Eucaryotic Expression of the Nucleocapsid Protein Gene of Porcine Circovirus Type 2 and Use of the Protein in an Indirect Immunofluorescence Assay for Serological Diagnosis of Postweaning Multisystemic Wasting Syndrome in Pigs

The purpose of this study was to develop a sensitive, rapid, and inexpensive immunofluorescence assay (IFA) using a recombinant porcine circovirus type 2 (PCV2) nucleocapsid protein for the serological detection of PCV2-specific antibodies in pig sera. The viral nucleocapsid protein encoded by the PCV2 ORF2 gene has recently been identified as the most immunoreactive viral protein that carries type-specific antigenic determinants. The ORF2 sequence of the IAF-2897 strain of PCV2 has been cloned into a pCEP5 eucaryotic expression vector under the control of the cytomegalovirus promoter, downstream of a polyhistidine sequence tag. The recombinant plasmid was used in transfection experiments with human epithelial kidney 293 cells that were further tested, and positive expression of the viral nucleocapsid protein was confirmed by IFA and Western blotting. Strong, specific fluorescence was observed in the nuclei of transfected cells. Test specificity to PCV2 was verified with several related infectious agents. Sensitivity was compared to that of standard IFA using PCV2-infected cells by evaluating the reactivities of 44 field serum samples from pigs on farms with a porcine population suffering from postweaning multisystemic wasting syndrome. The recombinant nucleocapsid-based test was able to detect 15 more positive-testing pigs than the PCV2-based IFA. Therefore, the relative sensitivity of the latter test was estimated at only 57.1% compared to that of the recombinant nucleocapsid-based test. The recombinant fusion protein has been purified by affinity chromatography and is being used to develop further sensitive serological tests.     

Epidemiology and genetic characterization of HIV-1 isolates in the general population of Djibouti (Horn of Africa)

During a national survey in 2002 in Djibouti, serum samples were collected using a valid sampling scheme from 2423 Djiboutians representing the general population of urban and rural districts. The HIV-1 seroprevalence was 2%. The HIV-1 polymerase gene from 53 untreated patients was amplified. Phylogenetic analysis of 34 isolates revealed a majority of subtype C (73%) as well as other subtypes, including CRF02_AG recombinants (18%), subtype D (6%), and subtype A (3%).     

HIV infection rapidly induces and maintains a substantial suppression of thymocyte proliferation

The supply of naive T cells by the thymus normally requires precursor T cell proliferation within the thymus and would be particularly important in the setting of HIV infection when both naive and memory T cells are progressively depleted. As a robust, quantitative index of intrathymic proliferation, the ratio of different T cell receptor excision circles (TRECs), molecular markers of distinct T cell receptor rearrangements occurring at different stages of thymocyte development, was measured in peripheral blood-mononuclear cells (PBMCs). This ratio has the virtue that it is a "signature" of thymic emigrants throughout their entire life and, thus, can be measured in peripheral cell populations that are easy to obtain. Using the new assay, we evaluated the effect of HIV infection on intrathymic precursor T cell proliferation by longitudinal analysis of PBMCs from recently infected individuals. Our findings reveal a substantial reduction in intrathymic proliferation. The analysis also indicates the existence of a compensatory mechanism acting to sustain the numbers of recent thymic emigrants (RTEs) in the periphery.     

Foscarnet salvage therapy efficacy is associated with the presence of thymidine-associated mutations (TAMs) in HIV-infected patients

BACKGROUND: Salvage therapy based on foscarnet plus a thymidine analog is effective in patients with advanced-stage HIV disease and viruses harbouring multiple drug-resistance mutations. OBJECTIVE: To identify viral genetic determinants associated with the virological efficacy of foscarnet salvage therapy. STUDY DESIGN: Thirteen patients received foscarnet at a fixed dose of 80mg/kg twice daily for 14 days, in combination with zidovudine or stavudine. RESULTS: The baseline median HIV viral load and CD4 cell count were 5.10log(10)copies/ml and 23cells/mm(3), respectively. Following foscarnet therapy, viral load fell by a median of 1.84log(10)copies/ml (range: -0.29 to -2.82), and by at least 1log(10)copies/ml in 11 patients, all of whom harboured viruses with at least three thymidine-associated mutations (TAMs). The two patients with smaller declines in viral load (<0.50log(10)copies/ml) harboured viruses with only one or zero TAMs. CONCLUSIONS: These findings corroborate, in vivo, the impact of TAMs on HIV susceptibility to foscarnet. The virological response to foscarnet salvage therapy in multiclass-experienced patients may thus differ according to the number of TAMs.     

Hypoesthesia after anterior cruciate ligament reconstruction: The relationship between proprioception and vibration perception deficits in individuals greater than one year post-surgery

Background

While surgical reconstruction restores mechanical stability following anterior cruciate ligament (ACL) rupture, many experience early-onset osteoarthritis despite surgery. Neurophysiological changes are hypothesized to contribute to knee osteoarthritis progression. Proprioceptive deficits have been reported following ACL injury/reconstruction; however, vibration perception threshold (VPT) has been less studied. This study explored relationships between pain, VPT, proprioception, function, and strength following ACL-reconstruction.

Inhibition of compound action potentials following administration of dental self-etching primer/adhesives

Self-etching dentin adhesive systems, a new generation of dental-bonding agents, were investigated for their ability to block nerve conductance in the rat sciatic nerve model. Clearfil SE Bond, Mac Bond and FL Bond were used as test materials. Isolated rat sciatic nerves obtained from female albino rats were placed between two electrodes in a bath containing Tyrode solution. The bonding agents were brought into contact with the nerves and the evoked compound action potentials were recorded versus time before and after contact with the materials. Statistical analysis was performed using the Friedman and Wilcoxon Signed-Ranks tests (P = 0.01) to compare the mean percentage of inhibition between test materials versus time. All test materials were able to block nerve transmission rapidly (3.3 +/- 0.5, 5.1 +/- 2.3 and 8.0 +/- 1.9 min for SE Bond, Mac Bond and FL Bond, respectively). Significant differences were found between FL Bond and SE and Mac Bond (P < 0.01). These results indicated that the bonded systems tested, intended primarily as dentinal tubule occluders, may also exert some direct desensitizing effect on intratubular or pulpal nerve terminals in extremely deep cavities or pulpal microexposures, contributing to decreased postoperative pain.     

Survival rates during the first trimester of multiple gestations achieved by ICSI: a report of 1448 consecutive multiples

BACKGROUND: The purpose of this study was to determine the rate of spontaneous gestational sac loss during the first trimester in women achieving multiple pregnancies by ICSI. METHODS: A retrospective analysis was performed of 1448 consecutive multiple pregnancies conceived by ICSI. RESULTS: Of the cohort of 1448 pregnancies, twin gestations constituted 59.6% (864), triplets 30.2% (438) and quadruplets 10.0% (146). During the first trimester, 69 (4.7%) patients miscarried, while 179 (12.3%) continued their pregnancies and had fewer gestational sacs at the end of the first trimester than at the beginning. The overall loss rate of any gestational sac during the first trimester in these multiple pregnancies was 10.1%. There was a significant difference in the frequency of spontaneous reduction to twin or singleton pregnancies in the first trimester between women carrying triplets (11.7%) and those carrying quadruplets (3.5%) [P = 0.004; odds ratio (OR) 3.5; 95% confidence interval (CI) 1.3-9.1]. The frequency of gestational sac loss was significantly greater among women >35 years old (20.9%) than in women less than 35 years old (15.9%) (P = 0.03; OR 1.4; 95% CI 1.0-1.9). CONCLUSION: In multiple pregnancies there is a significant risk of spontaneous loss of any embryo during the first trimester. These findings should be considered prior to any decision about selective embryo reduction.     

Increased spontaneous immunoglobulin secretion associated with cyclophosphamide-induced immune suppression

Spontaneous immunoglobulin (Ig) secretion by cells from multiple sclerosis (MS) patients (in the progressive phase) treated with monthly pulse doses of cyclophosphamide (CY) (1000–1600 mg/M²) was measured using the protein A plaque assay, to evaluate the effect of CY treatment on B-cell function. Surprisingly, an increase, rather than a decrease, in Ig-secreting cells was seen following CY treatment. CY-treated MS patients averaged 1380±535 spontaneous total (IgM+G+A) Ig plaque-forming cells (PFC) per 1×10⁶ peripheral blood mononuclear cells (MNC), measured at 15–22 days after monthly CY administration, while healthy adults had 280±47 Ig PFC/10⁶ MNC, and MS patients not treated with CY had 300±43 Ig PFC/10⁶ MNC. The observed increase was due to an increase in IgG and IgA PFC. PFC levels remained elevated for 4 weeks following CY treatment, decreasing to control levels by 7–8 weeks post-CY. A small increase in serum IgG level was noted after >12 months of pulse CY therapy; no increase was seen in CSF IgG levels. A preferential decrease in the number of CD4+ T cells was also seen in the CY-treated MS patients. We propose that the observed increase in the number of spontaneous Ig PFC was due to the CY-induced disruption of the CD4+ T cell-mediated control ofin vivo activated B cells.     

Detection of Vi-negative Salmonella enterica serovar typhi in the peripheral blood of patients with typhoid fever in the Faisalabad region of Pakistan

The synthesis and transportation proteins of the Vi capsular polysaccharide of Salmonella enterica serovar Typhi (serovar Typhi) are encoded by the viaB operon, which resides on a 134-kb pathogenicity island known as SPI-7. In recent years, Vi-negative strains of serovar Typhi have been reported in regions where typhoid fever is endemic. However, because Vi negativity can arise during in vitro passage, the clinical significance of Vi-negative serovar Typhi is not clear. To investigate the loss of Vi expression at the genetic level, 60 stored strains of serovar Typhi from the Faisalabad region of Pakistan were analyzed by PCR for the presence of SPI-7 and two genes essential for Vi production: tviA and tviB. Nine of the sixty strains analyzed (15%) tested negative for both tviA and tviB; only two of these strains lacked SPI-7. In order to investigate whether this phenomenon occurred in vivo, blood samples from patients with the clinical symptoms of typhoid fever were also investigated. Of 48 blood samples tested, 42 tested positive by fliC PCR for serovar Typhi; 4 of these were negative for tviA and tviB. Three of these samples tested positive for SPI-7. These results demonstrate that viaB-negative, SPI-7-positive serovar Typhi is naturally occurring and can be detected by PCR in the peripheral blood of typhoid patients in this region. The method described here can be used to monitor the incidence of Vi-negative serovar Typhi in regions where the Vi vaccine is used.