Virulence of an emerging pathogenic lineage of Vibrio nigripulchritudo is dependent on two plasmids

Vibrioses are the predominant bacterial infections in marine shrimp farms. Vibrio nigripulchritudo is an emerging pathogen of the cultured shrimp Litopenaeus stylirostris in New Caledonia and other regions in the Indo‐Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have revealed that recent pathogenic V. nigripulchritudo isolates from New Caledonia all cluster into a monophyletic clade and contain a small plasmid, pB1067. Here, we report that a large plasmid, pA1066 (247 kb), can also serve as a marker for virulent V. nigripulchritudo, and that an ancestral version of this plasmid was likely acquired prior to other virulence‐linked markers. Additionally, we demonstrate that pA1066 is critical for the full virulence of V. nigripulchritudo in several newly developed experimental models of infection. Plasmid pB1067 also contributes to virulence; only strains containing both plasmids induced the highest level of shrimp mortality. Thus, it appears that these plasmids, which are absent from non‐pathogenic isolates, may be driving forces, as well as markers, for the emergence of a pathogenic lineage of V. nigripulchritudo.     

Infections associated with monoclonal antibody and fusion protein therapy in humans

Monoclonal antibodies (mAbs), especially those that interact with immune or hematologic leukocyte membrane targets, have changed the outcome of numerous diseases. However, mAbs can block or reduce immune cells and cytokines, and can lead to increased risk of infection. Some of these risks are predictable and can be explained by their mechanisms of action. Others have been observed only after the mAbs were licensed and used extensively in patients. In this review, we focus on infectious complications that occur upon treatment with mAbs or Fc-containing fusion proteins targeting leukocyte membrane proteins, including CD52, CD20, tumor necrosis factor, VLA4, CD11a and CTLA4. We report their known infectious risks and the recommendations for their use. Although most of these drugs are clinically safe when the indications are respected, we emphasize the need for regular updating of pharmacovigilance data.Key words: monoclonal antibodies, infections, complication, human     

Selective inhibition of GluN2D-containing N-methyl-D-aspartate receptors prevents tissue plasminogen activator-promoted neurotoxicity both in vitro and in vivo

Background

Tau protein is the principal component of the neurofibrillary tangles found in Alzheimer's disease, where it is hyperphosphorylated on serine and threonine residues, and recently phosphotyrosine has been demonstrated. The Src-family kinase Fyn has been linked circumstantially to the pathology of Alzheimer's disease, and shown to phosphorylate Tyr18. Recently another Src-family kinase, Lck, has been identified as a genetic risk factor for this disease.

Results

In this study we show that Lck is a tau kinase. In vitro, comparison of Lck and Fyn showed that while both kinases phosphorylated Tyr18 preferentially, Lck phosphorylated other tyrosines somewhat better than Fyn. In co-transfected COS-7 cells, mutating any one of the five tyrosines in tau to phenylalanine reduced the apparent level of tau tyrosine phosphorylation to 25-40% of that given by wild-type tau. Consistent with this, tau mutants with only one remaining tyrosine gave poor phosphorylation; however, Tyr18 was phosphorylated better than the others.

Conclusions

Fyn and Lck have subtle differences in their properties as tau kinases, and the phosphorylation of tau is one mechanism by which the genetic risk associated with Lck might be expressed pathogenically.     

Targeted deletion of integrin-linked kinase reveals a role in T-cell chemotaxis and survival

Integrin-linked kinase (ILK) is a serine/threonine kinase that is important in cell-matrix interactions and cell signaling. To examine the role of ILK in leukocyte trafficking and survival, we generated T cell-specific ILK knockouts by breeding ILK(flox/flox) mice to transgenic mice expressing Cre recombinase under control of the Lck proximal promoter. Thymic T cells from Lck-Cre(+)/ILK(flox/flox) mice had a marked reduction (>95%) in ILK protein levels. Thymic cellularity was comparable in 3- to 4-week-old mice, but a threefold diminution of thymic T cells became evident by 6 to 8 weeks of age in the T cell-specific ILK knockout mice due to increased cell death of double-positive (DP) T cells. Analysis of peripheral T cells by quantitative PCR and by breeding Lck-Cre(+)/ILK(flox/flox) mice to a YFP-transgenic reporter strain demonstrated an approximate 20-fold enrichment of ILK-competent cells, suggesting these cells have a competitive advantage in trafficking to and/or survival in peripheral lymphatic organs. We explored mechanisms related to altered cell trafficking and survival that might explain the decreases in thymic cellularity and enrichment for ILK-competent cells in the spleen and lymph nodes. We observed a >50% reduction in chemotaxis of ILK-deficient T cells to the chemokines CXCL12 (stromal cell-derived factor [SDF]-1alpha) and CCL19 (macrophage inflammatory protein [MIP]-3beta), as well as enhanced apoptosis of ILK-deficient cells upon stress. Signaling studies in ILK-deficient T cells demonstrated diminished phosphorylation of Akt on the activating phosphorylation site, Ser 473, and a concordant decrease in Akt kinase activity following stimulation with the chemokine SDF-1. Rac1 activation was also markedly diminished in ILK-deficient T cells following chemokine stimulation. These data extend the role of ILK to immune-cell trafficking and survival via modulation of Akt- and Rac-dependent substrates, and have implications for cell recruitment in both homeostatic and pathological processes.     

Anchorage of synthetic peptides onto liposomes via hydrazone and alpha-oxo hydrazone bonds. preliminary functional investigations

Synthetic peptidoliposomes have been designed and prepared according to a chemoselective ligation. Two aldehyde-functionalized lipidic anchors were synthesized and incorporated into the lipidic bilayers of unilamellar liposomes during their preparation. Complementary hydrazino acetyl peptides were synthesized on the solid phase using N,N',N'-tri(tert-butyloxycarbonyl)-hydrazino acetic acid and further coupled to the aldehyde groups displayed at the surface of the vesicles. Coupling yields were measured by amino acid hydrolysis following total acid hydrolysis. The ligation methodology proved superior to the simple insertion of lipopeptides, which was performed for comparison in terms of yields, implementation, and reproducibility. To check whether the grafted-peptides were accessible and functional, cytoplasmic sequences of LAMP protein (lysosomal associated membrane protein), which is involved in intracellular membrane trafficking, have been selected. Using this model, we demonstrated in vitro the specific interaction of the synthetic LAMP-peptidoliposomes with the cytoplasmic adaptor protein AP-3, a result that contributes to the understanding of protein sorting in cells. Thus, these results clearly indicate the usefulness of such peptidoliposomes, easily prepared by hydrazone chemoselective ligation, as a tool for biological investigation.     

Novel cell-penetrating alpha-keto-amide calpain inhibitors as potential treatment for muscular dystrophy

Dipeptide-derived alpha-keto-amide compounds with potent calpain inhibitory activity have been identified. These reversible covalent inhibitors have IC(50) values down to 25nM and exhibit greatly improved activity in muscle cells compared to the reference compound MDL28170. Several novel calpain inhibitors have shown positive effects on histological parameters in an animal model of Duchenne muscular dystrophy demonstrating their potential as a treatment option for this fatal disease.     

Recognition of fresh human tumor by human peripheral blood lymphocytes transduced with a bicistronic retroviral vector encoding a murine anti-p53 TCR

The p53 protein is markedly up-regulated in a high proportion of human malignancies. Using an HLA-A2 transgenic mouse model, it was possible to isolate high-avidity murine CTLs that recognize class I-restricted human p53 epitopes. We isolated the alpha- and beta-chain of a TCR from a highly avid murine CTL clone that recognized the human p53(264-272) epitope. These genes were cloned into a retroviral vector that mediated high efficiency gene transfer into primary human lymphocytes. Efficiencies of >90% for gene transfer into lymphocytes were obtained without selection for transduced cells. The p53 TCR-transduced lymphocytes were able to specifically recognize with high-avidity, peptide-pulsed APCs as well as HLA-A2.1+ cells transfected with either wild-type or mutant p53 protein. p53 TCR-transduced cells demonstrated recognition and killing of a broad spectrum of human tumor cell lines as well as recognition of fresh human tumor cells. Interestingly, both CD8+ and CD4+ subsets were capable of recognizing and killing target cells, stressing the potential application of such a CD8-independent TCR molecule that can mediate both helper and cytotoxic responses. These results suggest that lymphocytes genetically engineered to express anti-p53 TCR may be of value for the adoptive immunotherapy of patients with a variety of common malignancies.     

Widespread Infection with Homologues of Human Parvoviruses B19, PARV4, and Human Bocavirus of Chimpanzees and Gorillas in the Wild

Infections with human parvoviruses B19 and recently discovered human bocaviruses (HBoVs) are widespread, while PARV4 infections are transmitted parenterally and prevalent specifically in injecting drug users and hemophiliacs. To investigate the exposure and circulation of parvoviruses related to B19 virus, PARV4, and HBoV in nonhuman primates, plasma samples collected from 73 Cameroonian wild-caught chimpanzees and gorillas and 91 Old World monkey (OWM) species were screened for antibodies to recombinant B19 virus, PARV4, and HBoV VP2 antigens by enzyme-linked immunosorbent assay (ELISA). Moderate to high frequencies of seroreactivity to PARV4 (63% and 18% in chimpanzees and gorillas, respectively), HBoV (73% and 36%), and B19 virus (8% and 27%) were recorded for apes, while OWMs were uniformly negative (for PARV4 and B19 virus) or infrequently reactive (3% for HBoV). For genetic characterization, plasma samples and 54 fecal samples from chimpanzees and gorillas collected from Cameroonian forest floors were screened by PCR with primers conserved within Erythrovirus, Bocavirus, and PARV4 genera. Two plasma samples (chimpanzee and baboon) were positive for PARV4, while four fecal samples were positive for HBoV-like viruses. The chimpanzee PARV4 variant showed 18% and 15% nucleotide sequence divergence in NS and VP1/2, respectively, from human variants (9% and 7% amino acid, respectively), while the baboon variant was substantially more divergent, mirroring host phylogeny. Ape HBoV variants showed complex sequence relationships with human viruses, comprising separate divergent homologues of HBoV1 and the recombinant HBoV3 species in chimpanzees and a novel recombinant species in gorillas. This study provides the first evidence for widespread circulation of parvoviruses in primates and enables future investigations of their epidemiology, host specificity, and (co)evolutionary histories.Autonomous parvoviruses known to infect humans comprise parvovirus B19 (18) and the recently discovered PARV4 (22) and human bocavirus (HBoV) (3). Members of the family Parvoviridae are genetically and biologically diverse and are classified into several genera or groups, showing marked differences in host range, pathology, and tissue/cellular tropisms (18). Human parvovirus B19, a member of the Erythrovirus genus, is transmitted primarily by the respiratory route but causes systemic infections. Erythroid progenitor cells are specifically targeted through expression of globoside P antigen, which acts as the B19 virus receptor for entry (5). In common with infections by most parvoviruses, B19 virus infections are acute; a period of intense viremia is followed by seroconversion for antibody to B19 virus and lifelong immunity from reinfection (29). Despite the clearance of viremia and seroconversion for antibody, lifelong persistence of viral DNA in tissues has been shown to occur (12, 20, 26, 28, 43, 58). Three genotypes of B19 virus have been described, differing in nucleotide sequence by approximately 13 to 14% (7, 21, 41, 53); genotypes 1 and 2 have been found in Europe, the United States, and other Western countries, while genotype 3 is restricted to sub-Saharan Africa and South America (7, 47, 49). B19 virus widely circulates in human populations worldwide; in Western countries, several studies have documented increasing frequencies of B19 virus seropositivity with age, rising to approximately 60 to 70% by adulthood (15, 39, 48, 61).Another human parvovirus, PARV4, shows markedly different epidemiology and transmission routes. It was originally detected in plasma from an individual with an “acute infection syndrome” resembling that of primary human immunodeficiency virus (HIV) infection (22). While this clinical presentation has not been observed again, infection with PARV4 is known to be widespread specifically in individuals with a history of parenteral exposure (injecting drug users [IDUs], hemophiliacs, polytransfused individuals), with a strikingly higher incidence in those infected with HIV-1 (13, 14, 30, 35, 54). These observations suggest that PARV4 is primarily transmitted though parenteral routes in Western countries (54, 56). In common with infection with the better-characterized human parvovirus B19, infection with PARV4 is associated with a period of acute viremia, followed by seroconversion for antibody and long-term persistence of viral DNA sequences in lymphoid and other tissue (33, 37, 52). Circulating variants of PARV4 have been classified into three distinct genotypes exhibiting approximately 8% nucleotide sequence divergence from each other. Genotypes 1 and 2 circulate in Western countries, while genotype 3 has to date been recorded only in sub-Saharan Africa (45, 55).The third human parvovirus, HBoV (3), shows a number of epidemiological and clinical attributes different from those of both B19 virus and PARV4. HBoV was originally found in the respiratory tract of young children and has been the subject of intense investigation as a potential cause of human respiratory disease (reviewed in references 1, 51, and 62). Although it is frequently detected by PCR in the nasopharynx of viremic individuals with primary infections with lower respiratory tract disease, other coinfecting respiratory viruses are frequently detected (19). HBoV additionally shows long-term, low-level carriage in the respiratory tract after primary infection, which further complicates PCR-based etiological studies (2, 38) and warrants the use of other diagnostic strategies, such as serology (30, 32, 59). In contrast to the rather minimal genetic diversity of B19 virus and PARV4 genotypes, bocaviruses infecting humans are now known to comprise three to four major genetic variants (termed types or species 1 to 4) (23, 24). HBoV1 and HBoV2 show 22%, 33%, and 20% amino acid sequence divergence from each other in the encoded viral nonstructural (NS), NP-1, and structural VP1/VP2 proteins, respectively, the latter potentially leading to antigenic diversity and some loss of antigenic cross-reactivity. A third type/species of HBoV is a chimeric form with a nonstructural gene region (NS, NP1) most similar to HBoV1, a recombination breakpoint in the intergenic region between NP1 and VP1, and structural genes related to those of HBoV2 (4, 23). Current data suggest that only HBoV1 is capable of infecting the respiratory tract; most published large-scale screening studies have failed to detect HBoV2 (or HBoV3) in respiratory samples (10, 11, 60), while all three types/species are detectable in fecal samples, indicating the existence of an alternative or additional site of virus replication (23). Despite extensive inquiry, the exact role of HBoV1 in respiratory disease remains unclear, as is the proposed etiological role of HBoV2 (and possibly HBoV3) in gastroenteritis (4, 11, 23, 50). Very recently, a fourth species/type, HBoV4, has been detected in fecal samples; genetically it also shows evidence for past recombination, with NS and NP1 region sequences grouping with HBoV2, while VP1/VP2 is more closely related to HBoV3 (23).We have little understanding of the past epidemiology, evolution, and origins of human parvoviruses. For both B19 virus and PARV4, evidence has been obtained for a temporal succession of genotypes over time (37, 43); in Europe, B19 virus genotype 1 largely replaced type 2 in the 1960 and 1970s (43), while current data indicate that a similar replacement of PARV4 genotypes occurred within the last 20 years (37). The highly restricted sequence diversity of currently circulating variants of PARV4 and B19 virus and of HBoV1 variants supports the hypothesis of a relatively recent emergence and spread of these viruses in human populations (36, 42, 64).The existence and evolution of parvoviruses on a much longer time scale is suggested by the observations that members of the Erythrovirus and Parvovirus genera both contain viruses that are highly host species specific and that the molecular phylogenies of both genera are largely congruent with those of their hosts (34). This has led to the hypothesis of long-term coevolution of parvoviruses with their host over the 90 million years of mammalian evolution and perhaps beyond. Among erythroviruses, simian homologues of B19 virus have been found in cynomolgus monkeys (44) and rhesus and pig-tailed macaques (16) and more genetically distant viruses have been characterized in chipmunks and cows (9, 63). Divergent homologues of PARV4 in pigs and cows have been described (31), while the bovine and canine parvoviruses distantly related to HBoV are the originally described members of the Bocavirus genus. However, the process of virus-host codivergence is known to be punctuated by occasional cross-species transmissions, including the well-documented spread of feline parvovirus to dogs (46). Based on serological evidence, the possible transmission of simian erythroviruses to animal handlers has been proposed (6).To gain further insights into the origins and evolution of human parvoviruses, we have performed large-scale serological and PCR-based screening of nonhuman primates (chimpanzees and gorillas) and of several species of Old World monkeys (OWMs) for evidence of infection with parvoviruses that are antigenically related to the human B19, PARV4, and HBoV viruses. By PCR, we have sought to genetically characterize homologues of the three autonomous human parvoviruses in apes and Old World monkey species and to analyze their evolutionary relationship to human and other mammalian homologues of these viruses.