign lesion if positioning variation results in altered coordinates of the region of initial en bloc tissue acquisition.In the current study, 89% of all appropriately selected ultrasound lesions were completely excised by the ultrasound-guided 8-gauge Mammotome® system is the desired and optimal method both for accurate diagnosis of any given suspicious ultrasound lesion and for complete removal of appropriately selected presumed benign ultrasound lesions. In this regard, and as emphasized by Sebag et al [® system allows for more rapid and larger volume tissue acquisition, resulting in more representative tissue sampling in cases of diagnostic breast biopsy and need for removal of fewer total Mammotome® cores in cases of attempted complete ultrasound lesion excision. Furthermore, as do all surgeons, we strongly advocate the adherence to sound oncologic principles for the complete surgical excision of malignant breast lesions.Therefore, when a breast lesion is sonographically visible, it is our opinion that the 8-gauge Mammotomeag et al , the 8-g® biopsy technique. Despite this lack of any exact evidence to allow one to set a finite size criteria for the successful complete excision of appropriately selected ultrasound breast lesions by the ultrasound-guided 8-gauge Mammotome® biopsy technique, the present authors arbitrarily recommend limiting any attempts at complete excision to that of such breast lesions which approximate a prolate ellipsoid  or a scalene ellipsoid  and to that of such breast lesions that have a maximum lesion length of 2.5 cm and maximum lesion width of 1.5 cm. In contrast, no upper limit for the maximum lesion height seems necessary when the attempted ultrasound-guided 8-gauge Mammotome® excision procedure is performed by appropriate placement of the Mammotome® device immediately beneath the ultrasound lesion. However, the most difficult breast lesions to excise are those breast lesions that approximate an oblate ellipsoid  in which the largest two nearly identical dimensions exceeds the 2.5 cm maximum size limit or those breast lesions that approximate a perfect sphere in which the diameter exceeds the 2.5 cm maximum size limit. These above recommendations are partially based on the experience of the present surgeon (SPP) in the currently reported series, as well as are based on the actual length of the tissue collection chamber (23 mm) and the actual inner diameter of the cutter blade (3.9 mm) for the 8-gauge Mammotome® biopsy device [Since multivariate analysis using logistic regression failed to show that any single original ultrasound breast lesion parameter could independently predict the ability to know whether a residual ultrasound breast lesion would be seen on interval follow-up ultrasound imaging, then one can not inexplicitly recommend a particular breast lesion size threshold below which any given ultrasound breast lesion can be successfully and completely excised by the ultrasound-guided 8-gauge Mammotomey device .® biopsy studies  declare that they have no competing interests.® system. He designed the current study, collected the data, and performed all data analyses. He organized, wrote, and edited all aspects of this manuscript. He approved the final version of this manuscript. REJ was the pathologist who was involved in reading the histopathology for many of the cases contained within this series. He edited all aspects of this manuscript and approved the final version of this manuscript.SPP was the surgeon who performed all the ultrasound-guided diagnostic biopsy and selective excision of breast lesions using the 8-gauge vacuum-assisted Mammotome