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INTRODUCTION The Saccharomyces cerevisiae 2.0 project (Sc2.0) aims to modify the yeast genome with a series of densely spaced designer changes. Both a synthetic yeast chromosome arm (synIXR) and the entirely synthetic chromosome (synIII) function with high fitness in yeast. For designer genome synthesis projects, precise engineering of the physical sequence to match the specified design is important for the systematic evaluation of underlying design principles. Yeast can maintain nuclear chromosomes as rings, occurring by chance at repeated sequences, although the cyclized format is unfavorable in meiosis given the possibility of dicentric chromosome formation from meiotic recombination. Here, we describe the de novo synthesis of synthetic yeast chromosome V (synV) in the “Build-A-Genome China” course, perfectly matching the designer sequence and bearing loxPsym sites, distinguishable watermarks, and all the other features of the synthetic genome. We generated a ring synV derivative with user-specified cyclization coordinates and characterized its performance in mitosis and meiosis. RATIONALE Systematic evaluation of underlying Sc2.0 design principles requires that the final assembled synthetic genome perfectly match the designed sequence. Given the size of yeast chromosomes, synthetic chromosome construction is performed iteratively, and new mutations and unpredictable events may occur during synthesis; even a very small number of unintentional nucleotide changes across the genome could have substantial effects on phenotype. Therefore, precisely matching the physical sequence to the designed sequence is crucial for verification of the design principles in genome synthesis. Ring chromosomes can extend those design principles to provide a model for genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders. RESULTS We chemically synthesized, assembled, and incorporated designer chromosome synV (536,024 base pairs) of S. cerevisiae according to Sc2.0 principles, based on the complete nucleotide sequence of native yeast chromosome V (576,874 base pairs). This work was performed as part of the “Build-A-Genome China” course in Tianjin University. We corrected all mutations found—including duplications, substitutions, and indels—in the initial synV strain by using integrative cotransformation of the precise desired changes and by means of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)–based method. Altogether, 3331 corrected base pairs were required to match to the designed sequence. We generated a strain that exactly matches all designer sequence changes that displays high fitness under a variety of culture conditions. All corrections were verified with whole-genome sequencing; RNA sequencing revealed only minor changes in gene expression—most notably, decreases in expression of genes relocated near synthetic telomeres as a result of design. We constructed a functional circular synV (ring_synV) derivative in yeast by precisely joining both chromosome ends (telomeres) at specified coordinates. The ring chromosome showed restoration of subtelomeric gene expression levels. The ring_synV strain exhibited fitness comparable with that of the linear synV strain, revealed no change in sporulation frequency, but notably reduced spore viability. In meiosis, heterozygous or homozygous diploid ring_wtV and ring_synV chromosomes behaved similarly, exhibiting substantially higher frequency of the formation of zero-spore tetrads, a type that was not seen in the rod chromosome diploids. Rod synV chromosomes went through meiosis with high spore viability, despite no effort having been made to preserve meiotic competency in the design of synV. CONCLUSION The perfect designer-matched synthetic chromosome V provides strategies to edit sequence variants and correct unpredictable events, such as off-target integration of extra copies of synthetic DNA elsewhere in the genome. We also constructed a ring synthetic chromosome derivative and evaluated its fitness and stability in yeast. Both synV and synVI can be circularized and can power yeast cell growth without affecting fitness when gene content is maintained. These fitness and stability phenotypes of the ring synthetic chromosome in yeast provide a model system with which to probe the mechanism of human ring chromosome disorders. Synthesis, cyclization, and characterization of synV . ( A ) Synthetic chromosome V (synV, 536,024 base pairs) was designed in silico from native chromosome V (wtV, 576,874 base pairs), with extensive genotype modification designed to be phenotypically neutral. ( B ) CRISPR/Cas9 strategy for multiplex repair. ( C ) Colonies of wtV, synV, and ring_synV strains.

Objective: The goal of this study was to develop and validate density-weighted arithmetic methods for the analysis of the dynamic maturational changes in random amplified polymorphic DNA (RAPD) polymorphisms in Cordyceps sinensis containing multiple fungi. Methods: Ten random primers were used for PCR amplification to monitor changes in the RAPD molecular marker polymorphisms in caterpillar body, stroma and ascocarp portion samples of C. sinensis collected at different stages of maturation. We compared (1) the density-unweighted Nei-Li similarity equation and new similarity equations considering the densities of DNA bands and (2) the density-unweighted and weighted arithmetic methods for cluster analysis. Results: The algorithm using the Nei-Li similarity equation did not account for the differences in the density of the matched randomly amplified amplicons, whereas the new similarity equations were capable of integrating different amplicon densities into the similarity computation. With improvements in the similarity computation and cluster construction, the new methods revealed dynamic maturational changes in the RAPD polymorphisms of molecular markers in C. sinensis caterpillar bodies and stromata. The polymorphism analysis revealed similarities of 74%-88% between the RAPD polymorphisms of the ascocarp portion versus premature and mature C. sinensis stromata, but low similarities of

Seronegative hepatitis—non-A, non-B, non-C, non-D, non-E hepatitis—is poorly characterized but strongly associated with serious complications. We collected 92 sera specimens from patients with non-A–E hepatitis in Chongqing, China between 1999 and 2007. Ten sera pools were screened by Solexa deep sequencing. We discovered a 3,780-bp contig present in all 10 pools that yielded BLASTx E scores of 7e-05–0.008 against parvoviruses. The complete sequence of the in silico -assembled 3,780-bp contig was confirmed by gene amplification of overlapping regions over almost the entire genome, and the virus was provisionally designated NIH-CQV. Further analysis revealed that the contig was composed of two major ORFs. By protein BLAST, ORF1 and ORF2 were most homologous to the replication-associated protein of bat circovirus and the capsid protein of porcine parvovirus, respectively. Phylogenetic analysis indicated that NIH-CQV is located at the interface of Parvoviridae and Circoviridae . Prevalence of NIH-CQV in patients was determined by quantitative PCR. Sixty-three of 90 patient samples (70%) were positive, but all those from 45 healthy controls were negative. Average virus titer in the patient specimens was 1.05 e4 copies/µL. Specific antibodies against NIH-CQV were sought by immunoblotting. Eighty-four percent of patients were positive for IgG, and 31% were positive for IgM; in contrast, 78% of healthy controls were positive for IgG, but all were negative for IgM. Although more work is needed to determine the etiologic role of NIH-CQV in human disease, our data indicate that a parvovirus-like virus is highly prevalent in a cohort of patients with non-A–E hepatitis.

The nervous and vascular systems, although functionally different, share many common regulators of function maintenance. Long non‐coding RNAs (lncRNAs) are important players in many biological processes and human disorders. We previously identified a role of MALAT1 in microvascular dysfunction. However, its role in neurodegeneration is still unknown. Here, we used the eye as the model to investigate the role of MALAT1 in retinal neurodegeneration. We show that MALAT1 expression is significantly up‐regulated in the retinas, Muller cells, and primary retinal ganglion cells (RGCs) upon stress. MALAT1 knockdown reduces reactive gliosis, Muller cell activation, and RGC survival in vivo and in vitro . MALAT1‐CREB binding maintains CREB phosphorylation by inhibiting PP2A‐mediated dephosphorylation, which leads to continuous CREB signaling activation. Clinical and animal experimentation suggests that MALAT1 dysfunction is implicated in neurodegenerative processes and several human disorders. Collectively, this study reveals that MALAT1 might regulate the development of retinal neurodegeneration through CREB signaling.

Vascular remodeling is an important pathological feature of hypertension, leading to increased vascular resistance and reduced compliance. Endothelial cell (EC) and vascular smooth muscle cell (VSMC) dysfunction is involved in vascular remodeling. Long noncoding RNAs are potential regulators of EC and VSMC function. Herein, we determined whether long noncoding RNA–growth arrest–specific 5 (GAS5) is involved in hypertension-related vascular remodeling. We revealed that GAS5 knockdown aggravated hypertension-induced microvascular dysfunction as shown by increased retinal neovascularization and capillary leakage. GAS5 regulated the remodeling of arteries, including caudal arteries, carotid arteries, renal arteries, and thoracic arteries. GAS5 was mainly expressed in ECs and VSMCs, and its expression was significantly downregulated in hypertension. GAS5 knockdown affected endothelial activation, endothelial proliferation, VSMC phenotypic conversion, and EC-VSMC communication in vivo and in vitro. Mechanistically, GAS5 regulated EC and VSMC function through β-catenin signaling. This study identified GAS5 as a critical regulator in hypertension and demonstrated the potential of gene therapy and drug development for treating hypertension.

Human parvovirus 4 (PARV4) is an emerging human virus, and little is known about the molecular aspects of PARV4 apart from its incomplete genome sequence, which lacks information of the termini. We analyzed the gene expression profile of PARV4 using a nearly full-length HPV4 genome in a replication competent system in 293 cells. We found that PARV4 utilizes two promoters to transcribe non-structural protein- and structural protein-encoding mRNAs, respectively, which were polyadenylated at the right end of the genome. Three major proteins, including the large non-structural protein NS1a, whose mRNA is spliced, and capsid proteins VP1 and VP2, were detected. Additional functional analysis of the NS1a revealed its capability to induce cell cycle arrest at G2/M phase in ex vivo-generated human hematopoietic stem cells. Taken together, our characterization of the molecular features of PARV4 suggests that PARV4 represents a new genus in the family Parvoviridae.

Human parvovirus B19 (B19V) is the only human pathogenic parvovirus. It causes a wide spectrum of human diseases, including fifth disease (erythema infectiosum) in children and pure red cell aplasia in immunocompromised patients. B19V is highly erythrotropic and preferentially replicates in erythroid progenitor cells (EPCs). Current understanding of how B19V interacts with cellular factors to regulate disease progression is limited, due to a lack of permissive cell lines and animal models. Here, we employed a recently developed primary human CD36(+) EPC culture system that is highly permissive for B19V infection to identify cellular factors that lead to cell cycle arrest after B19V infection. We found that B19V exploited the E2F family of transcription factors by downregulating activating E2Fs (E2F1 to E2F3a) and upregulating repressive E2Fs (E2F4 to E2F8) in the primary CD36(+) EPCs. B19V nonstructural protein 1 (NS1) was a key viral factor responsible for altering E2F1-E2F5 expression, but not E2F6-E2F8 expression. Interaction between NS1 and E2F4 or E2F5 enhanced the nuclear import of these repressive E2Fs and induced stable G₂ arrest. NS1-induced G₂ arrest was independent of p53 activation and increased viral replication. Downstream E2F4/E2F5 targets, which are potentially involved in the progression from G₂ into M phase and erythroid differentiation, were identified by microarray analysis. These findings provide new insight into the molecular pathogenesis of B19V in highly permissive erythroid progenitors.

The pre-mRNA processing strategy of the B19 virus is unique among parvoviruses. B19 virus-generated pre-mRNAs are transcribed from a single promoter and are extensively processed by alternative splicing and alternative polyadenylation to generate 12 transcripts. Blockage of the production of full-length B19 virus transcripts at the internal polyadenylation site [(pA)p] was previously reported to be a limiting step in B19 virus permissiveness. We show here that in the absence of genome replication, internal polyadenylation of B19 virus RNAs at (pA)p is favored in cells which are both permissive and nonpermissive for B19 viral replication. Replication of the B19 virus genome, however, introduced either by viral infection or by transfection of an infectious clone into permissive cells or forced by heterologous replication systems in nonpermissive cells, enhanced readthrough of (pA)p and the polyadenylation of B19 virus transcripts at the distal site [(pA)d]. Therefore, replication of the genome facilitates the generation of sufficient full-length transcripts that encode the viral capsid proteins and the essential 11-kDa nonstructural protein. Furthermore, we show that polyadenylation of B19 viral RNA at (pA)p likely competes with splicing at the second intron. Thus, we conclude that replication of the B19 virus genome is the primary limiting step governing B19 virus tropism.

Three full-length genomic clones (pB19-M20, pB19-FL and pB19-HG1) of parvovirus B19 were produced in different laboratories. pB19-M20 was shown to produce infectious virus. To determine the differences in infectivity, all three plasmids were tested by transfection and infection assays. All three clones were similar in viral DNA replication, RNA transcription, and viral capsid protein production. However, only pB19-M20 and pB19-HG1 produced infectious virus. Comparison of viral sequences showed no significant differences in ITR or NS regions. In the capsid region, there was a nucleotide sequence difference conferring an amino acid substitution (E176K) in the phospholipase A2-like motif of the VP1-unique (VP1u) region. The recombinant VP1u with the E176K mutation had no catalytic activity as compared with the wild-type. When this mutation was introduced into pB19-M20, infectivity was significantly attenuated, confirming the critical role of this motif. Investigation of the original serum from which pB19-FL was cloned confirmed that the phospholipase mutation was present in the native B19 virus.

The pathogenic parvovirus B19 (B19V) has an extreme tropism for human erythroid progenitor cells. In vitro, only a few erythroid leukemic cell lines (JK-1 and KU812Ep6) or megakaryoblastoid cell lines (UT7/Epo and UT7/Epo-S1) with erythroid characteristics support B19V replication, but these cells are only semipermissive. By using recent advances in generating large numbers of human erythroid progenitor cells (EPCs) ex vivo from hematopoietic stem cells (HSCs), we produced a pure population of CD36 + EPCs expanded and differentiated from CD34 + HSCs and assessed the CD36 + EPCs for their permissiveness to B19V infection. Over more than 3 weeks, cells grown in serum-free medium expanded more than 800,000-fold, and 87 to 96% of the CD36 + EPCs were positive for globoside, the cellular receptor for B19V. Immunofluorescence (IF) staining showed that about 77% of the CD36 + EPCs were positive for B19V infection, while about 9% of UT7/Epo-S1 cells were B19V positive. Viral DNA detected by real-time PCR increased by more than 3 logs in CD36 + EPCs; the increase was 1 log in UT7/Epo-S1 cells. Due to the extensive permissivity of CD36 + EPCs, we significantly improved the sensitivity of detection of infectious B19V by real-time reverse transcription-PCR and IF staining 100- and 1,000-fold, respectively, which is greater than the sensitivity of UT7/Epo-S1 cell-based methods. This is the first description of an ex vivo method to produce large numbers of EPCs that are highly permissive to B19V infection and replication, offering a cellular system that mimics in vivo infection with this pathogenic human virus.

In an attempt to experimentally define the roles of viral proteins encoded by the B19 genome in the viral life cycle, we utilized the B19 infectious clone constructed in our previous study to create two groups of B19 mutant genomes: (i) null mutants, in which either a translational initiation codon for each of these viral genes was substituted by a translational termination codon or a termination codon was inserted into the open reading frame by a frameshift; and (ii) a deletion mutant, in which half of the hairpin sequence was deleted at both the 5′ and the 3′ termini. The impact of these mutations on viral infectivity, DNA replication, capsid protein production, and distribution was systematically examined. Null mutants of the NS and VP1 proteins or deletion of the terminal hairpin sequence completely abolished the viral infectivity, whereas blocking expression of the 7.5-kDa protein or the putative protein X had no effect on infectivity in vitro. Blocking expression of the proline-rich 11-kDa protein significantly reduced B19 viral infectivity, and protein studies suggested that the expression of the 11-kDa protein was critical for VP2 capsid production and trafficking in infected cells. These findings suggest a previously unrecognized role for the 11-kDa protein, and together the results enhance our understanding of the key features of the B19 viral genome and proteins.

Human parvovirus B19 has a nonenveloped, icosahedral capsid packaging a linear single-stranded DNA genome of 5.6 kb with long inverted terminal repeats (ITR) at both the 5′ and 3′ end. Previous attempts to construct a full-length B19 clone were unsuccessful due to deletions in the ITR sequences. We cloned the complete parvovirus B19 genome with intact ITRs from an aplastic crisis patient. Sequence analysis of the complete viral genome indicated that both 5′ and 3′ ITRs have two sequence configurations and several base changes within the ITRs compared to previous published sequences. After transfection of the plasmid into permissive cells, spliced and non-spliced viral transcripts and viral capsid proteins could be detected. Southern blot analysis of the DNA purified from the plasmid-transfected cells confirmed parvovirus B19 DNA replication. Production of infectious virus by the B19 plasmid was shown by inoculation of cell lysate derived from transfected cells into fresh cells. Together, these results indicate the first successful production of an infectious clone for parvovirus B19 virus.

Anaplasma phagocytophila is an obligatory intragranulocytic bacterium that causes human granulocytic ehrlichiosis. Immunodominant 44-kDa outer membrane proteins of A. phagocytophila are encoded by a p44 multigene family. In the present study, expression profiles of p44 genes in the blood of acutely infected patients in the year 2000 were characterized. A single p44 gene was predominantly expressed in peripheral blood leukocytes from one patient, while up to 17 different p44 genes were transcribed without a single majority in the other two patients. The cDNA sequences of the central hypervariable region of several p44 genes were identical among the isolates from the three patients and a 1995 A. phagocytophila isolate. A. phagocytophila was isolated by cell culture from all of the three 2000 patients. Genomic Southern blot analysis of the three 2000 and two 1995 A. phagocytophila isolates with probes specific to the most dominant p44 transcript in each patient showed that the p44 loci in the A. phagocytophila genome were conserved. Analysis of the predicted amino acid sequences of 43 different p44 genes including 19 new sequences found in the present study, revealed that five amino acids were absolutely conserved. The hypervariable region was subdivided into five domains, including three extremely hypervariable central domains. These results suggest that variations in the sequences of p44 are not random but are restricted. Furthermore, several p44 genes are not hypermutatable in nature, based on the conservation of gene sequences and loci among isolates obtained 5 years apart.

Anaplasma ( Ehrlichia ) phagocytophila and Ehrlichia chaffeensis , the etiologic agents of granulocytic and monocytic ehrlichioses, respectively, are obligatory intracellular bacteria that cause febrile systemic illness in humans. We identified and characterized clusters of genes for a type IV secretion machinery in these two bacteria, and analyzed their gene expression in cell culture and mammalian hosts. Eight virB and virD genes were found in each bacterial genome, and all of the genes were transcribed in cell culture. Although the gene order and orientation were similar to those found in other bacteria, the eight virB and virD genes were clustered at two separate loci in each genome. Five of the genes ( virB8 , virB9 , virB10 , virB11 , and virD4 ) were located downstream from a ribA gene. These five genes in both A. phagocytophila and E. chaffeensis were polycistronically transcribed and controlled through at least two tandem promoters located upstream of the virB8 gene in human leukemia cell lines. The virB9 gene of A. phagocytophila was transcriptionally active in peripheral blood leukocytes from human ehrlichiosis patients and experimentally infected animals. Three of the remaining genes ( virB3 , virB4 , and virB6 ) of both A. phagocytophila and E. chaffeensis were arranged downstream from a sodB gene and cotranscribed with the sodB gene through one or more sodB promoters in human leukocytes. This suggests that transcription of the three virB genes in these two Anaplasma and Ehrlichia spp. is regulated by factors that influence the sodB gene expression. This unique regulation of gene expression for the type IV secretion system may be associated with intracellular survival and replication of Anaplasma and Ehrlichia spp. in granulocytes or monocytes.

Naccache et al. confirm our finding that NIH-CQV is a previously unidentified hybrid virus, with a replication-associated protein gene (rep) homologous to circoviruses and capsid protein gene (cp) homologous to parvoviruses (1⇓–3), and overall has a structure with characteristics of Parvoviridae (1).

Enzyme-linked immunosorbent assay (ELISA) for human granulocytic ehrlichiosis (HGE) using two different recombinant P44 proteins (rP44 and rP44-2hv) of the HGE agent as antigens was evaluated. Sera from a total of 72 healthy humans both from regions where HGE is nonendemic and regions where HGE is endemic were used as negative controls to determine the cutoff value for ELISA. Sera from a total of 14 patients (nine from whom the HGE agent was isolated and five who were HGE-PCR positive) were used as positive controls. One hundred nine sera from 72 patients in an area where HGE is endemic who were suspected of having HGE were examined by ELISA and indirect immunofluorescence assay (IFA). All IFA-negative sera were negative by both ELISAs. Of 39 sera that were IFA positive, 35 and 27 were positive by ELISA using rP44 and rP44-2hv, respectively, indicating that the use of rP44 is more sensitive. Western blot analysis of the four rP44-ELISA-negative IFA-positive sera using whole HGE agent as antigen suggests that these four sera were false IFA positive. There was no difference in results with or without the preabsorption of sera with Escherichia coli or with or without the cleavage of the fused protein derived from the vector. There was a significant positive correlation between IFA titers and optical densities of ELISAs. Four Ehrlichia chaffeensis -positive and 10 Borrelia burgdorferi -positive sera were negative by ELISA. However, two Babesia microti -positive sera showed strong cross-reactivity to the fused vector protein, which was eliminated after cleavage of the protein. Thus, ELISA using rP44 nonfusion protein would provide a simple, specific, and objective HGE serologic test which can be easily automated.

The pathogenic parvovirus B19 (B19V) has an extreme tropism for human erythroid progenitor cells. In vitro, only a few erythroid leukemic cell lines (JK-1 and KU812Ep6) or megakaryoblastoid cell lines (UT7/Epo and UT7/Epo-S1) with erythroid characteristics support B19V replication, but these cells are only semipermissive. By using recent advances in generating large numbers of human erythroid progenitor cells (EPCs) ex vivo from hematopoietic stem cells (HSCs), we produced a pure population of CD36+ EPCs expanded and differentiated from CD34+ HSCs and assessed the CD36+ EPCs for their permissiveness to B19V infection. Over more than 3 weeks, cells grown in serum-free medium expanded more than 800,000-fold, and 87 to 96% of the CD36+ EPCs were positive for globoside, the cellular receptor for B19V. Immunofluorescence (IF) staining showed that about 77% of the CD36+ EPCs were positive for B19V infection, while about 9% of UT7/Epo-S1 cells were B19V positive. Viral DNA detected by real-time PCR increased by more than 3 logs in CD36+ EPCs; the increase was 1 log in UT7/Epo-S1 cells. Due to the extensive permissivity of CD36+ EPCs, we significantly improved the sensitivity of detection of infectious B19V by real-time reverse transcription-PCR and IF staining 100- and 1,000-fold, respectively, which is greater than the sensitivity of UT7/Epo-S1 cell-based methods. This is the first description of an ex vivo method to produce large numbers of EPCs that are highly permissive to B19V infection and replication, offering a cellular system that mimics in vivo infection with this pathogenic human virus.

A 44-kDa major outer membrane protein of the human granulocytic ehrlichiosis (HGE) agent is an immunodominant antigen in human infection. A gene encoding this protein was cloned and sequenced. Southern blot results revealed the existence of multigenes homologous to the P44 gene in the genome of the HGE agent. The recombinant 44-kDa protein (rP44) was expressed by using expression vector pET30a. The reactivity of the affinity-purified rP44 was evaluated by Western immunoblot analysis and dot blot immunoassay. Western immunoblot analysis showed that mouse anti-rP44 serum reacted with 44- to 42-kDa proteins in six different HGE agent strains tested except strain 2, in which three proteins of 42, 40, and 38 kDa were recognized. Eleven HGE patient serum samples, a horse anti-HGE serum, and a horse anti- Ehrlichia equi serum recognized the rP44 protein. This suggests that rP44 is an HGE- E. equi group-specific antigen. Neither human anti- Ehrlichia chaffeensis serum nor rabbit anti- Borrelia burgdorferi serum reacted with rP44. Sera from two patients coinfected with the HGE agent and B. burgdorferi reacted positively with rP44 and the HGE agent. Sera from 20 HGE patients with indirect fluorescent-antibody (IFA) titers ranging from 1:20 to 1:2,560 gave distinct positive reactions in a dot immunoblot assay. There was a positive correlation between the color densities of the dot reactions and the IFA titers when greater than 50 ng of recombinant antigen per dot was used. The use of the affinity-purified rP44 protein as antigen would provide a more specific, consistent, and simpler serodiagnosis for HGE than the use of whole infected cells or purified HGE agents.

Several immunodominant major proteins ranging from 23 to 30 kDa were identified in the outer membrane fractions of Ehrlichia chaffeensis and Ehrlichia canis . The N-terminal amino acid sequence of a 28-kDa protein of E. chaffeensis (one of the major proteins) was determined. The gene ( p28 ), almost full length, encoding the 28-kDa protein was cloned by PCR with primers designed based on the N-terminal sequence of the E. chaffeensis 28-kDa protein and the consensus sequence between the C termini of the Cowdria ruminantium MAP-1 and Anaplasma marginale MSP-4 proteins. The p28 gene was overexpressed, and antibody to the recombinant protein was raised in a rabbit. The antibody and serum from a patient infected with E. chaffeensis reacted with the recombinant protein, three proteins (29, 28, and 25 kDa) of E. chaffeensis , and a 30-kDa protein of E. canis . Immunoelectron microscopy with the rabbit antibody revealed that the antigenic epitope of the 28-kDa protein was exposed on the surface of E. chaffeensis . Southern blot analysis with a 32 P-labeled p28 gene probe revealed multiple copies of genes homologous to p28 in the E. chaffeensis genome. Six copies of the p28 gene were cloned and sequenced from the genomic DNA by using the same probe. The open reading frames of these gene copies were tandemly arranged with intergenic spaces. They were nonidentical genes and contained a semivariable region and three hypervariable regions in the predicted protein molecules. One of the gene copies encoded a protein with an internal amino acid sequence identical to the chemically determined N-terminal amino acid sequence of a 23-kDa protein of E. chaffeensis . Immunization with the recombinant P28 protein protected mice from infection with E. chaffeensis . These findings suggest that the 30-kDa-range proteins of E. chaffeensis represent a family of antigenically related homologous proteins encoded by a single gene family.

A partial 16S rRNA gene was amplified in Ehrlichia canis-infected cells by nested PCR. The assay was specific and did not amplify the closely related Ehrlichia chaffeensis, Ehrlichia muris, Neorickettsia helminthoeca, and SF agent 16S rRNA genes. The assay was as sensitive as Southern hybridization, detecting as little as 0.2 pg of E. canis DNA. By this method, all blood samples from four dogs experimentally infected with E. canis were positive as early as day 4 postinoculation, which was before or at the time of seroconversion. One hundred five blood samples from dogs from Arizona and Texas (areas of E. canis endemicity) and 30 blood samples from dogs from Ohio (area of E. canis nonendemicity) were examined by nested PCR and immunofluorescent-antibody (IFA) test. Approximately 84% of dogs from Arizona and Texas had been treated with doxycycline before submission of blood specimens. Among Arizona and Texas specimens, 46 samples were PCR positive (44%) and 80 were IFA positive (76%). Forty-three of 80 IFA-positive samples (54%) were PCR positive, and 22 of 25 IFA-negative samples (88%) were negative in the nested PCR. None of the Ohio specimens were IFA positive, but 5 specimens were PCR positive (17%). Our results indicate that the nested PCR is highly sensitive and specific for detection of E. canis and may be more useful in assessing the clearance of the organisms after antibiotic therapy than IFA, especially in areas in which E. canis is endemic.

Human parvovirus B19 (B19V) is a member of the genusErythrovirusin the familyParvoviridae. In vitro, autonomous B19V replication is limited to human erythroid progenitor cells and in a small number of erythropoietin-dependent human megakaryoblastoid and erythroid leukemic cell lines. Here we report that the failure of B19V DNA replication in nonpermissive 293 cells can be overcome by adenovirus infection. More specifically, the replication of B19V DNA in the 293 cells and the production of infectious progeny virus were made possible by the presence of the adenovirus E2a, E4orf6, and VA RNA genes that emerged during the transfection of the pHelper plasmid. Using this replication system, we identified the terminal resolution site and the nonstructural protein 1 (NS1) binding site on the right terminal palindrome of the viral genome, which is composed of a minimal origin of replication spanning 67 nucleotides. Plasmids or DNA fragments containing an NS1 expression cassette and this minimal origin were able to replicate in both pHelper-transfected 293 cells and B19V-semipermissive UT7/Epo-S1 cells. Our results have important implications for our understanding of native B19V infection.

Anaplasma (formerly Ehrlichia) phagocytophilum, Ehrlichia chaffeensis, and Neorickettsia (formerly Ehrlichia) sennetsu are intracellular vector-borne pathogens that cause human ehrlichiosis, an emerging infectious disease. We present the complete genome sequences of these organisms along with comparisons to other organisms in the Rickettsiales order. Ehrlichia spp. and Anaplasma spp. display a unique large expansion of immunodominant outer membrane proteins facilitating antigenic variation. All Rickettsiales have a diminished ability to synthesize amino acids compared to their closest free-living relatives. Unlike members of the Rickettsiaceae family, these pathogenic Anaplasmataceae are capable of making all major vitamins, cofactors, and nucleotides, which could confer a beneficial role in the invertebrate vector or the vertebrate host. Further analysis identified proteins potentially involved in vacuole confinement of the Anaplasmataceae, a life cycle involving a hematophagous vector, vertebrate pathogenesis, human pathogenesis, and lack of transovarial transmission. These discoveries provide significant insights into the biology of these obligate intracellular pathogens., Synopsis Ehrlichiosis is an acute disease that triggers flu-like symptoms in both humans and animals. It is caused by a range of bacteria transmitted by ticks or flukes. Because these bacteria are difficult to culture, however, the organisms are poorly understood. The genomes of three emerging human pathogens causing ehrlichiosis were sequenced. A database was designed to allow the comparison of these three genomes to sixteen other bacteria with similar lifestyles. Analysis from this database reveals new species-specific and disease-specific genes indicating niche adaptations, pathogenic traits, and other features. In particular, one of the organisms contains more than 100 copies of a single gene involved in interactions with the host(s). These comparisons also enabled a reconstruction of the metabolic potential of five representative genomes from these bacteria and their close relatives. With this work, scientists can study these emerging pathogens in earnest.

Parvovirus B19 infection in adults is often associated with acute symmetrical polyarthropathy, but the mechanism is unknown. Recently, a secreted phospholipase A 2 (sPLA 2 ) motif was identified in the VPl-unique region (VPlu) of the B19 minor capsid protein. To investigate the role of this motif, we expressed VPlu with and without point mutations in the critical amino acids of sPLA 2 . Although high concentrations of B19 did not infect human fibroblast-like synoviocytes (HFLSs), there was a >3-fold increase in synoviocyte migration that could be blocked by phospholipase inhibitors. Recombinant proteins with intact VPlu demonstrated sPLA 2 activity and induced cell migration, whereas proteins with mutated VPlu were nonfunctional in both assays. The incubation of HFLSs with proteins that had intact VPlu, but not with proteins with mutated VPlu, increased the production of prostaglandin E 2 >100-fold. Expression of cyclooxygenase (COX)-2 mRNA transcripts, as determined by real-time reverse-transcription polymerase chain reaction, and COX-2 protein expression were both significantly increased after incubation with protein that had intact VPlu. Proteins with VPlu in noninfectious B19 may participate in the inflammatory response in the synovial compartment.

Human granulocytic ehrlichiosis (HGE), a tick-borne zoonosis, is caused by an obligatory intragranulocytic bacterium, the HGE agent, a strain ofAnaplasma phagocytophila. The equine model of HGE is considered valuable in understanding pathogenic and immune mechanisms of HGE. In the present study, cytokine mRNA expression by peripheral blood leukocytes (PBLs) in horses was examined during the course of infection by intravenous inoculation ofA. phagocytophilaor by allowing feeding by infected ticks. Thep44genes encoding the major outer membrane protein P44s ofA. phagocytophilawere detected by PCR in PBLs of all four horses from 4 to 20 days postexposure. During the 20-day infection period, interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) mRNA expression was upregulated in PBLs of all four horses, and IL-8 mRNA expression was upregulated in three horses. Gamma interferon, IL-10, and IL-12 p35 mRNAs were weakly expressed in only one horse each. IL-2, IL-4, IL-6, and IL-12 p40 mRNA expression , however, could not be detected in the PBLs of any of the four horses. These results suggest that IL-1β, TNF-α, and IL-8 generation duringA. phagocytophilainfection has a primary role in HGE pathogenesis and immunomodulation.

Objective To examine the maturational changes in proteomic polymorphisms resulting from differential expression by multiple intrinsic fungi in the caterpillar body and stroma of natural Cordyceps sinensis (Cs), an integrated micro-ecosystem. Methods The surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) biochip technique was used to profile the altered protein compositions in the caterpillar body and stroma of Cs during its maturation. The MS chromatograms were analyzed using density-weighted algorithms to examine the similarities and cluster relationships among the proteomic polymorphisms of the Cs compartments and the mycelial products Hirsutella sinensis (Hs) and Paecilomyces hepiali (Ph). Results: SELDI-TOF MS chromatograms displayed dynamic proteomic polymorphism alterations among samples from the different Cs compartments during maturation. More than 1,900 protein bands were analyzed using density-weighted ZUNIX similarity equations and clustering methods, revealing integral polymorphism similarities of 57.4% between the premature and mature stromata and 42.8% between the premature and mature caterpillar bodies. The across-compartment similarity was low, ranging from 10.0% to 18.4%. Consequently, each Cs compartment (i.e., the stroma and caterpillar body) formed a clustering clade, and the 2 clades formed a Cs cluster. The polymorphic similarities ranged from 0.51% to 1.04% between Hs and the Cs compartments and were 2.8- to 4.8-fold higher (1.92%–4.34%) between Ph and the Cs compartments. The Hs and Ph mycelial samples formed isolated clades outside of the Cs cluster. Conclusion Proteomic polymorphisms in the caterpillar body and stroma of Cs change dynamically during maturation. The proteomic polymorphisms in Hs and Ph differ from those in Cs, suggesting the presence of multiple Cs-associated fungi and multiple Ophiocordyceps sinensis genotypes with altered differential protein expression in the Cs compartments during maturation. In conjunction with prior mycological and molecular observations, the findings from this proteomic study support the integrated micro-ecosystem hypothesis for natural Cs.

Laboratory diagnosis of human ehrlichioses is routinely made by an indirect immunofluorescence assay (IFA) using cultured ehrlichia-infected whole cells as antigen. Concern has been raised that incorrect diagnoses of human monocytic ehrlichiosis (HME) or human granulocytic ehrlichiosis (HGE) may be made on the basis of serologic cross-reactivity between Ehrlichia chaffeensis and the agent of HGE. The present study examined whether two recombinant major outer membrane proteins, rP30 and rP44, that were previously shown to be sensitive and specific serodiagnostic antigens for HME and HGE, respectively, could be used to discriminate IFA dually reacting sera. Thirteen dually IFA-reactive sera, three sera that were IFA positive only with E. chaffeensis , and three sera that were IFA positive only with the HGE agent were examined by Western immunoblot analysis using purified whole organisms and recombinant proteins as antigens. All 16 E. chaffeensis IFA-positive sera reacted with rP30. However, none of these sera reacted with rP44, regardless of IFA reactivity with the HGE agent. The three HGE-agent-only IFA-positive sera reacted only with rP44, not with rP30. Western immunoblotting using purified E. chaffeensis and the HGE agent as antigens suggested that heat shock and other proteins, but not major outer membrane proteins, cross-react between the two organisms. Therefore, Western immunoblot analysis using rP44 and rP30 may be useful in discriminating dually HME and HGE IFA-reactive sera.

Human granulocytic ehrlichiosis (HGE) is caused by infection with an obligatory intracellular bacterium, the HGE agent. We previously cloned a gene encoding HGE agent 44-kDa major outer membrane protein and designated it p44. In this study, we (i) identified five different mRNAs that are transcribed from p44-homologous genes in the HGE agent cultivated in HL-60 cells; (ii) cloned genes corresponding to the mRNAs from the genomic DNA of the HGE agent; (iii) showed that the genes being expressed were not clustered in the HGE agent genome; (iv) estimated that a minimum copy number of the p44-homologous genes in the genome is 18; (v) detected two different P44-homologous proteins expressed by the HGE agent; and (vi) demonstrated existence of antibodies specific to the two proteins in sera from patients with HGE. These findings showed that p44 multigenes have several active expression sites and the expression is regulated at transcriptional level, suggesting a potentially unique mechanism for generating the diversity in major antigenic outer membrane proteins of the HGE agent. Characterization of p44-homologous genes expressed by the HGE agent in a tissue culture would assist in understanding a role of the p44 multigene family in pathogenesis and immune response in HGE.

A 30-kDa major outer membrane protein of Ehrlichia canis , the agent of canine ehrlichiosis, is the major antigen recognized by both naturally and experimentally infected dog sera. The protein cross-reacts with a serum against a recombinant 28-kDa protein (rP28), one of the outer membrane proteins of a gene ( omp-1 ) family of Ehrlichia chaffeensis . Two DNA fragments of E. canis were amplified by PCR with two primer pairs based on the sequences of E. chaffeensis omp-1 genes, cloned, and sequenced. Each fragment contained a partial 30-kDa protein gene of E. canis . Genomic Southern blot analysis with the partial gene probes revealed the presence of multiple copies of these genes in the E. canis genome. Three copies of the entire gene ( p30 , p30-1 , and p30a ) were cloned and sequenced from the E. canis genomic DNA. The open reading frames of the two copies ( p30 and p30-1 ) were tandemly arranged with an intergenic space. The three copies were similar but not identical and contained a semivariable region and three hypervariable regions in the protein molecules. The following genes homologous to three E. canis 30-kDa protein genes and the E. chaffeensis omp-1 family were identified in the closely related rickettsiae: wsp from Wolbachia sp., p44 from the agent of human granulocytic ehrlichiosis, msp-2 and msp-4 from Anaplasma marginale , and map-1 from Cowdria ruminantium . Phylogenetic analysis among the three E. canis 30-kDa proteins and the major surface proteins of the rickettsiae revealed that these proteins are divided into four clusters and the two E. canis 30-kDa proteins are closely related but that the third 30-kDa protein is not. The p30 gene was expressed as a fusion protein, and the antibody to the recombinant protein (rP30) was raised in a mouse. The antibody reacted with rP30 and a 30-kDa protein of purified E. canis . Twenty-nine indirect fluorescent antibody (IFA)-positive dog plasma specimens strongly recognized the rP30 of E. canis . To evaluate whether the rP30 is a suitable antigen for serodiagnosis of canine ehrlichiosis, the immunoreactions between rP30 and the whole purified E. canis antigen were compared in the dot immunoblot assay. Dot reactions of both antigens with IFA-positive dog plasma specimens were clearly distinguishable by the naked eye from those with IFA-negative plasma specimens. By densitometry with a total of 42 IFA-positive and -negative plasma specimens, both antigens produced results similar in sensitivity and specificity. These findings suggest that the rP30 antigen provides a simple, consistent, and rapid serodiagnosis for canine ehrlichiosis. Cloning of multigenes encoding the 30-kDa major outer membrane proteins of E. canis will greatly facilitate understanding pathogenesis and immunologic study of canine ehrlichosis and provide a useful tool for phylogenetic analysis.

The etiologic agent of human granulocytic ehrlichiosis (HGE) is an obligate intracellular bacterium. In 1996, blood specimens from 53 patients suspected of having HGE were examined by indirect fluorescent antibody (IFA) testing with the HGE agent no. 13 isolate as the antigen, by nested PCR, and by culture. All patients resided in Westchester County, N.Y. Twelve patient specimens were positive for IFA (titer > or = 1:40). Seven of these were also positive by PCR. Of the seven specimens positive by both IFA testing and PCR, the HGE agent was isolated from four (no. 2, 3, 6, and 11) and continuously cultured in HL-60 cells. These were confirmed as the HGE agent by sequencing of 16S rDNA. Both purified whole-cell organisms and the outer membrane fractions of the new isolates were compared with no. 13 isolate and a tick (USG) isolate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot analysis. No. 11 and 13 isolates had identical SDS-PAGE patterns with respect to 49- and 47-kDa proteins. No. 3 and USG isolates lacked the 47-kDa protein, and no. 6 isolate lacked the 49-kDa protein. Both 49- and 47-kDa bands were absent in no. 2 isolate. Western blot results with seven different sera, including five convalescent-phase sera from these patients, one dog anti-USG isolate, and one horse anti-BDS isolate, showed that all major antigens in six isolates were recognized by all sera. However, the molecular sizes and the numbers of major antigens recognized varied among the six isolates. Overall, HGE agent no. 3, 6, 11, and 13, and USG isolates had similar patterns, with 1 or 2 major antigens with molecular masses of around 49 and 47 kDa. No. 2 isolate was quite distinct in having a major antigen of 43 kDa. This indicates that although these antigenic epitopes are all cross-reactive among strains, the HGE agent has a strain pleomorphism in its major antigenic proteins. The major antigen profiles of the outer membrane protein fractions and of whole organisms of six HGE agent isolates were similar, suggesting that 49- and 47-kDa major antigens are the outer membrane proteins of the HGE agent.

A human granulocytic ehrlichiosis (HGE) agent with 16S rDNA sequence identical to the published sequence of HGE agents was isolated from a patient from New York State by inoculation of the blood leukocyte fraction directly into a human promyelocytic leukemia cell line HL-60. The HGE agent was also isolated from the leukocyte fraction of the blood and bone marrow of a mouse inoculated with the leukocyte fraction of the patient's blood. The isolate has been passaged in tissue culture 30 times over 8 months. Electron microscopy revealed pleomorphic coccobacilli with a thin and highly rippled outer membrane in the clear inclusion matrix. Comparison of IFA reactivity of antisera obtained from a variety of sources with the cell-cultured HGE agent revealed that 3 HGE agent strains (New York isolate, Wisconsin [BDS] isolate, and a tick-derived isolate) are highly cross-reactive and there are diverse antigenic cross-reactivities between HGE agent and Ehrlichia chaffeensis.

Campylobacters are important causes of enteritis worldwide. Campylobacter jejuni and C. coli are the species most commonly involved.3 Recently, several investigators have found an increased proportion of C. concisus, a Campylobacter mainly associated with the oral cavity, in faeces from patients with symptoms of enteritis.5,13 Since we incubate our campylobacter media with filters in an atmosphere containing 7% H2, a large number of C. concisus was isolated in our laboratory. The isolation rate of C. concisus for a 17 month period was 2.4% in children and 1.5% in adults.7 Most of the C. concisus culture-positive patients presented diarrhoea. However, the occasional isolation of C. concisus together with other enteric pathogens from faeces of patients with enteritis and the detection of the organism in many healthy individuals, do not support the etiologic role of C. concisus in patients with enteritis. To clarify the possible pathogenicity of C. concisus, a study of the immune response to C. concisus was needed. The aim of this study was to evaluate the antibody response in a large group of patients with C. concisus culture-positive enteritis using a solid-phase enzyme-linked immunosorbent assay and Western blot analysis.

Abstract 273 Seronegative hepatitis—non-hepatitis A, non-B, non-C, non-E—is poorly characterized but strongly associated with serious complications, especially aplastic anemia and fulminant hepatitis of childhood. Seronegative hepatitis is rare in the United States but more prevalent in Asia, constituting about 10–20% of acute cases. We applied next-generation sequencing to blood samples of patients from western China with seronegative hepatitis for virus discovery. A total of 92 plasma specimens were collected at Chongqing, China, between 1999 and 2007. Twenty-seven patients were diagnosed as having acute hepatitis by clinical and laboratory characteristics. Sixty-five patients had biopsy-proven chronic aggressive hepatitis, ten of which had cirrhosis. Serologic assays for hepatitis viruses A, B, C, E, HIV, Epstein-Barr virus and cytomegalovirus were all negative. Additional tests for antinuclear antibody, rheumatoid factor, anti-mitochondrial antibody also were normal. Ten plasma pools derived from 93 specimens of the patients were screened by Solexa deep sequencing. We discovered a 3780-bp contig present in all ten-pools that yielded tBLASTx E scores of 0.003 to 1.5 against parvoviruses. The sequence of the in silico assembled 3780-bp contig was confirmed by overlapping PCRs, indicating the contig that contained the nearly complete new virus genome indeed existed in the patient samples rather than being artificially generated by misassembly. The new virus is provisionally designated NIH-CQV. Further analysis revealed that the contig was composed of two major open reading frames (ORF). Protein Blast showed that ORF1 encoded a protein that contained a conserved P-loop NTPase domain, homologous to the replication-associated protein of bat circovirus (E score=4e-04). ORF2 was homologous to capsid protein of porcine parvovirus (E scores=7e-06). Phylogenetic analysis indicated that the NIH-CQV represents a new subfamily of parvovirus, located at the interface of Parvoviridae and Circoviridae (Figure 1). Prevalence of the NIH-CQV in hepatitis patients was investigated by qPCR. Sixty three out of 92 (69%) patient samples were positive, while all 45 healthy controls were negative. The average virus titer in the patients was 1.28 E4 copies/ul, and the highest one was 3.2 E4 copies/ul. Specific antibodies against NIH-CQV were sought by immunoblot using a recombinant capsid protein. No cross reactivity was detected between the capsid protein of NIH-CQV and other major human parvoviruses. Eighty five percent (78/92) of patients were positive for IgG, and 32% (29/92) of them were positive for IgM. In contrast, 78% (35/45) of healthy controls were positive for IgG and 16% (7/45) were positive for IgM. Viral particles were purified from IgM-positive patient plasma by ultracentrifugation through a 40% sucrose cushion and examined by electron microscopy: spherical, naked, parvovirus-like particles approximately 26–29 nm in diameter were visualized. There was no correlation between clinical diagnosis and the presence or absence of the viral DNA or specific antibodies. Although more work is needed to determine the etiologic role of NIH-CQV in human disease, our data indicate that a novel parvovirus-like virus is highly prevalent in a cohort of patients with seronegative hepatitis. Figure 1, whole-proteome tree of the new parvovirus and members of the families Parvoviridae and Circoviridae. Figure 1,. whole-proteome tree of the new parvovirus and members of the families Parvoviridae and Circoviridae. Disclosures: No relevant conflicts of interest to declare.

Objective Most continuous cell lines with erythroid characteristics are derived from patients with myelogenous leukemia or erythroleukemia. Among them, a few cell lines have been reported to be positive for CD36. We tried to establish a continuous erythroid cell line from the primary CD36 + erythroid progenitor cells (EPCs) by the lentivirus-mediated gene transduction system. Materials and Methods A lentiviral vector carrying SV40T , hTERT, or the human papillomavirus type 16 (HPV16) E6 and E7 ( E6 / E7 ) viral oncogenes, was introduced into CD36 + EPCs, singularly or combined. Transformed cells were characterized in terms of histology, phenotype, karyotype, and gene expression profile. Results The lentiviral vector carrying HPV16 E6/E7 genes successfully transformed CD36 + EPCs, creating a continuous cell line, CD36E. Immunophenotype analysis revealed that the CD36E cells had characteristics of erythroid progenitors, among which about 27% of the cell population produced hemoglobin. Colony-forming cell assay demonstrated that the CD36E cells were capable of forming erythroid colonies. Using cytokines or chemical agents, attempts were made to induce differentiation of the CD36E cells but were ineffective, indicating the irreversible erythroid lineage commitment of the cells. The gene expression profile of the CD36E cells displayed a marked difference from that of the CD36 + EPCs. Conclusions The continuous CD36E cell line is an erythroid progenitor cell line possessing the ability to produce hemoglobin. The CD36E cell line would be an excellent tool for applied research involving erythroid lineage cells and comparative studies with primary CD36 + EPCs.

Three-dimensional (3D) reconstruction of organs and tissues is a powerful tool to establish anatomical and functional relationships of microscopic structures. We developed whole-mount tissue processing methods for 3D in situ visualization of murine and human bone marrow; our methods are compatible with fluorescent labeling of different cell types and other structures of interest in the tissue microenvironment. The major technical problems addressed were the conditions for tissue fixation in the absence of permeabilization and sectioning; antibody penetration and binding; and the acquisition of high quality images by adequate laser scanning confocal microscope. For murine bone marrow, the sternum was bisected sagitally; for human tissue, 2–3 mm fragments of core biopsies were utilized. Bone marrow tissue and cells were exposed to fluorescence labeled nucleic acid dyes and antibodies, with or without prior chemical fixation. Single and double labeling of cells was feasible with combinations of various antibodies and direct and indirect immunofluorescent techniques. In some experiments, cells were visualized from transgenic mice with cell populations expressing green fluorescence protein (GFP). Series of two dimensional (xy) images 600 μm × 600 μm were collected along the z-axis at 5 μm z-intervals to depths of 60–100 μm using a Zeiss LSM 510 confocal microscope. Two dimensional images were assembled to reconstruct 3-dimensional volumes by Bitplane’s Imaris 3D computer software. Antigenicity was preserved, allowing simultaneous labeling of cell types and structures by immunohistochemistry or nuclear dyes. Different hematopoietic cell types as well as blood vessels, adipose cells, and extracellular matrix were visualized in complex 3-dimensional organization of intact bone marrow tissue revealing unknown features of multicellular architecture. Normal murine bone marrow, after brief fixation formaldehyde, is shown in Figure A. Rat anti-mouse basement-membrane monoclonal antibody (MAb) and fluorescent isothiocyanate (FITC)-labeled donkey anti-rat monoclonal antibody were used to visualize the extracellular matrix and micro-vessels (appearing green). Allophycocyanin (APC)-labeled rat anti-mouse CD45R cells permitted visualization of B lymphocytes (red). 4’,6-diamidino-2-phenylindole(DAPI) stained all nuclei (blue). Nests of lymphocytes appeared encased by extracellular matrix, fed by microvessels running from the bone edge. An example of the architecture of a human hematologic malignancy is shown in figure B, from a marrow biopsy of a patient with multiple myeloma prior to therapy. Mouse anti-human CD20 MAb and FITC-labeled donkey anti-mouse IgG were used to visualize mature B cells (green). APC-conjugated mouse anti-human CD38 MAb identified plasma cells (red). DAPI stained nuclei (blue). The large tumor cells appeared in unevenly distributed cell clumps. In mouse experiments, (not illustrated), marrow cells were easily observed in animals in which GFP was driven by the ubiquitin-C promoter. In humans (also not illustrated), we observed malignant cell populations stained with appropriate lineage-specific antibodies in patients with leukemia and compared CD34 cell numbers in normal with aplastic bone marrow. Confocal laser scanning microscopy, a powerful technique to generate serial sections of whole-mount tissue and their digital reassembly into virtual 3-dimensional structures, has been readily adapted to examination of murine and human bone marrow. The wide variety of MAbs available for specific antigens in combination with this imaging method should aid in conceptualizing microanatomical relationships among hematopoietic cells, stroma, blood vessels, and extracellular matrix in normal and diseased bone marrow. Figure Figure

Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne zoonosis caused by a strain of Anaplasma phagocytophila called the HGE agent, an obligatory intracellular bacterium. The agent expresses immunodominant 44-kDa outer membrane proteins (P44s) encoded by a multigene family. The present study established an experimental process for transmission of the HGE agent from infected mice (a reservoir model) to nymphal Ixodes scapularis ticks (a biological vector) and subsequently to horses (a patient model) by the adult infected ticks. Overall, a total of 20 different p44 transcripts were detected in the mammals, ticks, and cell cultures. Among them, a transcript from a p44 - 18 gene was major at acute stage in mice and horses but minor in ticks. Both mRNA and protein produced from the p44 - 18 gene were detected in the HGE agent cultivated in HL-60 cells at 37°C, but their expression levels decreased in the organisms cultivated at 24°C, suggesting that temperature is one of the factors that influence the expression of members of the p44 multigene family. Several additional p44 transcripts that were not detected in the mammals at the acute stage of infection were detected in ticks. Phylogenetic analysis of the 20 different p44 transcripts revealed that the major transcripts found in mammals and ticks were distinct, suggesting a difference in surface properties between populations of the HGE agent in different host environments. The present study provides new information for understanding the role of the p44 multigene family in transmission of the HGE agent between mammals and ticks.

Activated oncogene c-Ha-ras in stomach cancer tissues was identified by DNA transfection. The point mutations were determined in five DNA samples (from three solid tumors of patients with gastro carcinoma and two cell lines) out of twelve (eight solid tumors and four cell lines) by the restriction fragment length polymorphysm (RFLP) assay. The expressions of c-Ha-ras were enhanced to higher levels (10–20 times) in two cell lines. The lengths of variable tandem repetition (VTR) sequences downstream from the c-Ha-ras coding region were compared for different DNA samples. Their ability to enhance c-Ha-ras expression and their transforming ability are discussed.