bluuebunny/crossref_metadata_2025 huggingface dataset:
Xenogeneic human anti‐pig cytotoxicity mediated by activated natural killer cells
Authors: Seebach Jörg D. , Yamada Kazuhiko , McMorrow Isabel M. , Sachs David H. , DerSimonian Harout
Published: November 2008

Abstract:Even if hyperacute rejection, which is mediated by human natural antibodies (nAb) and complement, could be prevented, xenoreactive human anti‐pig cellular responses may lead to delayed and/or chronic xenograft rejection. Among the cell populations participating in such rejection, NK cells have been proposed as an important component. In this study we report the in vitro cytotoxic activity of natural killer (NK) cells obtained from healthy human donors against porcine target cells. Freshly isolated peripheral blood mononuclear cells (PBMC) and purified NK cells (CD16+/CD56+, CD3‐, CD20‐, CD33‐) exhibited little or no cytotoxic activity when tested on porcine phytohemagglutinin (PHA)‐stimulated lymphoblasts or bone marrow‐ or aortic‐derived endothelial cell lines in the presence of serum‐free medium. Killing was considerably higher in the presence of human decomplemented plasma, containing xenoreactive nAb, or purified Gal(α1,3)Gal‐reactive antibodies, suggesting that antibody dependent cell‐mediated cytotoxicity (ADCC) mediated by NK cells is an important mechanism involved in xenogeneic cytotoxicity. After incubation of human PBMC for 6 days in the presence of irradiated xenogeneic porcine or allogeneic stimulator cells, or in the presence of exogenous interleukin 2 (IL‐2), the cytotoxic activity of the bulk cultures as well as that of isolated NK cells (separated from stimulated bulk cultures) against xenogeneic targets increased considerably, and corresponded to an increased NK‐specific lysis of K562 target cells. Cell surface staining and flow cytometry showed that CD16+/CD56+, CD3‐ NK cells composed ca. 25% of short‐term (6 days) xenogeneic, allogeneic, or IL‐2 stimulated bulk cultures. In summary, these data suggest that, in contrast to allogeneic cell‐ mediated killing, xenogeneic human anti‐porcine cytotoxicity includes an important contribution from NK cells.
Porcine alveolar macrophage Mac‐1 (CD11b/CD18) adhesion molecule expression
Authors: Lee Jong‐Keuk , Schook Lawrence B. , Rutherford Mark S.
Published: November 2008

Abstract: The adhesion molecule Mac‐1 plays an important role during leukocyte recruitment into inflammatory sites and also serves as a complement receptor type 3 (CR3). Mac‐1 is a noncovalent heterodimer consisting of an α‐chain (CD11b) and a β‐chain (CD 18). We report the cloning and sequencing (GenBank accession number U40072) of the porcine CD 11b cDNA using the reverse transcriptase‐polymerase chain reaction (RT‐PCR). Porcine CD 11b cDNA and deduced amino acid sequences demonstrated 84% and 80% sequence identity, respectively, with human CD11b. The 4.7 kb porcine CD11b transcript was also similar to the size of the human CD11b transcript. Northern blot analyses detected CD11b transcripts in spleen cells and peripheral blood leukocytes, but not in lung lavage cells. In contrast to Northern blot results, 72% of freshly isolated alveolar macrophages stained positive using a cross‐reactive anti‐human CD11b antibody (TMG6–5). Compared to spleen and peripheral blood cells, alveolar macrophages expressed the highest levels of CD 18 mRNA. Using anti‐CD 18 monoclonal antibodies which cross‐react with porcine CD 18, two different porcine CD 18 epitopes were identified by flow cytometric antibody competition assays. However, the majority of the anti‐CD 18 antibodies tested competed for the same CD 18 epitope. Mac‐1 expression in porcine alveolar macrophages was not altered at either the mRNA or surface expression level by inflammatory stimuli, including fMLP, PMA, LPS, hIL‐1α, hIL‐6, hTNF‐α, and hIFN‐γ.
Acute cellular xenograft rejection
Authors: Korsgren Olle
Published: November 2008

Abstract:The acute cellular rejection process after xenotransplantation of vascularized organs, i.e. heart, kidney and liver, has so far been difficult to study since the implanted organs are lost in a hyperacute rejection before cellular rejection develops. Primarily non‐vascularized xenografts‐e.g. pancreatic islets‐escape the hyperacute rejection, but succumb to cell‐mediated rejection during the first week after transplantation in either mice, rats or cynomolugus monkeys. The present view on the mechanisms orchestrating islet xenograft rejection in these animal models will be summarized and in part generalized to give insight into the process of acute cellular xenograft rejection.
Utility of xenografts: Lack of correlation between PRA and natural antibodies to swine
Authors: Bartholomew A. , Latinne D. , Sachs D.H. , Arn J. Scotl , Gianello P. , de Bruyere ML , Sokal G. , Squifflet J. , Alexandre G. , Comerford C. , Saidman S. , Cosimi A.
Published: November 2008

Abstract: Among the patients that might potentially benefit from the availability of xenografts are those in kidney failure who demonstrate high levels of antibody reactivity to panels of typing lymphocytes. Such individuals with high PRA (panel reactive antibody) are unlikely to receive a renal allograft because they are highly sensitized to the vast majority of potential donors. In addition, all humans have demonstrable levels of natural antibodies reactive to distantly related species such as the pig. If there were a correlation between PRA and levels of natural antibodies, then such patients would also be at greater risk for hyperacute rejection of xenografts. We have therefore examined, in a blinded fashion, the porcine lymphocyte reactivity of sera from PRA positive donors. Subsets of the 105 sera tested were grouped by PRA level and analyzed for levels of natural antibodies detectable by a complement‐dependent cytotoxicity assay on porcine lymphocytes. There was no significant difference in the range of titers of natural antibodies between subsets. Thus, there was no demonstrable correlation between levels of PRA and levels of natural antibodies to porcine lymphocytes.

In addition, we studied the sera of 11 highly sensitized patients who received renal allografts and conventional triple drug immunosuppression in order to determine whether immunosuppression to maintain a vascularized allograft had an effect on PRA and/or xenoreactivity. Mean post‐operative PRA was significantly lower than mean pre‐operative PRA, while there was no significant difference in xenoreactivity. Thus, pre‐transplant immunosuppression may permit an increased opportunity to identify compatible human donors for highly sensitized recipients and will not help or hinder the pre‐transplant conditioning of a candidate for I xenotransplantation.
Rejection of hamster skin xenografts by rats in the absence of anti‐hamster antibody formation
Authors: Gourlay William A. , Maki Takashi , Monaco Anthony P.
Published: November 2008

Abstract: The hamster to rat xenograft combination is considered to be a “difficult” concordant combination. Although the rat lacks preformed anti‐hamster antibodies, there is rapid production of antibodies upon exposure to hamster antigens. When vascularized hamster xenografts are rejected in the rat, anti‐hamster antibodies are invariably detected in both the serum and in the rejected tissue. In allogeneic and other concordant xenogeneic (rat to mouse) combinations, rapamycin has been shown to markedly augment the survival of skin grafts in ALS‐treated, bone marrow injected mice. We investigated the ability of rapamycin to prolong the survival of skin grafts and block antibody production in the hamster to rat combination.

Outbred Golden Syrian hamsters served as skin and marrow donors and Lewis rats were recipients. ALS was administered on days ‐1, 0, and +2 relative to skin grafting. Rapamycin was administered at 3 mg/kg on alternate days from day +1 through day +13 (seven doses). Donor specific bone marrow was infused IV on day +4 at a dose of either 100 times 106 or 250 times 106 nucleated cells. Antibody titers were determined serially by using a complement dependent cytotoxicity assay. The administration of rapamycin alone (MST 8.5 days) and ALS alone (MST 10 days) prolonged graft survival slightly but significantly compared with untreated controls (MST 7 days). The addition of BM had no additional effect on graft survival beyond that achieved with ALS alone. Rapamycin markedly prolonged skin graft survival when added to ALS (MST 16 days), ALS+BM100 (MST 19 days), or ALS+BM250 (MST 20.5 days).

Rats receiving ALS or ALS+BM produced anti‐hamster antibodies in a slightly delayed fashion and at a slightly lower titer when compared to controls. Treatment with rapamycin alone substantially diminished but did not completely eliminate anti‐hamster antibody production. Rats receiving rapamycin with ALS or ALS+BM failed to produce anti‐hamster antibodies entirely, although they all eventually rejected the hamster skin grafts. Rapamycin, when used in combination with ALS or ALS plus BM is able to prolong skin graft survival and completely abolish antibody production in the hamster to rat combination. The eventual rejection of hamster skin grafts in this circumstance occurs in the absence of anti‐hamster antibodies.
Inhibition of complement‐mediated immune hemolysis by the constant domain of immunoglobulin
Authors: Kojima Taiki , Del Carpio Carlos A. , Hayashi Shuji , Yokoyama Itsuo , Takagi Hiroshi
Published: November 2008

Abstract: It has been reported that high‐dose administration of intravenous immunoglobulin (IVIG) is useful for the inhibition of complement‐dependent immune cytolysis, which is the first event of xenogeneic hyperacute rejection. We have found that monomeric peptide residues 289–292 and 282–292 from the second constant domain of the γ‐chain of the human IgG, part of the Clq binding sites, was also effective in inhibition of pig RBC lysis by human serum, and IgM‐rich IVIG (Pentaglobulin) was more active than IVIG in this assay.
Human CD8 responder cells can be directly activated to proliferate and to produce IL‐2 following stimulation by allogeneic, but not by xenogeneic, porcine blood mononuclear cells
Authors: Kawagishi Naoki , Möller Erna , Satake Masahiro
Published: November 2008

Abstract:In order to determine the precise nature of human T lymphocytes reactivity against porcine stimulator cells, purified CD4+and CD8+ human peripheral T lymphocytes have been tested for their responsiveness against porcine stimulator cells. In a xenogeneic mixed lymphocyte reaction (MLR), CD4+ T cells were capable of proliferating as a result of the recognition of porcine peripheral blood lymphocytes (PBL), whereas CD8+ T cells were unresponsive. A proliferative response of CD8+ T cells could be restored by treatment with human IL‐2, but not by IL‐lα, IL‐lβ, or IL‐6. Production of IL‐2 was not detected in the xenostimulated CD8+ responder cells, nor could IL‐2 production be restored by the addition of IL‐lα, IL‐1β, or IL‐6. The presence of human CD4+responder cells was crucial both for a xenoproliferative response and for IL‐2 synthesis. However, when the expression of the IL‐2 receptor (CD25) on the CD8+T cells was analyzed, no difference was detected between xenostimulated and allostimulated CD8+T cells. When the development of cytotoxic T cells in xenogeneic and allogeneic MLRs was compared, the cytotoxic activity exhibited by purified CD8+ T cells in xenogeneic MLR was significantly lower than that in the allogeneic combination. In the xenogeneic combination, exogenous IL‐2 reconstituted the cytotoxicity by purified CD8+T cells; however, IL‐lα, IL‐lβ, or IL‐6 did not.

Our results show that purified human CD4+T cells respond directly against pig PBMCs, whereas purified CD8+T cells do not. Furthermore, responsiveness in CD8+T cells is completely restored by the addition of human IL‐2.
High‐affinity anti‐Gal immunoglobulin G in chronic rejection of xenografts
Authors: Galili Uri , Minanov Oktavijan P. , MlchSer Robert E. , Stone Kevin R.
Published: November 2008

Abstract: The immune system in humans and monkeys responds vigorously against α‐gal (α‐galactosyl, Galα1‐3Galβ1‐4GIcNAc‐R) epitopes on xenografts by producing large amounts of high‐affinity anti‐Gal immunoglobulin G molecules. These antibody molecules may exacerbate chronic rejection of xenografts via several mechanisms including: antibody‐dependent cell cytotoxicity, chronic activation of endothelial cells, and increasing immunogenicity of xenograft‐specific antigens by targeting them to antigen‐presenting cells.
Direct gene replacement of the mouse α(1,3)‐galactosyltransferase gene with human α(1,2)‐fucosyltransferase gene: Converting α‐galactosyl epitopes into H antigens
Authors: Koike Chihiro , Katayama Akio , Kadomatsu Kenji , Muramatsu Takashi , Hiraiwa Nozomu , Kannagi Reiji , Nakashima Izumi , Yokoyama Itsuo , Takagi Hiroshi
Published: November 2008

Abstract: The chronic donor organ shortage has led to the production of transgenic animals. We assume that cells or organs derived from possible animal donors carrying a large amount of α‐galactosyl epitopes should not be transplanted into humans, because a corresponding amount of immunosuppressants would be needed to prolong the survival of such xenografts in the recipients. This may not only make the recipients compromised hosts but also introduce some unknown or uncontrollable pathogens into society at large. We also assume that gene manipulation itself should not be a detriment to possible transgenic animals. To explore possibilities that not only can minimize the possible detrimental factors to humans, such as α‐galactosyl epitopes, but also can minimize the possible detriment to transgenic animals, such as random integration of the extraneous genes with or without uncontrollable regulatory sequences, we have produced a DNA construct that replaces the mouse oc(1,3)‐galactosyltransferase gene (GT) with the human a(1,2)‐fucosyltransferase (FT) minigene (promoterless for the expression of FT) at the GT locus. The mouse fibrosarcoma cell line, L929, was transfected with the construct. Colonies were obtained after incubation with non‐heat‐inactivated human serum. Southern blot analysis demonstrated that one allele of the mouse GT gene was replaced with the FT minigene at the GT locus without integration of any selectable marker genes. The immunostaining analysis with lectins showed that the transfectants expressed H antigens, which suggested that H antigens were expressed by the intrinsic GT promoter. Thus gene replacement, knock‐in, of the mouse GT with the human FT without integration of any selectable marker genes in the GT locus was shown to be possible. This is especially important in producing transgenic animals for the clinical application of xenografts into humans.
A polycistronic retrovirus vector for expression of swine MHC class II DRaα/β heterodimers
Authors: Banerjee Papia T. , Kaynor G. Campbell , Muthukumar Shanfhini , Denaro Maria , Shimada Hideaki , Zhu Shaochun , Rosa Margaret D. , Sachs David H. , LeGuern Christian
Published: November 2008

Abstract: A recombinant polycistronic retroviral vector was generated to express multimeric proteins from a single transcript by using internal ribosomal entry sites which allow independent initiation of translation from internal cistrons. In addition, an enhancerless SV40 origin of replication was incorporated into the recombinant plasmids between the retroviral long terminal repeat sequences to facilitate the testing of expression of multiple proteins in a transient COS cell transfection assay.

With the ultimate goal of utilizing these vector types for the induction of transplantation tolerance via retrovirus‐mediated gene transfer (LeGuern et al. J Mol Med 1995: 42: 269 [1]; Sachs Transplant Sci 1993: 42: 59 [2]) we have incorporated miniature swine MHC class II DRA and DRB cDNAs which encode for the SLA‐DRα and SLA‐DRβ polypeptide chains, respectively, into the retroviral construct. The pro viral DNA integrated within the producer clone genome, was found to be stable over a 3‐month period and no loss of expression of DR heterodimers was observed on the cell surface of either the retrovirus producer cells or the retrovirally transduced fibroblast cell line. High titers (>1 times 106 CFU/ml) of recombinant particles, devoid of replication competent helper viruses, were obtained. Such vectors represent the first successful attempt to express multimeric proteins at the cell surface of transduced cells.
Expression of swine MHC class II genes in a cynomolgus monkey: Retro virus‐mediated gene therapy in a preclinical transplantation model
Authors: Banerjee Papia T. , Ierino Francesco L , Kaynor George C. , Giovino Maria , Hawley Robert , Rosa Margaret D. , LeGuern Christian , Sachs David H. , Monroy Rodney
Published: November 2008

Abstract: Immune reactivity against products of the major histocompatibility complex (MHC) is the major barrier to allotransplantation. Conversely, sharing of MHC class II antigens I appears to be of overwhelming importance in permitting the induction of immune tolerance to vascularized organ allografts, as demonstrated previously in miniature swine. Class II antigen has also been shown to be recognized predominantly in human anti‐pig xenoreactions in vitro. To achieve tolerance in the xenogeneic pig‐to‐primate model, we are therefore investigating an approach involving retrovirus‐mediated gene therapy to transfer swine MHC (SLA) class II genes into primate primitive hematopoietic stem cells, so that swine MHC class II antigens may participate in the education of the recipient's newly developing T cell repertoire. We report here the in vitro and in vivo use of a recombinant retrovirus containing a polycistronic retroviral vector which carries swine MHC class II DRA and DRB cDNA sequences to transduce CD34+ bone marrow cells (BMC) from a cynomolgus monkey. Transduction efficiency was assessed by reverse transcriptase‐polymerase chain reaction analysis of the colony‐forming unit progenitor colonies grown in the absence of gentamycin; 55% and 24% of the progenitor colonies were determined to express the retroviral transcript at 1 week and 3 weeks post‐transduction, respectively. These in vitro studies have been extended to the transplantation of retrovirally transduced autologous stem cells into a cynomolgus monkey prepared with a non‐myeloablative conditioning regimen. Prolonged expression of SLA‐DR transcripts in monkey peripheral blood mononuclear cells (PBMC) has been documented over a 56‐week period after transplantation of retrovirus‐transduced bone marrow cells. However, we could not detect any protein expression by FACS analysis on the surface of primate PBMC or bone marrow, using a porcine SLA‐DR‐specific antibody. Engraftment of hematopoietic cells with the transduced genes was further detected in the progenitor colonies grown from the bone marrow cells harvested at 4 weeks and 25 weeks after bone marrow transplantation. Our results thus document that long‐term engraftment of retrovirally transduced hematopoietic cells can be achieved in a primate model using a non‐myeloablative preparative I regimen.
Allogeneic and xenogeneic islets are rejected by different and specific mechanisms: A study in rodents using a mixed allogeneic‐xenogeneic islet transplantation model
Authors: Wennberg L. , Rafael E. , Liu J. , Sundberg B. , Wernersson A. , Korsgren O.
Published: November 2008

Abstract:Indirect evidence suggests that islet alio‐ and xeno‐graft rejections are two qualitatively, and not simply quantitatively, different processes. This, however, has never been clearly shown and a direct in vivo comparison of cell‐mediated alio‐ and xeno‐graft rejections has never been made. Therefore, Lewis rats were transplanted with either Wistar‐Furth rat islets or fetal porcine islet‐like cell clusters (ICC) under the kidney capsule. Other Lewis rats were transplanted with Wistar‐Furth rat islets and fetal porcine ICC, which were mixed together into one single graft. Animals were left untreated or were treated with cyclosporin A (CyA, 20 mg/kg b.w., p.o.) daily. Twelve days following transplantation, islet alio‐ and xeno‐graft survival was evaluated morphologically and any cellular infiltration into the grafts was characterized immunohistochemically. In untreated animals, both allogeneic and xenogeneic islets were destroyed by massive cellullar infiltration. The main infiltrating cells in islet allograft rejection were TCR a/(i‐positive T‐cells. In contrast, the main infiltrating cells in islet xenograft rejection were EDI‐ and ED2‐positive macrophages. In islet xenograft rejection, only a few T‐cells were found. In islet allografts removed from rats treated with CyA, many morphologically intact rat islets were found. Only a few infiltrating cells were detected and these were both EDI‐positive macrophages and TCR a/p‐positive T‐cells. In CyA‐treated rats transplanted with porcine ICC, we noted a massive infiltration with EDI‐ and ED2‐positive macrophages and TCR a/p‐positive T‐cells, similar to that observed in untreated animals. No endocrine cells were present. In mixed allogeneic‐xenogeneic islet grafts transplanted into CyA‐treated rats, many morphologically intact rat islets were found, as evidenced by positive staining for rat MHC class I. However, no porcine ICC remained. The xenogeneic islets in these mixed grafts were destroyed by massive cellular infiltration, similar to that observed after transplantation of xenogeneic islets alone. Obviously, the xenogeneic islets in these mixed grafts were rejected by a cell‐mediated process qualitatively different from that of cell‐mediated allograft rejection.
Species‐specific detection of porcine xenografts with an antibody against a novel epitope of the lymphocyte homing receptor CD44
Authors: Oettinger Henry F. , Sullivan Jennifer A. , Crosby Katherine E. , Kelley Julie A. , Jaooby Douglas B. , Dinsmore Jonathan , Zawadzka Agatha , Edge Albert S.B.
Published: November 2008

Abstract: We have isolated and characterized a murine monoclonal antibody, 10–14, that is reactive with an 85 kDa glycoprotein found on porcine peripheral blood lymphocytes, cultured aortic endothelial cells, fetal cardiomyocytes, and mixed neural/glial cell cultures. In contrast to the anti‐CD44 antibody Z062, which reacted with both porcine and murine CD44, the 10–14 antibody did not react with monkey, rabbit, rat, or murine cells, suggesting that 10–14 recognized a novel epitope found exclusively on the porcine CD44 molecule; 10–14 is thus a novel reagent for identification of xenotransplanted porcine cells. The reagent has been used in this study as an immunohistochemical probe to detect porcine neural cell grafts in the central nervous system of the rat and to label porcine fetal cardiomyocytes. Moreover, the antibody provides information on the expression of the CD44 molecule that is critical for lymphocyte adhesion and trafficking.
Association of the Ser704Cys DISC1 polymorphism with human hippocampal formation gray matter and function during memory encoding
Authors: Di Giorgio Annabella , Blasi Giuseppe , Sambataro Fabio , Rampino Antonio , Papazacharias Apostolos , Gambi Francesco , Romano Raffaella , Caforio Grazia , Rizzo Miriam , Latorre Valeria , Popolizio Teresa , Kolachana Bhaskar , Callicott Joseph H , Nardini Marcello , Weinberger Daniel R , Bertolino Alessandro
Published: November 2008

A common nonsynonymous single nucleotide polymorphism leading to a serine‐to‐cysteine substitution at amino acid 704 (Ser704Cys) in the DISC1 protein sequence has been recently associated with schizophrenia and with specific hippocampal abnormalities. Here, we used multimodal neuroimaging to investigate in a large sample of healthy subjects the putative association of the Ser704Cys DISC1 polymorphism with in vivo brain phenotypes including hippocampal formation (HF) gray matter volume and function (as assessed with functional MRI) as well as HF functional coupling with the neural network engaged during encoding of recognition memory. Individuals homozygous for DISC1 Ser allele relative to carriers of the Cys allele showed greater gray matter volume in the HF. Further, Ser/Ser subjects exhibited greater engagement of the HF together with greater HF–dorsolateral prefrontal cortex functional coupling during memory encoding, in spite of similar behavioral performance. These findings consistently support the notion that Ser704Cys DISC1 polymorphism is physiologically relevant. Moreover, they support the hypothesis that genetic variation in DISC1 may affect the risk for schizophrenia by modifying hippocampal gray matter and function.
Adenosine preconditions against ouabain but not against glutamate on CA1‐evoked potentials in rat hippocampal slices
Authors: Ferguson Alexandra L. , Stone Trevor W.
Published: November 2008

Hypoxic and ischaemic brain damage are believed to involve excessive release of glutamate, and recent work shows that glutamate‐induced damage in brain slices can be reduced by preconditioning with hypoxia or glutamate itself. Because adenosine is a powerful preconditioning agent, we have investigated whether adenosine could precondition against glutamatein vitro. In rat hippocampal slices, glutamate depolarization reduced the amplitudes of antidromic‐ and orthodromic‐evoked potentials, with only partial recovery. Applying adenosine before these insults failed to increase that recovery. Ouabain also produced depolarization with partial reversibility, but adenosine pretreatment increased the extent of recovery. The preconditioning effect of adenosine on ouabain responses was prevented by blocking receptors forN‐methyl‐d‐aspartate (NMDA), but not receptors for kainate or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), and was blocked by inhibiting nitric oxide synthase. Preconditioning was also abolished by the ATP‐dependent potassium channel blockers, glibenclamide (cytoplasmic) or 5‐hydroxydecanoate (mitochondrial). We conclude that adenosine does not precondition against glutamate in hippocampal slices, but that it does precondition against ouabain with a pharmacology similar to studiesin vivo. Ischaemic neuronal damage is a complex of many factors, and because adenosine can precondition against ischaemic neuronal damage, its failure to protect against glutamate highlights limitations of using glutamate alone as a model for ischaemia. Because damage following ischaemia, trauma or excitotoxicity also involves reduced Na+,K+‐ATPase activity, and adenosine can precondition against ouabain, we propose that ouabain‐induced damage represents an additional or alternative model for the contribution to cell damage of Na+,K+‐ATPase loss, this being more relevant to the mechanisms of preconditioning.
A chemical compound commonly used to inhibit PKR, {8‐(imidazol‐4‐ylmethylene)‐6H‐azolidino[5,4‐g] benzothiazol‐7‐one}, protects neurons by inhibiting cyclin‐dependent kinase
Authors: Chen Hsin‐Mei , Wang Lulu , D’Mello Santosh R.
Published: November 2008

Activation of the double‐stranded RNA‐dependent protein kinase (PKR) has been implicated in the pathogenesis of several neurodegenerative diseases. We find that a compound widely used as a pharmacological inhibitor of this enzyme, referred to as PKR inhibitor (PKRi), {8‐(imidazol‐4‐ylmethylene)‐6H‐azolidino[5,4‐g]benzothiazol‐7‐one}, protects against the death of cultured cerebellar granule and cortical neurons. PKRi also prevents striatal neurodegeneration and improves behavioral outcomes in a chemically induced mouse model of Huntington’s disease. Surprisingly, PKRi fails to block the phosphorylation of eIF2α, a downstream target of PKR, and does not reduce the autophosphorylation of PKR enzyme immunoprecipitated from neurons. Furthermore, neurons lacking PKR are fully protected from apoptosis by PKRi, demonstrating that neuroprotection by this compound is not mediated by PKR inhibition. Using in vitro kinase assays we investigated whether PKRi affects any other protein kinase. These analyses demonstrated that PKRi has no major inhibitory effect on pro‐apoptotic kinases such as the c‐Jun N‐terminal kinases, the p38 MAP kinases and the death‐associated protein kinases, or on other kinases including c‐Raf, MEK1, MKK6 and MKK7. PKRi does, however, inhibit the activity of certain cyclin‐dependent kinases (CDKs), including CDK1, CDK2 and CDK5 both in vitro and in low potassium‐treated neurons. Consistent with its inhibitory action on mitotic CDKs, the treatment of HT‐22 and HEK293T cell lines with PKRi sharply reduces the rate of cell cycle progression. Taken together with the established role of CDK activation in the promotion of neurodegeneration, our results suggest that PKRi exerts its neuroprotective action by inhibiting CDK.
Use of stable isotope‐labelled cells to identify active grazers of picocyanobacteria in ocean surface waters
Authors: Frias‐Lopez Jorge , Thompson Anne , Waldbauer Jacob , Chisholm Sallie W.
Published: November 2008

Prochlorococcus and Synechococcus are the two most abundant marine cyanobacteria. They represent a significant fraction of the total primary production of the world oceans and comprise a major fraction of the prey biomass available to phagotrophic protists. Despite relatively rapid growth rates, picocyanobacterial cell densities in open‐ocean surface waters remain fairly constant, implying steady mortality due to viral infection and consumption by predators. There have been several studies on grazing by specific protists on Prochlorococcus and Synechococcus in culture, and of cell loss rates due to overall grazing in the field. However, the specific sources of mortality of these primary producers in the wild remain unknown. Here, we use a modification of the RNA stable isotope probing technique (RNA‐SIP), which involves adding labelled cells to natural seawater, to identify active predators that are specifically consuming Prochlorococcus and Synechococcus in the surface waters of the Pacific Ocean. Four major groups were identified as having their 18S rRNA highly labelled: Prymnesiophyceae (Haptophyta), Dictyochophyceae (Stramenopiles), Bolidomonas (Stramenopiles) and Dinoflagellata (Alveolata). For the first three of these, the closest relative of the sequences identified was a photosynthetic organism, indicating the presence of mixotrophs among picocyanobacterial predators. We conclude that the use of RNA‐SIP is a useful method to identity specific predators for picocyanobacteria in situ, and that the method could possibly be used to identify other bacterial predators important in the microbial food‐web.
Volatile organic compounds: a potential direct long‐distance mechanism for antagonistic action of Fusarium oxysporum strain MSA 35
Authors: Minerdi Daniela , Bossi Simone , Gullino Maria Lodovica , Garibaldi Angelo
Published: November 2008

Fusarium oxysporum MSA 35 [wild‐type (WT) strain] is an antagonistic Fusarium that lives in association with a consortium of bacteria belonging to the genera Serratia, Achromobacter, Bacillus and Stenotrophomonas in an Italian soil suppressive to Fusarium wilt. Typing experiments and virulence tests provided evidence that the F. oxysporum isolate when cured of the bacterial symbionts [the cured (CU) form], is pathogenic, causing wilt symptoms identical to those caused by F. oxysporum f. sp. lactucae. Here, we demonstrate that small volatile organic compounds (VOCs) emitted from the WT strain negatively influence the mycelial growth of different formae speciales of F. oxysporum. Furthermore, these VOCs repress gene expression of two putative virulence genes in F. oxysporum lactucae strain Fuslat10, a fungus against which the WT strain MSA 35 has antagonistic activity. The VOC profile of the WT and CU fungus shows different compositions. Sesquiterpenes, mainly caryophyllene, were present in the headspace only of WT MSA 35. No sesquiterpenes were found in the volatiles of ectosymbiotic Serratia sp. strain DM1 and Achromobacter sp. strain MM1. Bacterial volatiles had no effects on the growth of the different ff. spp. of F. oxysporum examined. Hyphae grown with VOC from WT F. oxysporum f. sp. lactucae strain MSA 35 were hydrophobic whereas those grown without VOCs were not, suggesting a correlation between the presence of volatiles in the atmosphere and the phenotype of the mycelium. This is the first report of VOC production by antagonistic F. oxysporum MSA 35 and their effects on pathogenic F. oxysporum. The results obtained in this work led us to propose a new potential direct long‐distance mechanism for antagonism by F. oxysporum MSA 35 mediated by VOCs. Antagonism could be the consequence of both reduction of pathogen mycelial growth and inhibition of pathogen virulence gene expression.
Thrombolysis in recurrent lacunar stroke: a case example
Authors: Nguyen T. H. , Vo D. , Ngo M. B. , Truong A. L. T. , Bui C. Q. , Nguyen L. K. , Le T. V.
Published: November 2008

Background and purpose:  We report a case of intravenous thrombolysis in a patient with early recurrent stroke. A 62‐year‐old man recovered nearly completely after a lacunar infarct of the left putamen. He suffered stroke recurrence 7 days later due to a new infarct in the left internal capsule. Intravenous alteplase 0.9 mg/kg was administered 40 min after the symptom onset resulting in significant neurologic improvement.

Conclusion:  Intravenous thrombolysis may be safe for early recurrent lacunar stroke in patients with relatively small risk of hemorrhage.
Pregabalin as add‐on therapy induces REM sleep enhancement in partial epilepsy: a polysomnographic study
Authors: Romigi A. , Izzi F. , Marciani M. G. , Torelli F. , Zannino S. , Pisani L. R. , Uasone E. , Corte F. , Placidi F.
Published: November 2008

Background and purpose: To evaluate the effects of pregabalin (PGB) adjunctive therapy on sleepwake cycle and daytime somnolence in adult patients affected by partial epilepsy.

Methods: Twelve patients affected by partial epilepsy underwent a 24‐h ambulatory polysomnography and a subjective evaluation of daytime somnolence by means of the Epworth Sleepiness Scale (ESS), before and after 3 months treatment with PGB.

Results: Pregabalin therapy reduced seizures by >50% in 8 out of 12 patients. It induced a significant increase of REM sleep and a decrease of stage 2 NREM sleep (S2). A significant increase of the ESS score was observed without reaching the pathological cut‐off value (mean ESS score <10). No statistical correlation between REM sleep and seizure frequency was observed.

Discussion: Pregabalin seems to be effective and safe in partial epilepsy. The increase of REM sleep may be indicative of an improvement of nocturnal sleep quality considering the involvement of REM sleep in learning and memory processes. REM sleep enhancement may be the result of both a direct effect of PGB on sleep generators and an indirect effect due to its clinical efficacy. The increase of ESS score within normal range suggests that daytime somnolence is a minor adverse effect of PGB.
Locating Backup Facilities to Enhance Supply Chain Disaster Resilience
Authors: RATICK SAMUEL , MEACHAM BRIAN , AOYAMA YUKO
Published: November 2008

The use of emergency backup and storage facilities to supplement existing facilities in response to the potential effects of various natural and anthropogenic hazards (e.g., floods, fires, outages, and acts of malice) can be an effective way of reducing vulnerability and enhancing the resilience of supply chain and other logistics functions. Although there can be additional costs associated with utilizing emergency backup and storage facilities, they can be a particularly attractive and cost‐effective alternative in those cases where long‐term disruptions can, or should, be expected. In this paper we use set cover location modeling as a decision to determine the number of backup facilities to locate under varying cover, anticover, and complementary anticover distances. We then add the flexibility of allowing existing facilities to serve as backup facilities and explore the interrelationships among hazards, vulnerability, and location. Finally, these model formulations are applied to an example data set over 900 cities and towns in New England and New York.
Demography of a socially natural herd of Przewalski's horses: an example of a small, closed population
Authors: Tatin L. , King S. R. B. , Munkhtuya B. , Hewison A. J. M. , Feh C.
Published: November 2008

Owing to habitat loss and fragmentation, large mammal populations all over the world are becoming increasingly small and isolated. It is therefore a conservation priority to understand mechanisms influencing the demography of such populations, which can easily be driven to extinction. The Przewalski's horse Equus ferus przewalskii remains one of the world's most endangered species and reintroduced animals are still vulnerable. Over 9 years, we analysed factors affecting mortality and female fecundity at the individual level in a predator‐free, closed population of Przewalski's horses, which grew from 11 to 55 individuals. Similar to other wild equids, the annual growth rate of the population was r=0.169. Typically, adult mortality was much lower than juvenile mortality, the latter being correlated with neither inbreeding coefficient of foals nor population density. We found no link between female fecundity and operational sex ratio of the herd, or inbreeding coefficient, lactation status and body condition of the mares. Although food therefore seemed not to be limiting in this population, density (number of horses ha−1) clearly reduced fecundity, especially in subadult mares. Thus, our results show that space can slow the growth rate of a population before resources become limited, a potential source of concern for increasingly shrinking habitats of endangered large mammals. Possible mechanisms causing this may be found in incest avoidance or other social parameters. Finally, in large herbivores, population density is said to exert influence in a sequential order: juvenile survival first, followed by fecundity of young females, then adult females, and adult survival last. Although we observed no link between density and juvenile survival in the studied population, our results otherwise support this hypothesis.
Shaping the calcium signature
Authors: McAinsh Martin R. , Pittman Jon K.
Published: November 2008

In numerous plant signal transduction pathways, Ca2+is a versatile second messenger which controls the activation of many downstream actions in response to various stimuli. There is strong evidence to indicate that information encoded within these stimulus‐induced Ca2+oscillations can provide signalling specificity. Such Ca2+signals, or ‘Ca2+signatures’, are generated in the cytosol, and in noncytosolic locations including the nucleus and chloroplast, through the coordinated action of Ca2+influx and efflux pathways. An increased understanding of the functions and regulation of these various Ca2+transporters has improved our appreciation of the role these transporters play in specifically shaping the Ca2+signatures. Here we review the evidence which indicates that Ca2+channel, Ca2+‐ATPase and Ca2+exchanger isoforms can indeed modulate specific Ca2+signatures in response to an individual signal.

ContentsSummary275I.Introduction276II.Ca2+signalling pathways276III.Shaping Ca2+signatures278IV.Ca2+influx channels278V.Ca2+influx channels as modulators of Ca2+signatures281VI.Ca2+efflux transporters282VII.Ca2+efflux transporters as modulators of Ca2+signatures284VIII.The shaping of noncytosolic Ca2+signatures285IX.Future insights into the role of Ca2+oscillators from modelling studies287X.Conclusions and perspectives288Acknowledgements288References288
Relationship of CSF neurotransmitter metabolite levels to disease severity and disability in multiple sclerosis
Authors: Markianos Manolis , Koutsis Georgios , Evangelopoulos Maria‐Eleftheria , Mandellos Dimitrios , Karahalios Georgios , Sfagos Constantin
Published: November 2008

Axonal degeneration and brain tissue loss occur during disease progression in multiple sclerosis (MS) and are expected to influence neurotransmitter activities, with consequences on neurologic and psychiatric symptomatology. We searched for relationships of disease duration, disability, and severity of MS patients to CSF levels of the major metabolites of noradrenaline, dopamine, and serotonin, MHPG, methoxyhydroxyphenylglycol (MHPG), homovanillic acid, and 5‐hydroxyindoleacetic acid (5‐HIAA), respectively, in 39 patients with relapsing–remitting (RR) MS in remission, and 26 patients with progressive (PR) MS. Disability and Disease Severity were assessed by the Expanded Disability Status Scale (EDSS) and the Multiple Sclerosis Severity Score (MSSS). Compared with the levels of 50 control subjects, MHPG levels were not different in either MS group, correlated negatively to duration of illness and number of relapses in the RRMS group, but not to EDSS score or to MSSS. Homovanillic acid levels were significantly lower only in the PRMS group, with a negative correlation to duration of illness, and a strong negative correlation to EDSS score, but not to MSSS. 5‐HIAA was significantly lower in both RRMS and PRMS groups. In the RRMS group, 5‐HIAA levels were negatively related to EDSS and to MSSS. Multiple regression analyses revealed a significant association of MHPG to duration of illness, and a strong negative association of 5‐HIAA to MSSS rather than to EDSS. The strong negative correlation of MSSS to CSF 5‐HIAA levels in RRMS group of patients indicates that deficits in central serotonergic activity are related to the rate of disability accumulation in RRMS, and could be linked to the reported reduction of disease activity by serotonergic drugs.
Role of oxidative stress and caspase 3 in CD47‐mediated neuronal cell death
Authors: Xing Changhong , Lee Sunryung , Kim Woo Jean , Jin Guang , Yang Yong‐Guang , Ji Xunming , Wang Xiaoying , Lo Eng H.
Published: November 2008

CD47 or integrin‐associated protein promotes cell death in blood and tumor cells. Recently, CD47 signaling has been identified in neurons as well. In this study, we investigated the role of CD47 in neuronal cell death. Exposure of primary mouse cortical neurons to the CD47 ligand thrombospondin‐1 or the specific CD47‐activating peptide 4N1K induced cell death. Activation of CD47 elevated levels of active caspase 3 and increased the generation of reactive oxygen species (ROS) in a time‐dependent manner. Both ROS scavengers and caspase inhibitors attenuated cell death. But ROS scavenging did not reduce the activation of caspase 3, and combination treatments with a caspase inhibitor plus free radical scavenger did not yield additive protection. Taken together, these data suggest that parallel and redundant pathways of oxidative stress and caspase‐mediated cell death are involved. We conclude that CD47 mediates neuronal cell death through caspase‐dependent and caspase‐independent pathways.
Glutamate and nitric oxide modulate ERK and CREB phosphorylation in the avian retina: evidence for direct signaling from neurons to Müller glial cells
Authors: Socodato Renato Esteves da Silva , Magalhães Cristiane Rosa , Paes‐de‐Carvalho Roberto
Published: November 2008

Glutamate signaling in the mature retinal tissue is very important for accurate sensory decoding by retinal neurons and orchestrates the fine‐tuned output from the retina to higher‐order centers at the cerebral cortex. In this study, we show that glutamate induces a rapid extracellular‐regulated kinase and cAMP‐responsive element binding protein (CREB) phosphorylation in cultured developing retinal neurons. This process is reliant on α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptors and nitric oxide (NO) signaling and independent of NMDA receptors activation, as it is blocked by α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate/kainate antagonists as well as inhibiting NO synthase with NG‐nitro‐l‐arginine methyl ester but not by the NMDA channel blocker dizocilpine maleate. The effect of NO on extracellular‐regulated kinase and CREB is mediated by the classical NO/soluble guanylyl cyclase/protein kinase G pathways as it is inhibited by the soluble guanylyl cyclase blocker 1H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one and the protein kinase G inhibitor KT5823, respectively. Immunocytochemical data suggest that increased CREB phosphorylation in response to glutamate occurs in glial cell nuclei. We also have supporting evidence suggesting that neuronally produced NO directly reaches the glial cells and stimulates CREB phosphorylation. Hence, the results indicate the importance of neuronal–glial communication and glutamate/NO/CREB linkage during retinal development.
ROMANO‐EGYPTIAN RED LEAD PIGMENT: A SUBSIDIARY COMMODITY OF SPANISH SILVER MINING AND REFINEMENT*
Authors: WALTON M. S. , TRENTELMAN K.
Published: November 2008

Samples of red pigment from a group of seven Roman‐period Egyptian mummies, known as red‐shroud mummies, are investigated. Elemental analysis by inductively coupled plasma time‐of‐flight mass spectrometry (ICP–TOFMS) shows that the samples contain mostly Pb (83–92% by weight), along with 0.2–2.0% Sn. All of the samples are found to have similar trace element distributions when normalized to the continental crust, suggesting that they share a common geological origin. Lead isotope ratios are found to match the mixed lead sources typically associated with Rio Tinto, Spain – a site extensively mined for silver during the first centuryad. Raman microspectroscopy identifies the major phase of each sample to be red lead (Pb3O4) with a minor phase of lead tin oxide (Pb2SnO4). Lead tin oxide does not occur naturally, and its incidental occurrence within the sample indicates that the material was heated under oxidative conditions at temperatures in excess of 650°C. In archaeological contexts, the high‐temperature oxidative treatment of lead is typically associated with metallurgical refinement processes such as cupellation. Based on this evidence, it is argued that the pigment was produced out of litharge associated with silver cupellation at the Rio Tinto site.
International rivers and national security: The Euphrates, Ganges–Brahmaputra, Indus, Tigris, and Yarmouk rivers1
Authors: Zawahri Neda A.
Published: November 2008

To understand a state's incentives to invest in conflict or cooperation over their international rivers, this paper argues that it is necessary to appreciate the relationships a river can create and the national security threat riparians may confront. Rivers impose interdependent and vulnerable relationships, which can compromise a state's ability to respond effectively to floods and droughts, meet its domestic food and energy needs, dredge the river, maintain its drainage systems, and allocate its domestic water budget. The inability to accomplish these tasks can contribute to social, economic, and political losses that may threaten a state's territorial integrity. Regardless of whether a state is upstream or downstream, from these relationships it acquires leverage to manipulate the interdependence and vulnerability to inflict losses on its riparian neighbour. This argument challenges several assumptions within the existing literature, including the belief that a shortage of freshwater is the initial force producing a national security threat and that an upstream–downstream river bequeaths all advantages on the upstream state and leaves the downstream state purely dependent. As the paper shows, riparians confront a more complex relationship than captured by the existing literature.
Urokinase Immobilized on Medical Polymeric Materials: Fundamental and Clinical Studies
Authors: Ohshiro Takeshi , Kosaki Goro
Published: November 2008

One of the methods of preparing an antithrombogenic material is to immobilize a fibrinolytic enzyme on the surface of a carrier. The clinical trial of such a material must be subject to not only a basic study on the quality of a carrier, the technique of immobilization, and the method of disinfection, but also an in vivo study on its antithrombotic effect.

Reported herein is the evaluation of the fibrinolytic ability, at fundamental and clinical levels, of the urokinase that was immobilized on the surface of various polymeric materials. The results were favorable.