Morphological and molecular characterization of larval Echinocephalus sp. (Spirurida: Gnathostomatidae), a parasite of the greater lizard fish (Saurida undosquamis) and red porgy or common seabream (Pagrus pagrus).

Larvae of an unidentified Echinocephalus species were obtained from two fish species: red porgy or common seabream (Pagrus pagrus) and greater lizard fish (Saurida undosquamis) from the Red Sea. The prevalence of Echinocephalus sp. larvae in P. pagrus was 4.92% and 4.98% in S. undosquamis. The length, width, cephalic bulb, and spine shape and pattern of the larvae resembled Echinocephalus overstreeti. SSU gene sequences of larvae from P. pagrus and S. undosquamis were identical. Comparison of the SSU sequence to those available in GenBank showed that the larvae from P. pagrus and S. undosquamis are diagnosably distinct. Based on sequence similarity and published phylogenetic analysis, these larvae are most similar to an unknown species of Echinocephalus from an Australian sea snake (Hydrophis peronii). Despite morphological similarities of the Red Sea larvae to E. overstreeti, the SSU sequence differences show that they are not the same species.

CD28-Selective Inhibition Prolongs Non-Human Primate Kidney Transplant Survival.

Costimulation blockade using belatacept results in improved renal function after kidney transplant as well as decreased likelihood of death/graft loss and reduced cardiovascular risk; however, higher rates and grades of acute rejection have prevented its widespread clinical adoption. Treatment with belatacept blocks both positive (CD28) and negative (CTLA-4) T cell signaling. CD28-selective therapies may offer improved potency by blocking CD28-mediated costimulation while leaving CTLA-4 mediated coinhibitory signals intact. Here we test a novel domain antibody directed at CD28 (anti-CD28 dAb (BMS-931699)) in a non-human primate kidney transplant model. Sixteen macaques underwent native nephrectomy and received life-sustaining renal allotransplantation from an MHC-mismatched donor. Animals were treated with belatacept alone, anti-CD28 dAb alone, or anti-CD28 dAb plus clinically relevant maintenance (MMF, Steroids) and induction therapy with either anti-IL-2R or T cell depletion. Treatment with anti-CD28 dAb extended survival compared to belatacept monotherapy (MST 187 vs. 29 days, p=0.07). The combination of anti-CD28 dAb and conventional immunosuppression further prolonged survival to MST ∼270 days. Animals maintained protective immunity with no significant infectious issues. These data demonstrate CD28-directed therapy is a safe and effective next-generation costimulatory blockade strategy with a demonstrated survival benefit and presumed advantage over belatacept by maintaining intact CTLA-4 coinhibitory signaling.

PHYLOGENETIC ANALYSIS OF THE LUNGWORMS (NEMATODA: METASTRONGYLOIDEA) INFERRED USING NUCLEAR RIBOSOMAL AND MITOCHONDRIAL DNA SEQUENCES.

Phylogenetic relationships among the mammal-parasitic lungworms (Metastrongyloidea) were inferred using small- and large-subunit ribosomal DNA sequences together with 12S ribosomal mtDNA sequences. Maximum parsimony and Bayesian inference methods were used from optimal alignments and those filtered for alignment ambiguity. Analysis of 30 ingroup sequences using ribosomal DNA sequences yielded a single most parsimonious tree. Monophyly of the Metastrongyloidea was supported, but there was no support for monophyly of any of the 7 families as they have been traditionally defined. Parafilaroides decorus, an abursate lungworm of pinnipeds currently classified in the Filaroididae, was nested within a clade containing members of the Pseudaliidae, parasites of cetaceans. The tree also shows clades somewhat resembling the traditional familial divisions of the Metastrongyloidea, but in all groups, paraphyletic relationships were recovered. In a combined analysis of nuclear rDNA and 12S mtDNA, maximum parsimony and Bayesian analyses showed similar patterns to those observed with only nuclear rDNA sequences. Based on the phylogeny, the respiratory tract was inferred to be the ancestral predilection site for Metastrongyloidea, with multiple evolutionary invasions of extrapulmonary sites such as sinuses, circulatory system, and meninges. Similarly, the ancestral host was inferred to be a carnivore with subsequent colonization events into marsupial, rodent, artiodactyl, pinniped, and cetacean hosts.

The Many Lives of René Descartes.

A review essay of recent biographies of Descartes.

PHYLOGEOGRAPHY OF BAYLISASCARIS PROCYONIS (RACCOON ROUNDWORM) IN NORTH AMERICA.

Sequences of the mitochondrial cytochrome c oxidase 1 (COI) gene of 115 Baylisascaris procyonis individuals from 13 U.S. states and 1 Canadian province were obtained from 44 raccoon hosts to assess genetic variation and geographic structure. The maximum genetic distance between individuals was low (1.6%), consistent with a single species. Moderate COI haplotype (h = 0.60) and nucleotide (π = 0.0053) diversity were found overall. Low haplotype diversity was found among samples east of the Mississippi River (h = 0.036), suggesting that historical growth and expansion of raccoon populations in this region could be responsible for high parasite gene flow or a selective sweep of B. procyonis mtDNA. There was low genetic structure (average Φst = 0.07) for samples east of the continental divide, but samples from Colorado showed higher diversity and differentiation from midwestern and eastern samples. There was marked genetic structure between samples from east and west of the continental divide, with no haplotypes shared between these regions. There was no significant isolation by distance among any of these geographic samples. The phylogeographic patterns for B. procyonis are similar to genetic results reported for their raccoon definitive hosts. The phylogeographic divergence of B. procyonis from east and west of the continental divide may involve vicariance resulting from Pleistocene glaciation and associated climate variation.

Draft Genome Sequences of 28 Actinobacteria of the Family Microbacteriaceae Associated with Nematode-Infected Plants.

Draft genome sequences of 28 strains of Microbacteriaceae from plants infested by plant-parasitic nematodes were obtained using Illumina technology. The sequence data will provide useful baseline information for the development of comparative genomics and systematics of Microbacteriaceae and facilitate understanding of molecular mechanisms involved in interactions between plants and nematode-associated bacterial complexes.

Draft Genome Sequences of 13 Plant-Associated Actinobacteria of the Family Microbacteriaceae.

Draft genome sequences of 13 bacterial strains from the family Microbacteriaceae were generated using Illumina technology. The genome sizes varied from 3.0 to 4.8 Mb, and the DNA G+C content was 68.1 to 72.5%. The sequences obtained will contribute to the development of genome-based taxonomy and understanding of molecular interactions between bacteria and plants.

Host Population Expansion and the Genetic Architecture of the Pinniped Hookworm Uncinaria lucasi.

The long-term fidelity of pinniped hosts to their natal rookery site suggests the genetic architecture of their Uncinaria spp. hookworms should be strongly structured by host breeding biology. However, historical events affecting host populations may also shape parasite genetic structure. Sequences of the mitochondrial cytochrome c oxidase 1 (COI) gene of 86 Uncinaria lucasi individuals were obtained to assess genetic variation and structure of nematodes from 2 host species (68 hookworms from northern fur seals; 18 hookworms from Steller sea lions) and rookeries from 3 widely separated geographic regions: the western Bering Sea and Sea of Okhotsk, eastern Bering Sea, and the eastern Pacific Ocean. High COI haplotype (h = 0.96-0.98) and nucleotide (π = 0.014) diversity was found. The haplotype network showed a star-shaped pattern with a large number of haplotypes separated by few substitutions. The network did not show separation of U. lucasi by geographic region or host species. Fst values between U. lucasi individuals representing geographic regions showed no differentiation, consistent with the absence of genetic structure. At face value, this lack of genetic structure in U. lucasi suggests high gene flow but could also be explained by recent (post-glacial) population expansions of northern fur seals and their hookworms.

Complete and Draft Genome Sequences of 12 Plant-Associated Rathayibacter Strains of Known and Putative New Species.

Complete and draft genome sequences of 12 Rathayibacter strains were generated using Oxford Nanopore and Illumina technologies. The genome sizes of these strains are 3.21 to 4.61 Mb, with high G+C content (67.2% to 72.7%) genomic DNA. Genomic data will provide useful baseline information for natural taxonomy and comparative genomics of members of the genus Rathayibacter.

Vertical Transmission of a Nematode from Female Lizards to the Brains of Their Offspring.

Parasites have evolved a diversity of lifestyles that exploit the biology of their hosts. Some nematodes that parasitize mammals pass via the placenta or milk from one host to another. Similar cases of vertical transmission have never been reported in avian and nonavian reptiles, suggesting that egg laying may constrain the means of parasite transmission. However, here we report the first incidence of transovarial transmission of a previously undescribed nematode in an egg-laying amniote, the common wall lizard (Podarcis muralis). Nematodes enter the developing brain from the female ovary early in embryonic development. Infected lizard embryos develop normally and hatch with nematodes residing in their braincase. We present a morphological and molecular phylogenetic characterization of the nematode and suggest that particular features of lizard biology that are absent from birds and turtles facilitated the evolutionary origin of this novel life history.

Spinoza and Menasseh ben Israel: Facts and Fictions.

An examination of what has been claimed about the relationship between the seventeenth-century philosopher Bento (Benedictus) Spinoza and Menasseh ben Israel, a rabbi of the Amsterdam Portuguese-Jewish community. The article shows that, despite claims by scholars that Menasseh was Spinoza's teacher and intellectual mentor, in fact there is no evidence for the former claim, and much evidence that the two men were, intellectually, diametrically opposed on certain important substantive questions.

CD28 Signaling Controls Metabolic Fitness of Pathogenic T Cells in Medium and Large Vessel Vasculitis.

BACKGROUND: In giant cell arteritis, vessel-wall infiltrating CD4 T cells and macrophages form tissue-destructive granulomatous infiltrates, and the artery responds with a maladaptive response to injury, leading to intramural neoangiogenesis, intimal hyperplasia, and luminal occlusion. Lesion-residing T cells receive local signals, which represent potential therapeutic targets. OBJECTIVES: The authors examined how CD28 signaling affects vasculitis induction and maintenance, and which pathogenic processes rely on CD28-mediated T-cell activation. METHODS: Vasculitis was induced by transferring peripheral blood mononuclear cells from giant cell arteritis patients into immunodeficient NSG mice engrafted with human arteries. Human artery-NSG chimeras were treated with anti-CD28 domain antibody or control antibody. Treatment effects and immunosuppressive mechanisms were examined in vivo and in vitro applying tissue transcriptome analysis, immunohistochemistry, flow cytometry, and immunometabolic analysis. RESULTS: Blocking CD28-dependent signaling markedly reduced tissue-infiltrating T cells and effectively suppressed vasculitis. Mechanistic studies implicated CD28 in activating AKT signaling, T-cell proliferation and differentiation of IFN-γ and IL-21-producing effector T cells. Blocking CD28 was immunosuppressive by disrupting T-cell metabolic fitness; specifically, the ability to utilize glucose. Expression of the glucose transporter Glut1 and of glycolytic enzymes as well as mitochondrial oxygen consumption were all highly sensitive to CD28 blockade. Also, induction and maintenance of CD4+CD103+ tissue-resident memory T cells, needed to replenish the vasculitic infiltrates, depended on CD28 signaling. CD28 blockade effectively suppressed vasculitis-associated remodeling of the vessel wall. CONCLUSIONS: CD28 stimulation provides a metabolic signal required for pathogenic effector functions in medium and large vessel vasculitis. Disease-associated glycolytic activity in wall-residing T-cell populations can be therapeutically targeted by blocking CD28 signaling.

Discovery of a JAK1/3 Inhibitor and Use of a Prodrug To Demonstrate Efficacy in a Model of Rheumatoid Arthritis.

The four members of the Janus family of nonreceptor tyrosine kinases play a significant role in immune function. The JAK family kinase inhibitor, tofacitinib 1, has been approved in the United States for use in rheumatoid arthritis (RA) patients. A number of JAK inhibitors with a variety of JAK family selectivity profiles are currently in clinical trials. Our goal was to identify inhibitors that were functionally selective for JAK1 and JAK3. Compound 22 was prepared with the desired functional selectivity profile, but it suffered from poor absorption related to physical properties. Use of the phosphate prodrug 32 enabled progression to a murine collagen induced arthritis (CIA) model. The demonstration of a robust efficacy in the CIA model suggests that use of phosphate prodrugs may resolve issues with progressing this chemotype for the treatment of autoimmune diseases such as RA.

Plagiorchis sp. in small mammals of Senegal and the potential emergence of a zoonotic trematodiasis.

Trematodes of the genus Plagiorchis have a wide geographical distribution and can exploit a variety of hosts. The occurrence and zoonotic potential of Plagiorchis spp. have been characterised across several countries in Asia; in contrast, information on Plagiorchis parasites in Africa remains anecdotal. We isolated a previously undescribed Plagiorchis species from the biliary tract and small intestine of 201 out of 427 small mammals collected in the region of Lake Guiers, Senegal, with local prevalence ranging from 38.6% to 77.0%. Conversely, Plagiorchis isolates were not observed in the 244 small mammals sampled in and around the town of Richard Toll, Senegal. Molecular phylogenetics of the internal transcribed spacer region, nuclear ribosomal DNA, and of the cytochrome c oxidase subunit 1 gene, mitochondrial DNA, supported the monophyly and multi-host spectrum of this newly discovered West African Plagiorchis species. Sequencing of individual cercariae shed by Radix natalensis (Gastropoda: Lymnaeidae) suggested that these freshwater snails may act as suitable first intermediate hosts. Phylogenetic analysis yielded a highly resolved topology indicating two different clades, one composed by Plagiorchis spp. infecting rodents, insectivores, and birds, while the other included parasites of bats. Our findings showed the low host specificity and high prevalence of the isolated Plagiorchis sp. in the Lake Guiers region, with Hubert's multimammate mice (Mastomys huberti) appearing to play a primary role in the epidemiology of this parasite. The results raise concern about the zoonotic potential of Plagiorchis sp. in local communities of the Lake Guiers region, and highlight food-borne trematodiases and their link to land-use change as a neglected public health issue in regions of West Africa.

Molecular phylogenetics and species-level systematics of Baylisascaris.

Nucleotide sequences representing nine genes and five presumptive genetic loci were used to infer phylogenetic relationships among seven Baylisascaris species, including one species with no previously available molecular data. These genes were used to test the species status of B. procyonis and B. columnaris using a coalescent approach. Phylogenetic analysis based on combined analysis of sequence data strongly supported monophyly of the genus and separated the species into two main clades. Clade 1 included B. procyonis, B. columnaris, and B. devosi, species hosted by musteloid carnivores. Clade 2 included B. transfuga and B. schroederi from ursids, B. ailuri, a species from the red panda (a musteloid), and B. tasmaniensis from a marsupial. Within clade 2, geographic isolates of B. transfuga, B. schroederi (from giant panda), and B. ailuri formed a strongly supported clade. In certain analyses (e.g., some single genes), B. tasmaniensis was sister to all other Baylisascaris species rather than sister to the species from ursids and red panda. Using one combination of priors corresponding to moderate population size and shallow genetic divergence, the multispecies coalescent analysis of B. procyonis and B. columnaris yielded moderate support (posterior probability 0.91) for these taxa as separate species. However, other prior combinations yielded weak or no support for delimiting these taxa as separate species. Similarly, tree topologies constrained to represent reciprocal monophyly of B. columnaris and B. procyonis individuals (topologies consistent with separate species) were significantly worse in some cases, but not others, depending on the dataset analyzed. An expanded analysis of SNPs and other genetic markers that were previously suggested to distinguish between individuals of B. procyonis and B. columnaris was made by characterization of additional individual nematodes. The results suggest that many of these SNPs do not represent fixed differences between nematodes derived from raccoon and skunk hosts.

Renal Cell Carcinoma (RCC) Tumors Display Large Expansion of Double Positive (DP) CD4+CD8+ T Cells With Expression of Exhaustion Markers.

Checkpoint inhibitors target the inhibitory receptors expressed by tumor-infiltrating T cells in order to reinvigorate an anti-tumor immune response. Therefore, understanding T cell composition and phenotype in human tumors is crucial. We analyzed by flow cytometry tumor-infiltrating lymphocytes (TILs) from two independent cohorts of patients with different cancer types, including RCC, lung, and colon cancer. In healthy donors, peripheral T cells are usually either CD4+ or CD8+ with a small percentage of CD4+ CD8+ DP cells (<5%). Compared to several other cancer types, including lung, and colorectal cancers, TILs from about a third of RCC patients showed an increased proportion of DP CD4+CD8+ T cells (>5%, reaching 30-50% of T cells in some patients). These DP T cells have an effector memory phenotype and express CD38, 4-1BB, and HLA-DR, suggesting antigen-driven expansion. In fact, TCR sequencing analysis revealed a high degree of clonality in DP T cells. Additionally, there were high levels of PD-1 and TIM-3 expression on DP T cells, which correlated with higher expression of PD-1 and TIM-3 in conventional single positive CD8 T cells from the same patients. These results suggest that DP T cells could be dysfunctional tumor-specific T cells with the potential to be reactivated by checkpoint inhibitors.

Circulating T Cell Subpopulations Correlate With Immune Responses at the Tumor Site and Clinical Response to PD1 Inhibition in Non-Small Cell Lung Cancer.

Agents targeting the PD1-PDL1 axis have transformed cancer therapy. Factors that influence clinical response to PD1-PDL1 inhibitors include tumor mutational burden, immune infiltration of the tumor, and local PDL1 expression. To identify peripheral correlates of the anti-tumor immune response in the absence of checkpoint blockade, we performed a retrospective study of circulating T cell subpopulations and matched tumor gene expression in melanoma and non-small cell lung cancer (NSCLC) patients. Notably, both melanoma and NSCLC patients whose tumors exhibited increased inflammatory gene transcripts presented high CD4+ and CD8+ central memory T cell (CM) to effector T cell (Eff) ratios in blood. Consequently, we evaluated CM/Eff T cell ratios in a second cohort of NSCLC. The data showed that high CM/Eff T cell ratios correlated with increased tumor PDL1 expression. Furthermore, of the 22 patients within this NSCLC cohort who received nivolumab, those with high CM/Eff T cell ratios, had longer progression-free survival (PFS) (median survival: 91 vs. 215 days). These findings show that by providing a window into the state of the immune system, peripheral T cell subpopulations inform about the state of the anti-tumor immune response and identify potential blood biomarkers of clinical response to checkpoint inhibitors in melanoma and NSCLC.

Transuterine infection by Baylisascaris transfuga: Neurological migration and fatal debilitation in sibling moose calves (Alces alces gigas) from Alaska.

Larval Baylisascaris nematodes (L3), resulting from transuterine infection and neural migration, were discovered in the cerebrum of sibling moose calves (Alces alces gigas) near 1-3 days in age from Alaska. We provide the first definitive identification, linking morphology, biogeography, and molecular phylogenetics, of Baylisascaris transfuga in naturally infected ungulates. Life history and involvement of paratenic hosts across a broader assemblage of mammals, from rodents to ungulates, in the transmission of B. transfuga remains undefined. Neural infections, debilitating young moose, may seasonally predispose calves to predation by brown bears, facilitating transmission to definitive hosts. Discovery of fatal neurological infections by L3 of B. transfuga in mammalian hosts serves to demonstrate the potential for zoonotic infection, as widely established for B. procyonis, in other regions and where raccoon definitive hosts are abundant. In zones of sympatry for multi-species assemblages of Baylisascaris across the Holarctic region presumptive identification of B. procyonis in cases of neurological larval migrans must be considered with caution. Diagnostics in neural and somatic larval migrans involving species of Baylisascaris in mammalian and other vertebrate hosts should include molecular-based and authoritative identification established in a phylogenetic context.

Phylogenetic Relationships of Spiruromorph Nematodes (Spirurina: Spiruromorpha) In North American Freshwater Fishes.

Nematodes are common in the parasite communities of North American freshwater fishes, and the majority of them belong to 1 conventional order, Spirurida Chitwood, 1933. Within the Spirurida, the superfamilies Habronematoidea Chitwood and Wehr, 1932 and Thelazioidea Sobolev, 1949 have undergone considerable diversification. The dominant families of these 2 superfamilies, Cystidicolidae Skrjabin 1946 and Rhabdochonidae Railliet, 1916, respectively, are particularly common, widely distributed, and diverse, especially in North America, yet their phylogenetic relationships remain largely unexplored. In this study, we use near complete sequences of the 18S rRNA genes ( SSU rDNA) from species in 6 genera ( Capillospirura Skrjabin, 1924, Cystidicola Fischer, 1798, Salmonema Moravec, Santos and Brasil-Sato, 2008, Rhabdochona Railliet, 1916, Spinitectus Fourment, 1883, and a putative new cystidicolid in mooneyes, Hiodontidae), along with a species of Hedruris Nitzsch, 1812 from newts as a surrogate for the fish parasite Hedruris tiara VanCleave and Mueller, 1932, to explore their phylogenetic relationships. These sequences, together with available sequences from a range of other nematodes, including fish nematodes in other groups (Camallanoidea and 'Seuratoidea'), were analyzed using Bayesian inference and maximum likelihood. The results from both analyses indicate, for the first time, support for the close relationships of the sturgeon parasite Capillospirura with Ascarophis van Beneden, 1871 and Cystidicola; the relationship of the cystidicolid from Hiodontidae with Salmonema of salmonid fishes; the monophyly of the 2 dominant spiruridan genera of fishes, Rhabdochona and Spinitectus; and for previous relationships among Nearctic Spinitectus spp. The results also indicate a closer relationship of Rhabdochona and Spinitectus than is suggested by their conventional classification and reject the monophyly of Habronematoidea, Thelazioidea, and Cystidicolidae. Hedruridae appears to be an early branching lineage of spirurins. Finally, the pattern of association between the fish parasites in this study and their hosts indicates, with few exceptions, ecologically driven diversification events involving host shifting not related to the phylogenetic relationships of their hosts.

The bipartite mitochondrial genome of Ruizia karukerae (Rhigonematomorpha, Nematoda).

Mitochondrial genes and whole mitochondrial genome sequences are widely used as molecular markers in studying population genetics and resolving both deep and shallow nodes in phylogenetics. In animals the mitochondrial genome is generally composed of a single chromosome, but mystifying exceptions sometimes occur. We determined the complete mitochondrial genome of the millipede-parasitic nematode Ruizia karukerae and found its mitochondrial genome consists of two circular chromosomes, which is highly unusual in bilateral animals. Chromosome I is 7,659 bp and includes six protein-coding genes, two rRNA genes and nine tRNA genes. Chromosome II comprises 7,647 bp, with seven protein-coding genes and 16 tRNA genes. Interestingly, both chromosomes share a 1,010 bp sequence containing duplicate copies of cox2 and three tRNA genes (trnD, trnG and trnH), and the nucleotide sequences between the duplicated homologous gene copies are nearly identical, suggesting a possible recent genesis for this bipartite mitochondrial genome. Given that little is known about the formation, maintenance or evolution of abnormal mitochondrial genome structures, R. karukerae mtDNA may provide an important early glimpse into this process.