Natural infection of a homozygous delta24 CCR5 red-capped mangabey with an R2b-tropic simian immunodeficiency virus

A homozygous 24-bp deletion (Delta24) was found in the CC chemokine receptor 5 (CCR5) of 11 out of 15 red-capped mangabeys (RCMs), Cercocebus torquatus torquatus, both in Africa and in an American zoo. The CCR5 Delta24 defect encompassed eight amino acids in frame in the fourth transmembrane region. Unexpectedly, RCM-009, one of 11 homozygotes (Delta24CCR5/ Delta24CCR5), was found to be naturally infected with a divergent simian immunodeficiency virus (SIV) strain, which was not R5-tropic, but used CCR2b (R2b) as its major coreceptor. SIVrcmGab1 was the only R2b-tropic SIV among other divergent SIVs tested. Cells transfected with the Delta24 CCR5 did not support entry of R5-tropic SIVmac, SIVcpz, SIVmne, HIV-2, or HIV-1, and were also inactive in signal transduction mediated by beta-chemokines. At 86.6%, the Delta24 allelic frequency was significantly higher than that of the 32-bp deletion found in humans. The Delta24 frequency was 4.1% in 34 sooty mangabeys (SMs), a geographically isolated subspecies that was naturally infected with R5-tropic SIV. Finding identical deletions in two mangabey subspecies separated for 10,000 years or more dates the Delta24 CCR5 deletion as ancient. However, the source of the selective pressure for the high rate of CCR5 deletion in RCMs remains to be determined. The high allelic frequency of the Delta24 CCR5 in RCMs, in comparison to that of SMs, suggests that R2b-tropism may have been acquired by SIVrcm, as an adaptation to CCR5 genetic defects appeared in its host.     

Natural history of adhesional small bowel obstruction: counting the cost

BACKGROUND: The aim of this study was to identify patients admitted with adhesional obstruction to determine if there was an identifiable pattern to the type of initial operation, the type of treatment used for the obstructive episode and the subsequent need for further treatment. METHODS: Patients with adhesional obstruction were identified retrospectively in a cross-sectional study using ICD codes relating to admissions in the years 1990 to 1996. The case notes were used to assess their outcome. RESULTS: Fifty-nine case notes from a total of 175 identified initially satisfied the inclusion criteria. These patients had a mean age at presentation of 51 (range 16-88) years and had undergone a total of 122 operations. Thirty-one patients (53 per cent) had a single previous operation with a median time to presentation with obstruction of 5.5 years (range 11 days to 34.7 years); 33 patients (56 per cent) were treated conservatively on their first admission. There was no statistically significant difference in the outcome in patients who received either conservative or surgical treatment. The length of stay in patients treated surgically (median 11 (range 2-47) days) was significantly longer than that for those treated conservatively (median 6 (range 1-39) days) (P< 0.001). A flow chart was constructed demonstrating the eventual outcome of the patients in the study, enabling the cost of adhesional obstruction to be calculated. CONCLUSION: This type of approach could be used to assess the potential effect of different treatment strategies for adhesional obstruction.     

Analysis of major histocompatibility complex class I, TAP expression, and LMP2 epitope sequence in Epstein-Barr virus-positive Hodgkin's disease

The Epstein-Barr virus (EBV)-encoded latent membrane proteins, LMP1 and LMP2, are consistently expressed by the malignant Hodgkin/Reed-Sternberg (HRS) cells of EBV-associated Hodgkin's disease (HD). Cytotoxic T lymphocyte (CTL) responses to both of these proteins have been shown in the blood of EBV-seropositive individuals, yet in HD the apparent failure of the CTL response to eliminate HRS cells expressing LMP1 and LMP2 in vivo has given rise to the suggestion that HD may be characterized by the presence of defects in antigen processing/presentation or in CTL function. This study has used immunohistochemistry to show high-level expression of major histocompatibility complex (MHC) class I molecules by the HRS cells of EBV-associated HD and either low level or absence of expression of MHC class I molecules on HRS cells of EBV-negative tumors. In addition, HRS cells expressed high levels of transporter-associated proteins (TAP-1, -2), irrespective of the presence of latent EBV infection. These results suggest that global downregulation of MHC class I molecules does not account for the apparent ability of EBV-infected HRS cells to evade CTL responses, but may be important in the understanding of EBV-negative disease. We have also sequenced an epitope in LMP2A (CLGGLLTMV) that is restricted through HLA A2.1, a relatively common allele in Caucasian populations, and showed that this epitope is wild type in a small group of EBV-associated HLA A2.1-positive HD tumors. This result may be relevant to proposed immunotherapeutic approaches for EBV-positive HD patients that target CTL epitopes.     

Effect of human C-reactive protein on chemokine and chemotactic factor-induced neutrophil chemotaxis and signaling

C-reactive protein (CRP) is a unique serum pentraxin and the prototype acute phase reactant. CRP is a ligand for specific receptors on phagocytic leukocytes, and mediates activation reactions of monocytes/macrophages, but inhibits the respiratory burst of neutrophils (PMN). Since CRP selectively accumulates at inflammatory sites in which IL-8 is also produced, we tested the effects of CRP on the responsiveness of PMN to IL-8 and the bacterial chemotactic peptide, FMLP-phenylalanine (FMLPP). Purified human CRP inhibited the chemotactic response of PMN to IL-8 and FMLPP. A mouse IgM mAb that was generated against the leukocyte CRP receptor (CRP-R) also inhibited the chemotactic response. Incubation of purified CRP with activated PMN generated CRP-derived peptides that also inhibited chemotaxis. A synthetic CRP peptide (residues 27-38) that binds to the CRP-R had weak chemotactic activity, whereas two other CRP synthetic peptides (residues 174-185 and 191-205) inhibited chemotaxis of PMNs to both IL-8 and FMLPP. CRP did not alter receptor-specific binding of IL-8, but exerted its effect at the level of signaling. CRP augmented both IL-8- and FMLPP-induced mitogen-activated protein kinase (extracellular signal-regulated kinase-2) activity. CRP at acute phase levels increased both agonist-induced and noninduced phosphatidylinositol-3 kinase activity. The results suggest a role for CRP as a regulator of leukocyte infiltration at inflammatory sites.     

Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment

When microbes evolve in a continuous, nutrient-limited environment, natural selection can be predicted to favor genetic changes that give cells greater access to limiting substrate. We analyzed a population of baker's yeast that underwent 450 generations of glucose-limited growth. Relative to the strain used as the inoculum, the predominant cell type at the end of this experiment sustains growth at significantly lower steady-state glucose concentrations and demonstrates markedly enhanced cell yield per mole glucose, significantly enhanced high-affinity glucose transport, and greater relative fitness in pairwise competition. These changes are correlated with increased levels of mRNA hybridizing to probe generated from the hexose transport locus HXT6. Further analysis of the evolved strain reveals the existence of multiple tandem duplications involving two highly similar, high-affinity hexose transport loci, HXT6 and HXT7. Selection appears to have favored changes that result in the formation of more than three chimeric genes derived from the upstream promoter of the HXT7 gene and the coding sequence of HXT6. We propose a genetic mechanism to account for these changes and speculate as to their adaptive significance in the context of gene duplication as a common response of microorganisms to nutrient limitation.     

Results of treatment of inferior vena cava syndrome with expandable metallic stents

A 73-year-old male was admitted to our hospital because of detection of Shigella flexneri 2a from his stool. Antimicrobial treatment with levofloxacin (LVFX) was started, but could not eliminate the organism in the stool. In the examination of drug susceptibility, this strain was highly resistant to all new quinolones. The minimal inhibitory concentration of norfloxacin, ofloxacin and ciprofloxacin to this strain was 12.5 micrograms/ml, 6.25 micrograms/ml and 6.25 micrograms/ml, respectively. The dual mutations were detected in the codon 83 and 87 of the gyrA gene by sequencing the quinolone-resistance determining region (QRDR). There was, however, no significant difference between the intracellular uptake of ciprofloxacin in this strain and in the ciprofloxacin-sensitive strain. The amount of ciprofloxacin in this strain unchanged when carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was added. These results suggest that the advanced resistance in Shigella flexneri against new quinolones could be acquired by only this dual mutations without the change of the active efflux mechanism.     

Preparation and in vitro characterization of gentamycin-impregnated biodegradable beads suitable for treatment of osteomyelitis

A new method for preparing poly(L-lactide) (PLA) biodegradable beads impregnated with an ionic aminoglycoside, gentamycin, is described. The process employs hydrophobic ion pairing to solubilize gentamycin in a solvent compatible with PLA, followed by precipitation with a compressed antisolvent (supercritical carbon dioxide). The resulting precipitate is a homogeneous dispersion of the ion-paired drug in PLA microspheres. The microspheres are approximately 1 microm in diameter and can be compressed into beads (3-6 mm in diameter) strung on surgical sutures for implantation. The bead strings exhibit no significant change in release kinetics upon sterilization with a hydrogen peroxide plasma (Ster-Rad). The kinetics of gentamycin release from the PLA beads are consistent with a matrix-controlled diffusion mechanism. While nonbiodegradable poly(methyl methacrylate) (PMMA) beads initially release gentamycin in a similar manner, the drug release from PMMA ceases after 8 or 9 weeks, while the PLA beads continue to release drug for over 4 months. Moreover, only 10% of the gentamycin is released from the PMMA beads, while PLA beads release more than 60% of their load, if serum is present in the release medium. The PLA system displays improved release kinetics relative to PMMA, is biodegradable, is unaltered by gas sterilization, can be used for a range of antibiotics, and can be manipulated without disintegration. These are all desirable properties for an implantable drug delivery system for the prevention or treatment of osteomyelitis.     

Hinge bending within the cytokine receptor superfamily revealed by the 2.4 A crystal structure of the extracellular domain of rabbit tissue factor

Tissue factor (TF), a member of the cytokine receptor superfamily, is the obligate cofactor of coagulation factor VIIa (FVIIa), and has a pivotal role in initiating the extrinsic pathway of blood coagulation through formation of the TF x FVIIa complex. The crystal structure of the extracellular portion of rabbit TF has been solved at 2.35 A resolution and refined to a crystallographic R-value of 19.1% (free R-value, 27.7%). Like the human homologue, the extracellular portion consists of two fibronectin type III domains connected by a short alpha-helical segment. Unexpectedly, the two molecules in the crystallographic asymmetric unit differ in their relative domain-domain orientation, revealing unsuspected hinge motion consisting of a rotation of about 12.7 degrees around an axis intersecting the linker segment at residue 106. Superposition of rabbit tissue factor with free and bound human tissue factor allows for the detection of an identical, albeit smaller, hinge motion in human TF induced upon binding of FVIIa. This raises the possibility that a very similar hinge axis may be present in other members of the cytokine receptor superfamily.     

Digital subtraction indocyanine green angiography of occult choroidal neovascularization

OBJECTIVE: This study aimed to use a new technique for ocular imaging, digital subtraction indocyanine green angiography (DS-ICGA), to evaluate patients with occult choroidal neovascularization (CNV). DESIGN: The design was a cross-sectional study of patients with occult CNV. PARTICIPANTS: A total of 31 eyes of 31 patients were studied. INTERVENTION: Digital subtraction of sequentially acquired indocyanine green angiographic frames was used to image the progression of the dye front in eyes with occult CNV. A method of pseudocolor imaging of the choroid was developed that allows differentiation and identification of underlying choroidal arteries and veins. RESULTS: The DS-ICGA of occult CNV showed consistent findings. Occult CNV was imaged with greater detail and in a shorter period of time than with conventional indocyanine green angiography. The fundus landmarks were retained with DS-ICGA much better than with conventional indocyanine green angiography. CONCLUSIONS: The DS-ICGA uses time to dissect the choroidal circulation. With DS-ICGA, occult CNV could be imaged more quickly and in greater detail than with conventional imaging techniques. The DS-ICGA may improve the authors ability to image, and subsequently treat, occult CNV.