Microvascular and systemic responses to novel PEGylated carboxyhaemoglobin-based oxygen carrier in a rat model of vaso-occlusive crisis.

Hypoxia drives sickle cell disease (SCD) by inducing sickle cell haemoglobin to polymerize and deform red blood cells (RBC) into the sickle shape. A novel carboxyhaemoglobin-based oxygen carrier (PEG-COHb; PP-007) promotes unsickling in vitro by relieving RBC hypoxia. An in vivo rat model of vaso-occlusive crisis (VOC) capable of accommodating a suite of physiological and microcirculatory measurements was used to compare treatment with PEG-COHb to a non-oxygen carrying control solution (lactated ringer's [LRS]). Male Sprague-Dawley rats were anesthetized and surgically prepared to monitor microvascular interstitial oxygenation (PISFO2), cardiovascular parameters and blood chemistry. Human homozygous SCD RBCs were isolated and exchange transfused into the rats until the distal microcirculation of the exteriorized spinotrapezius muscle was hypoxic and RBC aggregates were visualized. VOC was left untreated (Sham) or treated 15 min later with PEG-COHb or LRS and observed for up to 4 h. Treatment with PEG-COHb showed better improvement of PISFO2, end-point lactate, mean arterial pressure and survival duration compared to Sham and LRS. Restoring PISFO2 was associated with relieving the RBC aggregates driving VOC, which then affected other study metrics. Compared to LRS, PEG-COHb's oxygen-carrying properties were key to improved outcomes.

Quantifying the relationship between HIV-1 susceptibility to CCR5 antagonists and virus affinity for antagonist-occupied co-receptor.

Previous studies have demonstrated that HIV-1 develops resistance to CCR5 antagonists by gaining the ability to use drug-occupied co-receptor. However, the effects of CCR5 antagonists on the affinity of virus-co-receptor interactions have been difficult to quantify. We developed a pharmacological model for allosteric interaction at G-protein coupled receptors to analyze the effect of different CCR5 antagonists on infection by three laboratory adapted viruses with low, moderate and high susceptibility to the inhibitors. Infection data for these viruses fitted a model in which susceptibility to inhibition by CCR5 antagonists was directly related to fold reduction in virus affinity for CCR5. Dissociation constants for CCR5 antagonists calculated from the modeled data were consistent with values obtained by standard methods, suggesting that this approach can quantify pharmacologically relevant changes in co-receptor:ligand affinity in the context of infection of whole cells by authentic HIV-1 particles.

Antiviral activity of transiently expressed IFN-kappa is cell-associated.

Most type I interferons (IFNs) are expressed by the majority of cell types in response to viral infection. In contrast, IFN-kappa has been reported to have a cellular distribution limited to keratinocytes and certain lymphoid cell populations. Recombinant expressed IFN-kappa has been shown previously to possess weak antiviral activity when directly compared with IFN-beta. In order to expand on the antiviral potential of IFN-kappa, we transiently transfected human cell lines to circumvent the need to purify recombinant proteins and to avoid the possible loss of biologic activity by the purification process. We evaluated the transcriptional signaling and antiviral activity of IFN-kappa in parallel with IFN-alpha2b with mammalian expression vectors to express each protein transiently. Both IFN-kappa and IFN-alpha2b exhibited comparable transcriptional and antiviral activities. However, in contrast to IFN-alpha2b transcriptional signaling and antiviral activity, IFN-kappa activity was not detectable in conditioned cell culture medium. Subsequent experiments revealed there was a direct relationship between IFN-kappa-expressing cells and antiviral activity. These results were confirmed in immunocytochemical studies. Furthermore, IFN-kappa exhibited cell-associated antiviral activity against a hepatitis C virus (HCV) replicon cell line. This novel IFN signaling strategy may represent an important distinct and divergent mechanism for limiting viral infections.