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R J Cooper

Publications and source records attributed to R J Cooper.

At least 37 records · Page 2Linked to original sources
PubMed2000

PCR and restriction endonuclease analysis for rapid identification of human adenovirus subgenera.

Subgenus identification of adenoviruses is of clinical importance and is as informative as identification by serotype in most clinical situations. A PCR-based identification of adenovirus subgenera A, B, C, D, E, and F and sometimes serotypes is described. The PCR uses nonnested primer pair ADRJC1-ADRJC2, which targets a highly conserved region of the adenovirus hexon gene, has a sensitivity of 10 to 40 copies of adenovirus type 2 (Ad2) DNA, and generates 140-bp PCR products from adenovirus serotypes representative of all the subgroups. The PCR products of all subgroups can be differentiated on the basis of the restriction fragment patterns produced by a total of five restriction endonucleases. In addition, serotypes Ad40 and Ad41 (subgroup F) and important serotypes of subgroup D (Ad8, Ad10, Ad19, and Ad37) can easily be differentiated, but serotypes within subgroups B and C cannot. The method was assessed by blind subgenus identification of 56 miscellaneous clinical isolates of adenoviruses. The identities of these isolates at the subgenus level by the PCR correlated 91% (51 of 56) with the results of serotyping by the neutralization test, and 9% (5 of 56) of clinical isolates produced discordant results.

Adenovirus Infections, Human↗
PubMed2000

Multiplex polymerase chain reaction for diagnosis of viral and chlamydial keratoconjunctivitis.

PURPOSE: To develop a multiplex polymerase chain reaction (PCR) for the detection of adenovirus, herpes simplex virus, and Chlamydia trachomatis in conjunctival swabs. METHODS: Oligonucleotide primers for detection of the 3 agents were combined in one reaction and evaluated for optimal performance using control DNAs of adenovirus type 2, herpes simplex virus, and C. trachomatis plasmid. The multiplex PCR was evaluated prospectively against its corresponding uniplex PCRs, virus isolation, Chlamydia Amplicor PCR, and an immunoassay technique (immune dot blot test) in a total of 805 conjunctival swabs from patients with suspected viral and chlamydial keratoconjunctivitis. RESULTS: The multiplex PCR was as sensitive as uniplex PCRs for the detection of the agents in clinical specimens. In the prospective study, 48 of 49 (98%) clinical specimens were positive for adenovirus by the multiplex PCR compared with 26 of 49 (53%) by adenovirus isolation. For herpes simplex virus detection, the multiplex PCR had a sensitivity of 92% (34/37) compared with 94.5% (35/37) by cell culture. The multiplex PCR produced identical results to the Amplicor PCR (21/21; 100%) compared with 71% (15/21) by the immune dot blot test. CONCLUSIONS: With clinical specimens the multiplex PCR was as sensitive as its respective uniplex PCRs but more sensitive than adenovirus isolation and as sensitive as herpes simplex virus isolation or C. trachomatis Amplicor PCR. It has the potential to replace several diagnostic tests with consequent savings in cost. The test also reduces the risk of misdiagnosis by the clinicians.

Adenovirus Infections, Human↗
PubMed1999

Costimulation of transduced T lymphocytes via T cell receptor-CD3 complex and CD28 leads to increased transcription of integrated retrovirus.

Primary human T lymphocytes were transduced at high efficiency with the Moloney murine leukemia virus (Mo-MuLV) vector, LNC-mB7-1, in which an internal cytomegalovirus (CMV) promoter drives expression of the murine B7-1 cDNA. Compared with transduced T cells expanded in IL-2 or reactivated with soluble antibodies to CD3 or CD28, transgene expression was significantly increased after activation on immobilized anti-CD3 antibodies (CD3i) or by simultaneous activation on immobilized anti-CD3 and anti-CD28 antibodies (CD3i/CD28i). A similar pattern of transgene expression was observed in T cells transduced with Mo-MuLV LNC-EGFP. Proviral copy number was maintained in LNC-mB7-1-transduced T cells expanded in IL-2 or reactivated on CD3i/CD28i. Substantial increases in LNC-mB7-1 steady state mRNA in reactivated T lymphocytes, compared with those maintained in IL-2, correlated with increased transcription of the LNC-mB7-1 proviral DNA. Furthermore, T cells transduced with the Mo-MuLV ZIPPGK-mADA, in which the mADA cDNA is driven by an internal human phosphoglycerate kinase (PGK) promoter, showed increases in steady state ZIPPGK-mADA RNA on reactivation. High levels of transgene expression were evident irrespective of cell cycle position in both CD4+ and CD8+ lymphocytes. After reactivation, increases in LNC-mB7-1 mRNA were observed in the presence of the protein synthesis inhibitor cycloheximide, indicating that proteins involved in upregulating transgene expression preexisted in transduced lymphocytes. Induction of transgene expression on CD3i/CD28i showed a dose-dependent decrease in transgene expression when incubated with selective protein kinase inhibitors. These data provide new insights into the mechanisms governing transgene expression driven by Mo-MuLV constructs containing internal promoters in transduced primary T lymphocytes.

Antibodies↗
PubMed1999

Effects of imposed feed intake variation on acidosis and performance of finishing steers.

Four metabolism and two finishing trials were conducted to determine the effects of imposed feed intake variation on acidosis and performance of finishing steers. In Metabolism Trial 1, four ruminally fistulated steers were limit-fed and subjected to either a constant amount of feed per day (C) or low intake variation of .7 kg/d (LV). No treatment differences were found for intake or measures of acidosis. Metabolism Trial 2 was conducted similarly to Metabolism Trial 1 with treatments of C and high intake variation of 1.4 kg/d (HV). Treatment HV increased (P < .05) acidosis, as indicated by the area of ruminal pH below 5.6. In Metabolism Trial 3, four steers were fed at ad libitum levels of intake and subjected to three levels of intake variation: ad libitum intake with no imposed intake variation (AL), LV of .7 kg/d, and HV of 1.4 kg/d. No treatment differences were found. In Metabolism Trial 4, six ruminally fistulated steers were fed at ad libitum levels and subjected to three levels of intake variation: AL, LV of .9 kg/d, and HV of 1.8 kg/d. Average ruminal pH increased (P < .05) and area of ruminal pH below 5.6 decreased (P < .05) as level of intake variation was increased. In Finishing Trial 1, 75 steers were assigned to eight pens and two treatments: AL or HV of 1.8 kg/d. Dry matter intake increased (P < .05) from AL to HV. Daily gain and gain/feed were not affected by treatment. In Finishing Trial 2, 94 steers were assigned to 12 pens and two treatments: AL or HV of 1.8 kg/d. No treatment differences were noted in DMI, daily gain, or gain/ feed. Therefore, results of these trials indicate that intake variation of up to 1.8 kg/d does not increase acidosis or decrease performance of finishing steers fed at ad libitum levels of intake.

Acidosis↗
PubMed1999

Adenovirus polymerase chain reaction assay for rapid diagnosis of conjunctivitis.

PURPOSE: To evaluate newly designed primers in a polymerase chain reaction (PCR) for the detection of adenovirus DNA in conjunctival swabs. METHODS: Oligonucleotides were derived from the adenovirus hexon gene and modified such that a maximum of only two mismatches occurred with adenovirus types 2 through 5, 7, and 16. Specificity was determined against adenovirus types 2 through 4, 7, 8 through 11, 14, 19, 37, 40, and 41 and from non-adenoviral DNA and the sensitivity by PCR amplification of purified adenovirus type 2 DNA. The assay was compared retrospectively with cell culture and a PCR with different primers on 59 stored conjunctival swab samples. The new PCR also was used prospectively in comparison with cell culture on 2743 conjunctival swabs. RESULTS: The 140-bp product was amplified from all the adenovirus serotypes tested except types 40 and 41, which have not been isolated from the eye. There were no amplified products from the non-adenoviral DNA tested. With adenovirus type 2 DNA, despite two deliberate mismatches, 40 copies of the target were detectable after PCR and ethidium bromide-staining. In the retrospective study, 51 of 55 (92.7%) were positive by this new PCR compared with 42 of 55 (76.4%) by the older PCR and 40 of 55 (72.7%) by cell culture. In the prospective study, the new PCR detected 386 of 415 (93%) adenovirus-positive specimens compared with 248 of 415 (59.8%) by cell culture. Of 167 specimens positive for herpes simplex virus by cell culture, none were positive by the adenovirus PCR. CONCLUSIONS: PCR with the newly designed primers shows a much increased sensitivity over cell culture and previous PCRs for the detection of adenoviruses in conjunctival swabs.

Adenovirus Infections, Human↗
PubMed1998

Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse as a model system to study the engraftment and mobilization of human peripheral blood stem cells.

Mobilized CD34(+) cells from human peripheral blood (PB) are increasingly used for hematopoietic stem-cell transplantation. However, the mechanisms involved in the mobilization of human hematopoietic stem and progenitor cells are largely unknown. To study the mobilization of human progenitor cells in an experimental animal model in response to different treatment regimens, we injected intravenously a total of 92 immunodeficient nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with various numbers of granulocyte colony-stimulating factor (G-CSF) -mobilized CD34(+) PB cells (ranging from 2 to 50 x 10(6) cells per animal). Engraftment of human cells was detectable for up to 6.5 months after transplantation and, depending on the number of cells injected, reached as high as 96% in the bone marrow (BM), displaying an organ-specific maturation pattern of T- and B-lymphoid and myeloid cells. Among the different mobilization regimens tested, human clonogenic cells could be mobilized from the BM into the PB (P = .019) with a high or low dose of human G-CSF, alone or in combination with human stem-cell factor (SCF), with an average increase of 4.6-fold over control. Therefore, xenotransplantation of human cells in NOD/SCID mice will provide a basis to further study the mechanisms of mobilization and the biology of the mobilized primitive human hematopoietic cell.

Animals↗
PubMed1996

Prolonged administration of granulocyte colony-stimulating factor (filgrastim) to patients with Fanconi anemia: a pilot study.

This report examines the effect of filgrastim (granulocyte colony-stimulating factor, [G-CSF] in 12 patients with neutropenia [absolute neutrophil count [ANC] < 1,000/mm3]) caused by Fanconi anemia (FA). Two of 14 patients who were evaluated for study entry were ineligible because of unsuspected cytogenetic abnormalities in their bone marrow (BM). G-CSF was started at 5 micrograms/kg/d. All patients had an increase in their ANC at week 8 (mean increase = 15,664/mm3). The median ANC during therapy was 5,030/mm3. Eight of 10 patients who completed 40 weeks on study maintained an ANC > 1,500/mm3 on G-CSF given every-otherday. Four patients had an increase in their platelet count by week 8 without transfusion (maximum increase = 23,000 to 45,000/mm3); however, platelet counts fell toward baseline levels as the G-CSF dose was reduced. BM CFU-MK were increased at week 8 in three of four evaluable patients. Four patients who did not receive red blood cell transfusions had an increase in their hemoglobin level of at least 2.0 g/dL. A fifth patient had a red blood cell transfusion in week 2 and then had a similar increase in hemoglobin level without subsequent transfusion. Eight of 10 patients who completed 40 weeks of treatment showed increases in the percentage of BM CD34+ cells measured by flow cytometry. The same proportion showed increases in peripheral blood CD34+ cells. Increased BM cellularity and myeloid hyperplasia were constant findings and were associated with increased expression of the proliferating cell nuclear antigen. Adverse experiences were mild fever (1 patient) and a new BM cytogenetic abnormality at week 40 (1 patient). This study shows that prolonged administration of G-CSF exerts a stimulatory effect on the BM of FA patients and may be used to maintain a clinically adequate ANC in these patients. G-CSF has beneficial effects on multiple hematopoietic lineages in some patients and may be a good candidate for use in combination cytokine protocols for FA patients with progressive aplastic anemia. G-CSF use results in an increase in circulating CD34+ cells, a finding with important implications for future gene transfer protocols.

Adolescent↗
PubMed1996

Comparison of primer sets for detection of fecal and ocular adenovirus infection using the polymerase chain reaction.

Adenoviruses of subgenus F (types 40 and 41) cause infantile gastroenteritis and adenoviruses principally of types 1-7 are found in feces during respiratory or generalized infections. Adenoviruses (mostly types 3, 4, 8, 19, or 37) are also linked with follicular or epidemic conjunctivitis. The diagnostic efficiency of the polymerase chain reaction (PCR) for adenoviruses was assessed using genus-reactive primers H1 and H2 or JCH1 and JCH2 or subgenus F-specific primers F1a and F2a. With diarrheal stool specimens containing subgenus F adenoviruses, F1a/F2a PCR achieved at least as high a positivity rate (75/76 [99%]) as electron microscopy (72/76 [95%]) and was more sensitive than polyclonal antibody-based immune electron microscopy (IEM) for subgenus identification (75/76 [99%] vs. 66/76 [87%], P = 0.008). Twenty-three subgenus F strains untypeable by monoclonal antibody-based IEM were typed as 40 (n = 4) or 41 (n = 19) by Hha I digestion of the PCR product. The genus-reactive primer pairs provided DNA amplification assays of generally equal efficiency on conjunctival swab specimens though possible nucleic acid degradation in DNA extracts during storage could have meant that JCH1/JCH2 PCR was truly the more sensitive. The use of either genus-reactive primer set on fecal specimens cannot be recommended because, although the positivity rates with subgenus F PCR positive specimens were high (70/75 [93%] for H1 and H2, 14/15 [93%] for JCH1 and JCH2), the detection rates were disappointing with similar specimens yielding nonsubgenus F adenoviruses.

Adenoviridae Infections↗
PubMed1996

The effect on chick osteoclasts of infection with paramyxoviruses.

The detection of virus in osteoclasts from Pagetic patients is now well known, but it has yet to be shown convincingly that the presence of virus in Pagetic osteoclasts influences their behaviour. In this study, osteoclasts from embryonic chick tibiae were infected with canine distemper virus or measles virus and compared with mock-infected controls. Infection was confirmed using virus-specific fluorescent antibodies. It was found that virus infection did not alter osteoclast morphology or tartrate-resistant acid phosphatase (TRAP) activity. Both infected and mock-infected osteoclasts produced resorption pits on bovine bone slices; these could be divided into two distinct size classes with a computer-based measuring system. Virus infection significantly increased the proportion of the larger size class of resorption pit. These results suggest that virus infection can increase bone resorption by osteoclasts, lending further support to the hypothesis that viruses play a role in Paget's disease of bone.

Acid Phosphatase↗
PubMed1996

Multiplex polymerase chain reaction for adenovirus and herpes simplex virus in eye swabs.

Adenoviruses and herpes simplex virus (HSV) can cause clinically indistinguishable episodes of acute eye disease. Adenovirus infection is associated with nosocomial outbreaks and HSV may result in episodes of recurrent ocular inflammation. In a comparison of multiplex PCR for the two viral DNAs and virus isolation in cell culture, identical results were obtained for 18 of 20 specimens (positive for adenovirus in 5, HSV in 5, and negative in 8). One specimen was falsely negative for each viral DNA. Inclusion of human beta-globin primers in the adenovirus-HSV reaction was precluded by a consequential 10--100-fold reduction in sensitivity for the two viral targets and by the failure of beta-globin DNA amplification at the annealing temperature (45 degrees C) required to ensure detection of adenoviruses of serotypes 7 and 11 with the selected adenovirus primers. A single-target beta-globin PCR gave positive results with 19 of the 20 specimens prepared by treatment with proteinase K lysis buffer, indicating the effectiveness of this simple DNA extraction procedure. Nonetheless, the availability of effective antiviral therapy for HSV made monitoring for extraction failure using human primers crucial to avoid false-negative results for HSV DNA. Adenovirus-HSV PCR has considerable potential for the rapid diagnosis of viral eye disease particularly if beta-globin primers can be included in the reaction.

Adenovirus Infections, Human↗
PubMed1996

Polymerase chain reaction for rapid diagnosis of respiratory adenovirus infection.

Endemic (type 1, 2, 5 and 6) and epidemic (type 3, 4 and 7) respiratory adenovirus infections are associated with upper respiratory tract symptoms, pharyngoconjunctival fever, and pneumonia. Improved methods of diagnosis are needed, particularly in immunocompromized patients. We examined 93 throat swabs or nasopharyngeal aspirates from patients with acute respiratory disease using virus isolation and an adenovirus-specific polymerase chain reaction (PCR) based on consensus primers H1 and H2 derived from the hexon region DNA sequences of serotypes 2 and 5. Specimens which yielded viruses other than adenovirus in cell culture (n = 23) or which were negative for infectious viruses (n = 25) were negative in the PCR. The sensitivity of DNA amplification was 76% (34/45) in comparison with virus culture, being markedly lower with subgenus B (types 3 and 7) strains than with subgenus C (type 1, 2, 5 and 6) isolates (8/16 (50%)) vs. 26/28 (93%). P = 0.004) despite the use of a low annealing temperature to maximize detection of adenoviruses belonging to subgenera other than C. Of the 11 samples falsely negative in a single-round PCR but yielding adenovirus type 1 (n = 1), type 2 (n = 1). type 3 (n = 7), type 7 (n = 1), or untyped isolates (n = 1) in cell culture, nine (82%) gave positive results after nested DNA amplification. Possible approaches to further improving the performance of adenovirus PCR with respiratory specimens are discussed.

Adenoviridae Infections↗
PubMed1996

Detection of herpes simplex virus DNA in donor cornea culture medium by polymerase chain reaction.

AIMS/BACKGROUND: Herpes simplex virus (HSV) may establish latent infection in the cornea and therefore be transmissible by corneal transplantation. Monitoring of donor cornea culture medium was evaluated for HSV infection. METHODS: HSV was sought using virus isolation in cell culture, and its DNA was amplified to detectable levels using the polymerase chain reaction (PCR). RESULTS: Virus isolation in cell culture was negative on neat, cell pellet, and cell free supernatant prepared from the spent culture media of 80 corneas. Three cell pellets (3.8%) were positive for HSV DNA. The PCR positive culture negative results might have reflected latent rather than active HSV infection of the cornea. Post transplant follow up of the three recipients of corneas with HSV PCR positive organ culture media revealed no evidence of HSV induced eye disease or primary graft failure. CONCLUSION: Screening of corneal culture medium for HSV by virus culture or for HSV DNA by PCR could not be recommended.

Aged↗
PubMed1996

Effects of recombinant human interleukin-11 (Neumega rhIL-11 growth factor) on megakaryocytopoiesis in human bone marrow.

We examined the effects of recombinant human interleukin 11 (rhIL-11) on in vivo human hematopoiesis. Twelve women with advanced breast cancer and no evidence of bone marrow (BM) involvement were treated with 10, 25, 50, or 75 micrograms/kg/day of rhIL-11 administered subcutaneously for 14 consecutive days. Examination of bone marrow trephine biopsies obtained before and after rhIL-11 treatment revealed unchanged BM cellularity at all doses, and a statistically significant increase in megakaryocyte (MK) frequencies (from 0.5 +/- 0.1% to 1.0 +/- 0.3%) following administration of the two highest doses (p < 0.001). The BM biopsies also showed an increased proportion of immature myeloid and erythroid precursors following 14 days of treatment in all cases. The mean proportion of marrow cells stained with PC10, a monoclonal antibody (mAb) that recognizes the proliferating cell nuclear antigen (PCNA), increased from 16.3 +/- 5.7% to 45.8 +/- 17.1% (p < 0.001) following the two highest treatment doses. Most of the PC10+ cells were promyelocytes and proerythroblasts. In this same group, the proportion of PC10+ MKs increased from 28.3 +/- 11.5% to 56.8 +/- 24.3% (p < 0.01) after treatment, while megakaryocyte ploidy analysis revealed a greater number of higher ploidy (64N) megakaryocytes following rhIL-11 treatment (p < 0.012). Numbers of BM and peripheral blood (PB) CD34+, CD34+DR+, and CD34+DR- cells did not change following rhIL-11 treatment. Following rhIL-11 therapy at the highest dose studied, a 3- and 10-fold increase in the number of committed BM MK progenitor cells (CFU-MK) was observed in two of three patients, while no change was seen in the number of the other BM or PB progenitor cells examined. rhIL-11 administration was also associated with an increase in BM reticulin content (fibrosis grade 1-2) in 7 patients. These results indicate that the administration of rhIL-11 to patients with normal hematopoiesis stimulates MK endoreduplication, PCNA expression, and, at high doses, increases MK and CFU-MK progenitor cell numbers. In addition, rhIL-11 was able to stimulate precursor cells of different marrow lineages without affecting the number of assayable progenitor cells.

Administration, Cutaneous↗
PubMed1995

Polymerase chain reaction for rapid detection of ocular adenovirus infection.

Adenoviruses are associated with endemic and epidemic acute conjunctivitis, large nosocomial outbreaks reflecting virus transmission on unwashed hands or inadequately sterilised ophthalmic instruments. The polymerase chain reaction (PCR) proved more sensitive than antigen detection by immune dot-blot test for the rapid diagnosis of ocular adenovirus infection (sensitivities in a retrospective study 112/123 (91%) versus 72/123 (59%), P < 0.001). Indeed, in a prospective comparison, DNA amplification and virus isolation generated similar numbers of positive results (34 versus 32), though five PCR positive results were possibly false positives. The sensitivity of the PCR was largely independent of adenovirus subgenus or serotype, though reduced sensitivity with subgenus B strains could not be excluded. Specimen preparation for DNA amplification using a simple lysis buffer proved more effective than phenol-chloroform extraction. The immune dot-blot test gave unavoidable false positive results, but with the PCR this problem could be minimized by technical modifications. The PCR could replace antigen detection and virus isolation as the initial test for adenoviruses in conjunctival swabs, with cell culture only being retained for adenovirus serotyping in PCR positive specimens and for other viruses such as herpes simplex.

Adenoviridae↗
PubMed1995

Multiplex polymerase chain reaction for human herpesvirus-6, human cytomegalovirus, and human beta-globin DNA.

Human cytomegalovirus and human herpesvirus-6 are closely related viruses which cause similar diseases, have similar cellular repositories of latent infection, and may be detected largely in the same types of clinical specimens. DNA amplification appears likely to play an increasing role in the diagnosis of recent and remote infection with these agents. A sensitive multiplex polymerase chain reaction was therefore developed for the two viruses and for human beta-globin DNA. Optimization of parameters such as the primers, primer concentrations, magnesium concentration, and buffer constituents was crucial in achieving a sensitive assay. Preliminary results indicated that the assay could simultaneously monitor DNA extraction from clinical specimens and allow detection of HCMV or HHV-6 in patients with diseases possibly caused by either pathogen.

Base Sequence↗