A novel 86-bp indel of the motilin receptor gene is significantly associated with growth and carcass traits in Gushi-Anka F2 reciprocal cross chickens.

1. A previous whole-genome association analysis has identified the motilin receptor gene (MLNR), which regulates gastrointestinal motility and gastric emptying, as a candidate gene related to chicken growth.2. MLNR mRNA was expressed in all tissues tested, and the expression level in digestive tissues was greater than in other tissues. Expression levels in the pancreas, duodenum and glandular stomach at day old and one, two and three weeks of age indicated a possible correlation with the digestive system. This suggested that the MLNR gene plays a central role in gastrointestinal tract function and affects the growth and development of chickens. Moreover, there was a significant difference in expression in the glandular stomach tissue between Ross 308 and Gushi chickens at six weeks of age.3. Re-sequencing revealed an 86-bp insertion/deletion polymorphism in the downstream region of the MLNR gene. The mutation locus was genotyped in 2,261 individuals from nine different chicken breeds. MLNR expression levels in the glandular stomach of chickens with DD genotypes were greater than those in chickens with the ID and II genotypes. The DD genotype was the most dominant genotype in commercial broiler's (Ross 308 and Arbor Acres broilers), and the D allele frequency in these breeds exceeded 91%. The deletion mutation tended towards fixation in commercial broilers.4. Association with growth and carcass traits analysed in a Gushi-Anka F2 intercrossed population, showed that the DD genotype was significantly associated with the greatest growth and carcass trait values, whereas values associated with the II genotype were the lowest in the F2 reciprocal cross chickens.5. The results suggest that the mutation is strongly associated with growth related traits and it is likely to be useful for marker-assisted selection of chickens.

A cryptic paracentric inversion of MSH2 exons 2-6 causes Lynch syndrome.

Lynch syndrome is an autosomal dominant disorder that predisposes carriers of DNA mismatch repair (MMR) gene mutations to early-onset cancer. Germline testing screens exons and splice sites for mutations, but does not examine introns or RNA transcripts for alterations. Pathogenic mutations have not been detected in ~30% of suspected Lynch syndrome cases with standard screening practices. We present a 38-year-old male with a clinicopathological and family history consistent with Lynch syndrome, including loss of MSH2 expression in his tumor. Germline testing revealed normal MSH2 coding sequence, splice sites and exon copy number, however, cDNA sequencing identified an aberrant MSH2 transcript lacking exons 2-6. An inversion PCR on germline DNA identified an ~18kb unbalanced, paracentric inversion within MSH2, with breakpoints in a long terminal repeat in intron 1 and an Alu repeat in intron 6. The 3' end of the inversion had a 1.2 kb deletion and an 8 bp insertion at the junction with intron 6. Screening of 55 additional Australian patients presenting with MSH2-deficient tumors who were negative in germline genetic tests for MSH2 mutations identified another inversion-positive patient. We propose an Alu-mediated recombination model to explain the origin of the inversion. Our study illustrates the potential value of cDNA screening to identify patients with cryptic MMR gene rearrangements, clarifies why standard testing may not detect some pathogenic alterations, and provides a genetic test for screening individuals with suspected Lynch syndrome that present with unexplained MSH2-deficient tumors.

Increased expression of T cell immune response cDNA 7 in patients with acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) has become the important complication post-allogeneic hematopoietic stem cell transplantation. Abnormally activated T cells might play an important role in the pathogenesis of aGVHD. But its exact mechanism remains poorly understood. T cell immune response cDNA 7 (TIRC7) has been identified to be essential in T cell activation; however, the role of TIRC7 in aGVHD remains unclear. The purpose of this study was to measure the expression of TIRC7 and T helper (Th) cells in patients with aGVHD before and after treatment. We showed that TIRC7 levels in aGVHD patients were higher than those of healthy controls and markedly declined after treatment. The levels of IFN-γ (Th1), IL-17 (Th17), and IL-22 (Th22) were in accordance with the grade of aGVHD. In addition, TIRC7 levels were also associated with the severity of aGVHD. In conclusion, TIRC7 might be involved in the pathogenesis of aGVHD and TIRC7 level might be an indicator to evaluate the response of patients with aGVHD to treatment.

A combination of Flt3 ligand cDNA and CpG oligodeoxynucleotide as nasal adjuvant elicits protective secretory-IgA immunity to Streptococcus pneumoniae in aged mice.

Our previous study showed that a combination of a plasmid-expressing Flt3 ligand (pFL) and CpG oligodeoxynucleotides (CpG ODN) as a combined nasal adjuvant elicited mucosal immune responses in aged (2-y-old) mice. In this study, we investigated whether a combination of pFL and CpG ODN as a nasal adjuvant for a pneumococcal surface protein A (PspA) would enhance PspA-specific secretory-IgA Ab responses, which could provide protective mucosal immunity against Streptococcus pneumoniae infection in aged mice. Nasal immunization with PspA plus a combination of pFL and CpG ODN elicited elevated levels of PspA-specific secretory-IgA Ab responses in external secretions and plasma in both young adult and aged mice. Significant levels of PspA-specific CD4(+) T cell proliferative and PspA-induced Th1- and Th2- type cytokine responses were noted in nasopharyngeal-associated lymphoreticular tissue, cervical lymph nodes, and spleen of aged mice, which were equivalent to those in young adult mice. Additionally, increased numbers of mature-type CD8, CD11b-expressing dendritic cells were detected in mucosal inductive and effector lymphoid tissues of aged mice. Importantly, aged mice given PspA plus a combination of pFL and CpG ODN showed protective immunity against nasal S. pneumoniae colonization. These results demonstrate that nasal delivery of a combined DNA adjuvant offers an attractive possibility for protection against S. pneumoniae in the elderly.

Direct loop-mediated isothermal amplification from Plasmodium chabaudi infected blood samples: inability to discriminate genomic and cDNA sequences.

Loop-mediated isothermal amplification (LAMP) has been increasingly used for diagnosis and quantification of pathogens. Since the Bst DNA polymerase used in this assay is highly resistant to PCR inhibitors present in blood, direct analysis of blood samples without DNA or RNA extraction is possible. Indeed, the presence of Plasmodium chabaudi specific nucleic acids was easily detectable using primer sets for P. chabaudi 18S rRNA and the cir 1 mRNA. Despite the fact that primers for cir 1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and actin II mRNAs were used that spanned an intron, selective amplification of mRNA in the presence of contaminating genomic DNA was not possible. Optimization of the reaction temperature could only improve discrimination when low complexity templates (target sequences cloned in a plasmid vector) were used. Placing different LAMP primers across intron exon boundaries did not prevent amplification in the absence of reverse transcriptase. Probably due to the high A+T content and low number of introns only a very limited number of possible primer sets spanning introns could be identified in the target genes and no reaction conditions could be established that would allow quantification of RNA levels in the presence of DNA directly from blood samples.

Safety levels of systemic IL-12 induced by cDNA expression as a cancer therapeutic.

Aim: The aim of this work is to utilize a gene expression procedure to safely express systemic IL-12 and evaluate its effects in mouse tumor models. Materials & methods: Secondary lymphoid organs and tumors from EL4 and B16 tumor-bearing mice were analyzed by supervised and unsupervised methods. Results: IL-12 cDNA induced systemic IL-12 protein levels lower than the tolerated dose in patients. Control of tumor growth was observed in subcutaneous B16 and EL4 tumors. Systemic IL-12 expression induced a higher frequency of both total tumor-infiltrated CD45+ cells and proliferative IFN-γ+CD8+ T cells along with a lower frequency of CD4+FOXP3+ and CD11b+Gr-1+ cells. Conclusion: This approach characterizes the systemic effects of IL-12, helping to improve treatment of metastases or solid tumors.

Lay abstract IL-12 has emerged as a potent cytokine in mediating antitumor activity in preclinical models of cancer. However, this antitumor response has not yet been translated into the clinic because of toxic side effects. The aim of our work is to analyze the effects of IL-12 in mouse tumor models. We demonstrate that one injection of IL-12 cDNA can induce systemic IL-12 levels in serum even lower than the tolerated dose in patients. At this dose, an efficient control of tumor growth can be observed. We found a higher frequency of both total tumor-infiltrated leukocytes and IFN-γ-producing CD8⁺ T cells along with a lower frequency of regulatory CD4⁺FOXP3⁺ and CD11b⁺Gr1⁺ cells. Our work demonstrates that IL-12 cDNA can safely be used to treat cancer.

Feline adiponectin: molecular structures and plasma concentrations in obese cats.

Adiponectin is an adipokine that is specifically expressed in adipose tissues, directly sensitizes the body to insulin via specific receptors and its decreased plasma concentration is responsible for insulin resistance in obese humans. Diabetes is an important problem also in veterinary medicine, and feline diabetes is very similar to human type 2 diabetes, in which obesity is an important risk factor. In the present study, We obtained cDNA clones corresponding to feline adiponectin and adiponectin receptor 1 (AD-R1), whose nucleotide and deduced amino acid sequences were highly identical to those of other species, especially, the extra-cellular domain of feline AD-R1 was almost identical to that of human AD-R1. Adiponectin mRNA was exclusively detected in the adipose tissue, but AD-R1 was in all tissues tested in this study. Next, plasma samples were collected from 22 cats visiting veterinary practices. They were divided to 2 groups based on a five-point scale body condition score (BCS), such as normal group (BCS ranged from 2.5 through 3.5) and obese group (BCS ranged from 4.0 through 5.0). Plasma adiponectin in obese cats (7.2 +/- 1.5 microg/ml) was significantly lower than that of normal cats (18.0 +/- 3.2 microg/ml). These results suggest that adiponectin may be responsible for insulin function also in the cat, and it can be a target molecule for treatment of obesity and diabetes in cats.

Comprehensive cDNA study and quantitative transcript analysis of mutant OPA1 transcripts containing premature termination codons.

Autosomal dominant optic atrophy (adOA) is most commonly caused by mutations in the OPA1 gene. There is a considerable allelic heterogeneity among adOA-associated OPA1 mutations, however these mutations have mostly been identified and studied only at the genomic DNA level. Here we report the identification of 22 novel OPA1 mutations and their analysis at the cDNA level along with 15 already known OPA1 mutations. We found that 18 of these mutations cause splice defects that involve either skipping of the adjacent exon or the activation of a cryptic splice site. We also observed a reduced level of the mutant transcript in several adOA subjects. Allele-specific quantification of the transcript steady-state level was performed for 13 different OPA1 mutations applying pyrosequencing to a RT-PCR amplified cSNP (c.2109C>T) in OPA1. Using this new assay we could demonstrate that the majority of OPA1 mutations that lead to a premature termination codon (PTC) undergo nonsense-mediated mRNA decay (NMD). Mutant transcript levels were reduced between 1.25- and 2.5-fold and varied between PTC containing mutations, and between subjects. Our results emphasize the value of cDNA analysis in the characterization of OPA1 mutations and further strengthen the model of haploinsufficiency as a major pathomechanism in OPA1-associated adOA.

Detection of HIV antigen and cDNA among antibody-negative blood samples in Nigeria.

In developing countries as many as 50% of patients for whom a transfusion is indicated are at risk of dying immediately if transfusion is withheld. It is therefore important that blood transfusion is made as safe as possible. This study was designed to assess the safety of blood transfusion in two large blood banks in Ibadan, Nigeria. Aliquots of 250 samples already screened and passed as negative for HIV-1 and -2 were collected from each of the blood banks. Samples were tested for the presence of HIV-1 antigen (ELAVIA Ag I) and the antigen-positive samples tested for the presence of specific HIV-1 antibodies by Western blot (BioRad, France). All antigen-positive samples were also subjected to PCR. HIV-1 antigen was detected in 6 (1.2%) of the 500 samples, of which 4 (0.8%) and 3 (0.6%) were Western blot-indeterminate and PCR-positive, respectively. Transfusion of HIV-contaminated blood may be contributing significantly to the spread of the virus in Nigeria. There is therefore an urgent need for an organized blood-banking system with facilities for more sensitive assays for the detection of HIV in blood to prevent transmission through transfusion.

Sensitization against anticancer drugs by transfection with UBE2I variant gene into ras-NIH3H3 mouse fibroblasts.

We previously performed SEREX (serological identification of antigens by recombinant expression cloning) using the sera of patients with esophageal squamous cell carcinoma (SCC), and isolated a variant clone (AK093616) of ubiquitin-conjugating enzyme E21 (UBE2I). This clone was tentatively designated as UBE2I-v5 and analyzed for biological function by transient transfection of the cDNA into activated Ha-ras-transformed NIH3T3 (ras-NIH) mouse fibroblasts. Chemosensitivity to 92 cytotoxic drugs was compared between UBE2I-v5-transfected cells and the parental ras-NIH cells. The UBE2I-v5-transfected cells were more sensitive than the parental cells to anticancer drugs such as vincristine (VCR), mitoxantrone (MIT) and etoposide (VP16). The regression analysis of the total chemosensitivity pattern of UBE2I-vS-transfected cells revealed that the function of UBE2I-v5 was positively related to RPA2 (replication protein A2), Rho-GDI (Rho guanine nucleotide dissociation inhibitor a), FUS (putative tumor suppressor) and TKT (transketolase) but negatively related to Per-1 (period-I), Ran (nuclear Ras-related protein), PTEN (phosphatase and tensin homolog), C/EBPalpha (CCAAT/enhancer binding protein a) and the tumor suppressor p53. Thus, it is possible that UBE21-v5 plays a role in carcinogenesis by suppressing the function of CIEBPa and/or p53 via RPA2-like activity.

Two functional assays employed to detect an unusual mutation in the oligomerisation domain of p53 in a Li-Fraumeni like family.

Previous investigations of a Li - Fraumeni like family (Barnes et al., 1992) demonstrated that both the proband and her mother had elevated p53 protein levels in both tumour tissue and normal tissue at sites distant from the tumour, although no mutation was found in the p53 gene. In the present study two recently described functional assays for p53, an apoptotic assay and the functional assay for the separation of alleles in yeast (FASAY), have been employed to study the functional activity of p53 from this patient. The results of the apoptotic assay demonstrated that this patient had a p53 functional defect and the FASAY result suggested that this defect was in fact a germline mutation of the p53 gene. A point mutation of codon 337, which results in an amino acid substitution of a cysteine for an arginine, was demonstrated initially in cDNA and was confirmed by sequencing of genomic DNA. This is an unusual mutation as it is in the oligomerisation domain of p53, in contrast to the majority of p53 mutations which are in the core DNA binding domain. This mutation results in a protein which still retains partial transactivational activity in the FASAY. The mutation of codon 337 is only the second reported case of a germline missense mutation occurring in the oligomerisation domain of p53.

Differential expression of carcinoembryonic antigen (CEA) splice variants in whole blood of colon cancer patients and healthy volunteers: implication for the detection of circulating colon cancer cells.

Quantification of circulating cancer cells in whole blood samples by real time quantitative RT-PCR might be of clinical value for monitoring therapeutic effectiveness. In colon cancer patients, carcinoembrynic antigen (CEA) and cytokeratin 20 (CK20) have been frequently used for RT-PCR based tumor cell detection, but the specificity in particular for CEA has been questioned. In this study, we compared real-time RT-PCR for CEA and CK20 and analysed patients with metastatic disease (n=32) and healthy volunteers (n=17). CK20 mean values were elevated in cancer patients (P<0.001) and defined a subgroup (38%) who showed CK20 levels at least 100-fold above the highest value of the healthy control group. In contrast, only two cancer patients (6%) showed elevated CEA levels. Samples of the healthy control group showed exclusively a CEA-PCR product of 79 degrees C melting temperature. Thirty per cent of the colon cancer patients showed an additional product of 82 degrees C melting temperature. The 82 degrees C product was identical with the amplification product of CEA-cDNA and cDNA from different colon cancer cell lines. Colon cancer cells were spiked into normal blood in 10-fold dilutions that resulted in a dose dependent shift of the melt curve from 79 degrees C to the 82 degrees C. Sequencing of the PCR products showed that white blood cells express a splice variant of CEA, which hinders detection of tumor cell cDNA in whole blood samples. Our findings have implications for the use of CEA as a diagnostic molecule (e.g. by RT-PCR). The discovery of a physiologically expressed CEA splice variant might lead to a better understanding of the biological function of CEA and its family members.

Inducible and reversible lentiviral and Recombination Mediated Cassette Exchange (RMCE) systems for controlling gene expression.

Manipulation of gene expression to invoke loss of function (LoF) or gain of function (GoF) phenotypes is important for interrogating complex biological questions both in vitro and in vivo. Doxycycline (Dox)-inducible gene expression systems are commonly used although success is often limited by high background and insufficient sensitivity to Dox. Here we develop broadly applicable platforms for reliable, tightly controlled and reversible Dox-inducible systems for lentiviral mediated generation of cell lines or FLP Recombination-Mediated Cassette Exchange (RMCE) into the Collagen 1a1 (Col1a1) locus (FLP-In Col1a1) in mouse embryonic stem cells. We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs. We demonstrate the usefulness of these platforms in ectopic gene expression or gene knockdown in multiple cell lines, primary neurons and in FLP-In Col1a1 mouse embryonic stem cells. We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs. These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function.

Autoantibodies against topoisomerase I detected with the natural enzyme and overlapping recombinant peptides.

Antibodies against topoisomerase I (anti-topo I, anti-Scl-70) are regarded as a marker of systemic sclerosis. The various frequencies of anti-topo I detected in those patients depends at least in part on the test design and the kind of the antigen used. We therefore analyzed three overlapping recombinant topo I fragments (N-terminal, center and C-terminal part of the molecule) covering the full length of the enzyme for substitution of highly purified natural antigens (n-topo I) in ELISA for antibody screening. 49 of 50 sera reacting with n-topo I in ELISA also recognized the recombinant C-terminal topo I fragment under identical test conditions, 37 sera recognized the recombinant center and two sera the recombinant N-terminal peptide. All sera reactive with the N-terminal and center peptide reacted with the recombinant C-terminus which therefore may substitute for the natural antigen. In immunoblot assays 92% (46/50) of the sera reacted with n-topo I and 86% (43/50) with the recombinant C-terminal peptide. Immunoblots therefore seem to be less sensitive for detecting anti-topo I antibodies than ELISA regardless the source of the antigen used. In a screening of 696 sera submitted for routine antibody tests the recombinant peptide ELISA on the other hand detected two sera which did not react with n-topo I in ELISA. Because of the high rate of agreement within the results obtained with the two antigens, n-topo I can be substituted by the recombinant peptide ELISA allowing better standardization and interlaboratory comparison of results.

Expression of nitric oxide synthase in human peripheral blood mononuclear cells and neutrophils.

It has been clearly demonstrated in rodents that nitric oxide (NO) plays an important role in host defense and immunity. However, evidence that human leukocytes express inducible nitric oxide synthase (iNOS) or its products has been inconclusive and a source of controversy. We report that iNOS could not be detected in human monocytes, HL-60 cells, neutrophils, and T cells by Western blotting analysis (< or = 10 pg) or by radiolabeled L-arginine-to-L-citrulline conversion (< or = 20 pmol L-citrulline) under conditions sufficient to induce iNOS in the rodent system and in human hepatocytes, which include activation with cytokines, endotoxins, and/or chemoattractants. However, sensitive methods such as RT-PCR and Northern blot analysis show "constitutively expressed" iNOS mRNA from human monocytes, neutrophils, Jurkat cells, and HL-60 cells. This iNOS mRNA is 4.4 kb and is similar to that seen in human hepatocytes and rodent macrophages. In spite of the constitutive expression of mRNA in neutrophils and the lack of detectable NOS activity (based on Western blotting and L-arginine-to-L-citrulline conversion assay), stimulation of human neutrophils unit FMLP in vitro induced the ADP-ribosylation of an intracellular NO target, glyceraldehyde-3-PO4 dehydrogenase (GAPDH), in a NO-dependent manner. These studies indicate that low levels of NOS protein are expressed in neutrophils (and perhaps T cells and monocytes) and produce NO following stimulation. The data indicate that, in addition to its phagocytic and tumoricidal activity. NO may also function as an autacoid signaling molecule within the cells.

Nonradiometric ELISA-based quantitation and validation of polymerase chain reaction-amplified DNA, including detection of point mutations, without allele-specific amplification, or ligation.

We describe two simple novel procedures, one direct and the other involving hybridization, for the enzyme-linked immunosorbent assay (ELISA)-based detection, quantitation, and validation of polymerase chain reaction (PCR)-amplified DNA. Both procedures are applicable to any PCR reaction, and do not require specially synthesized or enzyme-tagged oligonucleotides. We obtained accurate quantitation of PCR-amplified human cc10kDa cDNA with a sensitivity of about 0.6 fmoles. This cDNA was also used to detect single-base insertions, deletions, and substitutions specifically. Additionally, we could readily distinguish zinc-finger Y chromosome-specific genomic sequences in mixtures of male and female cells and normal and mutant cystic fibrosis transmembrane conductance regulator (CFTR) gene sequences in crude fibroblast lysates. To our knowledge, this is the first report of point mutation detection in solution by ELISA without allele-specific amplification or ligation. These novel procedures have vast potential for basic and clinical applications, including gene expression studies, rapid screening of genetic diseases, detection of oncogene and anti-oncogene mutations, and identification of pathogens (e.g., HIV-1) in clinical specimens.

Minimal illegitimate levels of cytokeratin K19 expression in mononucleated blood cells detected by a reverse transcription PCR method (RT-PCR).

Cytokeratin K19 (CK19) expression was evaluated by a reverse transcription PCR method (RT-PCR) in the RNA obtained from peripheral blood stem cell collections (PBSC) from four patients with breast cancers (BC) and 34 mononucleated blood cell (MBC) negative controls (17 PBMC from normal subjects 12 PBSC from different types of leukaemias--M3, M4Eo, M2, etc.--and two from patients with Hodgkin's lymphoma; and three bone marrow (BM) collections). Two BC tissues were taken as positive controls. The method studied (Datta YH, Paul T, Adams PT, Drobyski WR. Sensitive detection of occult breast cancer by reverse transcription polymerase chain reaction. J Oncol 1994;12:475-8) is sensitive enough to allow the detection of CK19 transcripts in a 10(-6) dilution of cDNA reverse transcribed from 1 microgram of BC RNA, but CK19 transcripts were also detected in 64% of the RNA obtained from the MBC controls. However, the amplified product detected in the control samples represents the transcript of the CK19 gene as confirmed by the results of Mae III digestion. It should be pointed out that although the CK19 expression was detected, the levels of expression in PBMC were almost negligible for they disappeared at 1:5 cDNA dilution. Moreover, a direct relationship between the number of BC cells added to PBMC and the increasing dilution levels of the cDNA necessary to prevent CK19 expression was observed. This allows us to conclude that the cDNA dilutions make it possible to distinguish the false from the true positive samples and that, in addition, the cDNA dilutions inform about the degree of BC cell contamination.

Plasma cell-free DNA and its DNA integrity as biomarker to distinguish prostate cancer from benign prostatic hyperplasia in patients with increased serum prostate-specific antigen.

OBJECTIVES: To investigate whether plasma cell-free DNA (cfDNA) or its integrity could differentiate prostate cancer from benign prostate hyperplasia (BPH) in patients with serum prostate-specific antigen (PSA) ≥ 4 ng/ml. METHODS: Ninety-six patients with prostate cancer and 112 patients with BPH were enrolled. cfDNA levels in plasma before prostate biopsy were quantified by real-time PCR amplification of ALU gene (product size of 115 bp), and quantitative ratio of ALU (247 bp) to ALU (115 bp) reflected the integrity of cfDNA. RESULTS: In patients with serum PSA ≥ 4 ng/ml, there were significant differences in plasma cfDNA or its integrity between the patients with prostate cancer (19.74 ± 4.43, 0.34 ± 0.05) and patients with BPH (7.36 ± 1.58, 0.19 ± 0.03; P < 0.001, P < 0.001). Prostate cancer could be differentiated with a sensitivity of 73.2 % and a specificity of 72.7 % by cfDNA (AUC = 0.864). The integrity of cfDNA had a sensitivity of 81.7 % and a specificity of 78.8 % for the distinguishing prostate cancer from BPH (AUC = 0.910). CONCLUSIONS: cfDNA and its integrity could be applied to differentiate prostate cancer from BPH in patients with serum PSA ≥ 4 ng/ml.

Differential innate immune responses to low or high dose oral SIV challenge in Rhesus macaques.

Mucosal transmission of HIV predominately occurs during sexual intercourse or breast-feeding and generally results in a successful infection from just one or few founder virions. Here we assessed the impact of viral inoculum size on both viral and immune events within two groups of Rhesus macaques that were non-traumatically, orally inoculated with either multiple low (1000 to 4000 TCID(50)) or high (100,000 TCID(50)) doses of SIV. In agreement with previous studies, more diverse SIV variants were observed in macaques following infection with high dose oral SIV compared to a low dose challenge. In peripheral blood cells, the immune gene transcript levels of CXCL9, IFNγ, TNFα and IL10 remained similar to uninfected macaques. In contrast, OAS and CXCL10 were upregulated following SIV infection in both the high and low dosed macaques, with a more rapid kinetics (detectable by 7 days) following the high SIV dose challenge. In peripheral lymph nodes, an increase in CXCL10 was observed irrespective of viral dose while CXCL9 and OAS were differentially regulated in the two SIV dosed groups. Magnetic bead sorting of CD3+, CD14+ and CD3- /CD14- cells from peripheral blood identified the increase in OAS expression primarily within CD14+ monocytes, whereas the CXCL10 expression was primarily in CD3+ T cells. These findings provide insights into the impact of SIV challenge dose on viral and innate immune factors, which has the potential to inform future SIV/HIV vaccine efficacy trials in which vaccinated hosts have the potential to be infected with a range of viral challenge doses.

Transcription of Alu DNA elements in blood cells of sporadic Creutzfeldt-Jakob disease (sCJD).

Alu DNA elements were long considered to be of no biological significance and thus have been only poorly defined. However, in the past Alu DNA elements with well-defined nucleotide sequences have been suspected to contribute to disease, but the role of Alu DNA element transcripts has rarely been investigated. For the first time, we determined in a real-time approach Alu DNA element transcription in buffy coat cells isolated from the blood of humans suffering from sporadic Creutzfeldt-Jakob disease (sCJD) and other neurodegenerative disorders. The reverse transcribed Alu transcripts were amplified and their cDNA sequences were aligned to genomic regions best fitted to database genomic Alu DNA element sequences deposited in the UCSC and NCBI data bases. Our cloned Alu RNA/cDNA sequences were widely distributed in the human genome and preferably belonged to the "young" Alu Y family. We also observed that some RNA/cDNA clones could be aligned to several chromosomes because of the same degree of identity and score to resident genomic Alu DNA elements. These elements, called paralogues, have purportedly been recently generated by retrotransposition. Along with cases of sCJD we also included cases of dementia and Alzheimer disease (AD). Each group revealed a divergent pattern of transcribed Alu elements. Chromosome 2 was the most preferred site in sCJD cases, besides chromosome 17; in AD cases chromosome 11 was overrepresented whereas chromosomes 2, 3 and 17 were preferred active Alu loci in controls. Chromosomes 2, 12 and 17 gave rise to Alu transcripts in dementia cases. The detection of putative Alu paralogues widely differed depending on the disease. A detailed data search revealed that some cloned Alu transcripts originated from RNA polymerase III transcription since the genomic sites of their Alu elements were found between genes. Other Alu DNA elements could be located close to or within coding regions of genes. In general, our observations suggest that identification and genomic localization of active Alu DNA elements could be further developed as a surrogate marker for differential gene expression in disease. A sufficient number of cases are necessary for statistical significance before Alu DNA elements can be considered useful to differentiate neurodegenerative diseases from controls.