USPIO-related T1 and T2 mapping MRI of cartilage in a rabbit model of blood-induced arthritis: a pilot study.

Ultrasmall paramagnetic iron oxide (USPIO)-enhanced MRI is promising for evaluating inflammation. The aims of this study were to investigate the effect of USPIO on cartilage T1 and T2 mapping, and to evaluate a proposed rapid vs. conventional T2 map method for imaging cartilage in a blood-induced arthritis model. Knees of nine arthritic (induction by intra-articular autologous blood injection) and six control rabbits were imaged over time (baseline, weeks 1, 5, 10) by 1.5 T MRI. All rabbits had USPIO (35-75 µmol Fe/kg)-enhanced MRI at each time point. T1 and T2 (conventional and rapid) maps and signal-to-noise ratios (SNR) were obtained pre- and post-USPIO administration. Cartilage biochemistry and histology were compared with MRI. Excellent correlations were noted between T1 map values and histologic scores at week 10 pre-USPIO (medial, r = 0.93, P = 0.0007; lateral, r = 0.87, P = 0.005) in the arthritic group, but not between T2 map and histology. Marginally and significant differences were observed between pre- and post-USPIO T2 values at weeks 5 (P = 0.06) and 10 (P = 0.02), but only with the administration of high USPIO doses in the arthritic group using the conventional method. No significant differences were noted between pre- and post-USPIO T1 values at any imaging time points. Cartilage T2 maps with short-TR and conventional protocols provided similar T2 values [(decreased trend)] (P > 0.05). Concomitant use of USPIO to T1 and T2 mapping of cartilage would not impair the identification of interval changes of T1 and T2 maps. Rapid T2 map provides similar results compared to conventional method, but its validation warrants further investigation.

Herpes simplex virus type 2 and HIV infection among US military personnel: implications for health prevention programmes.

US military personnel are routinely screened for HIV infection. Herpes simplex virus type 2 (HSV-2) is a risk factor for HIV acquisition. To determine the association between HSV-2 and HIV, a matched case-control study was conducted among US Army and Air Force service members with incident HIV infections (cases) randomly matched with two HIV-uninfected service members (controls) between 2000 and 2004. HSV-2 prevalence was significantly higher among cases (30.3%, 138/456) than among controls (9.7%, 88/912, P < 0.001). HSV-2 was strongly associated with HIV in univariate (odds ratio [OR] = 4.2, 95% confidence interval [CI] = 3.1-5.8) and multiple analyses (adjusted [OR] = 3.9, 95% CI = 2.8-5.6). The population attributable risk percentage of HIV infection due to HSV-2 was 23%. Identifying HSV-2 infections may afford the opportunity to provide targeted behavioural interventions that could decrease the incidence of HIV infections in the US military population; further studies are needed.

The ABC transporter genes of Plasmodium falciparum and drug resistance.

The seminal observations that (a) chloroquine-resistant Plasmodium falciparum strains accumulate less drug than more sensitive parasites, and (b) chloroquine resistance could be modulated in vitro by the classic multidrug-resistance (MDR) modulator verapamil, suggested not only that parasite resistance to multiple drugs may be similar to the MDR phenotype described in mammalian cancer cells, but that homologous proteins may be involved. These findings prompted search for MDR-like genes in the parasite. To date, three full-length ABC transporter genes have been isolated from P. falciparum: two P-glycoprotein-like homologues, pfmdr1 and pfmdr2, and a homologue of the yeast GCN20 gene, pfgcn20.

Episodic evolution mediates interspecies transfer of a murine coronavirus.

Molecular mechanisms permitting the establishment and dissemination of a virus within a newly adopted host species are poorly understood. Mouse hepatitis virus (MHV) strains (MHV-A59, MHV-JHM, and MHV-A59/MHV-JHM) were passaged in mixed cultures containing progressively increasing concentrations of nonpermissive Syrian baby hamster kidney (BHK) cells and decreasing concentrations of permissive murine DBT cells. From MHV-A59/MHV-JHM mixed infection, variant viruses (MHV-H1 and MHV-H2) which replicated efficiently in BHK cells were isolated. Under identical treatment conditions, the parental MHV-A59 or MHV-JHM strains failed to produce infectious virus or transcribe detectable levels of viral RNA or protein. The MHV-H isolates were polytrophic, replicating efficiently in normally nonpermissive Syrian hamster smooth muscle (DDT-1), Chinese hamster ovary (CHO), human adenocarcinoma (HRT), primate kidney (Vero), and murine 17Cl-1 cell lines. Little if any virus replication was detected in feline kidney (CRFK) and porcine testicular (ST) cell lines. The variant virus, MHV-H2, transcribed seven mRNAs equivalent in relative abundance and size to those synthesized by the parental virus strains. MHV-H2 was an RNA recombinant virus containing a crossover site in the S glycoprotein gene. At the molecular level, episodic evolution and positive Darwinian natural selection were apparent within the MHV-H2 S and HE glycoprotein genes. These findings differ from the hypothesis that neutral changes are the predominant feature of molecular evolution and argue that changing ecologies actuate episodic evolution in the MHV spike glycoprotein genes that govern interspecies transfer and spread into alternative hosts.

Biochemical analysis of the response in rat bone marrow cell cultures to mechanical stimulation.

Bone marrow cells obtained from rat femora were subjected to primary culture with 15% fetal bovine serum in the presence of 10(-8) M dexamethasone, and following trypsin treatment 5 days later were seeded on Petriperm dishes which have a flexible bottom. After a 2-day subculture, a cyclic stress consisting of a 1 s stretch (0.3% strain. 0.5 Hz) and a 1 s relaxation for 30 min every day was started. Culture tissue was removed on day 2 of the subculture (immediately prior to start of stimulation), and then on days 5 and 8 (3 and 6 days after the start of stimulation, respectively), at which times dry weight, DNA, alkaline phosphatase (ALP) activity, and bone Gla protein (BGP, osteocalcin) were measured. Both the dry weight and DNA showed a significant increase in the stimulated group by day 8, while the ALP activity showed a significant increase by day 5. The BGP began to increase in the stimulated group on day 5 in contrast to the control group in which it only increased on day 8. These results support the contention that mechanical stimulation promotes the differentiation of osteogenic cells and enhances bone formation. Since in this experimental model the acceleration of bone formation by mechanical stimulation can be reproduced in vitro, it is extremely useful for investigating the mechanisms underlying mechanical stimulation.

A strong association between mefloquine and halofantrine resistance and amplification, overexpression, and mutation in the P-glycoprotein gene homolog (pfmdr) of Plasmodium falciparum in vitro.

Stepwise selection for increased mefloquine resistance in a line of Plasmodium falciparum in vitro resulted in increased resistance to halofantrine and quinine, increased sensitivity to chloroquine, and amplification and overexpression of the P-glycoprotein gene homolog (pfmdr1). A point mutation (tyrosine to phenylalanine) noted at amino acid 86 in pfmdr1 in the mefloquine-resistant line W2mef was amplified in more resistant lines derived from it by in vitro selection pressure with mefloquine. Conversely, lines selected for increased chloroquine resistance exhibited a revertant phenotype that was sensitive to mefloquine and halofantrine. These lines also demonstrated increased sensitivity to quinine, loss of amplification of pfmdr1, loss of the mefloquine/halofantrine phenylalanine-86 mutation, and selection for a tyrosine-86 mutation previously associated with chloroquine resistance. These findings provide strong evidence for pfmdr1 mediating cross-resistance to halofantrine and mefloquine in P. falciparum in vitro.

Isolation and identification of six Pneumocystis carinii genes utilizing codon bias.

Pneumocystis carinii pneumonia (PCP) is a leading cause of death among AIDS patients in the United States. Our analysis of P. carinii protein-coding genes has revealed a significant A + T codon bias. Polymerase chain reaction (PCR) was utilized to isolate and identify the genes encoding calmodulin, beta-tubulin, DNA polymerase II, and RNA polymerases I, II and III from P. carinii. Primer pairs were designed to incorporate P. carinii codon preference to known conserved protein regions from other organisms. This strategy should be useful for a large variety of P. carinii genes and assist in the comprehensive analysis of the genomic structure of this important pathogen.

Derivation of highly mefloquine-resistant lines from Plasmodium falciparum in vitro.

Serial passage of a multidrug-resistant clone of Plasmodium falciparum in concentrations of mefloquine hydrochloride ranging from 30 to 2,400 ng/ml resulted in the derivation of increasingly resistant parasite lines in vitro. Parasite lines isolated in mefloquine concentrations greater than 300 ng/ml demonstrated increased vacuolization, enhanced pigment production, and increased growth rates as compared with the progenitor clone, W2-mef. Although microdilution incorporation assays demonstrated that the 50% inhibitory concentration (IC50) of mefloquine were similar for all lines, the IC90, IC95, and IC99 levels were significantly increased. Growth rate assays performed in 5% hematocrit suspensions demonstrated different levels of mefloquine resistance among these lines. Under these conditions the most resistant line, Mef 2.4, grew efficiently in approximately 10-fold higher concentrations of mefloquine than the progenitor clone W2-mef. Analysis of drug susceptibility profiles to mefloquine hydrochloride, chloroquine diphosphate, quinine sulfate, and halofantrine hydrochloride indicated that selection for high levels of mefloquine resistance had resulted in significant increases in resistance to halofantrine and increased sensitivity to chloroquine. The phenotypic changes demonstrated in the most resistant line, Mef 2.4, reflect a multidrug resistant-like phenotype, and appear to mimic changes recently reported in drug susceptibility profiles of recrudescent isolates following mefloquine treatment failures in Thailand.

Studies into the mechanism for MHV transcription.

Previous studies have demonstrated that the MHV genome is divided into seven transcriptional units which are transcribed from highly conserved intergenic start sites (UCU/CAAAC) into mRNA containing a common leader RNA at the 5' end and a coterminal 3' end. In this manuscript, we provide evidence that an additional transcriptional unit is encoded at the 3' end of the MHV genome and is transcribed from a perfect intergenic region into a leader-containing approximately 800 nt mRNA. This mRNA could potentially encode a small 17-18 kDa protein which is identical to the C-terminal third of the nucleocapsid gene.