CCR5 blockade modulates inflammation and alloimmunity in primates.

Pharmacologic antagonism of CCR5, a chemokine receptor expressed on macrophages and activated T cells, is an effective antiviral therapy in patients with macrophage-tropic HIV infection, but its efficacy in modulating inflammation and immunity is only just beginning to be investigated. In this regard, the recruitment of CCR5-bearing cells into clinical allografts is a hallmark of acute rejection and may anticipate chronic rejection, whereas conventionally immunosuppressed renal transplant patients homozygous for a nonfunctional Delta32 CCR5 receptor rarely exhibit late graft loss. Therefore, we explored the effects of a potent, highly selective CCR5 antagonist, Merck's compound 167 (CMPD 167), in an established cynomolgus monkey cardiac allograft model. Although perioperative stress responses (fever, diminished activity) and the recruitment of CCR5-bearing leukocytes into the graft were markedly attenuated, anti-CCR5 monotherapy only marginally prolonged allograft survival. In contrast, relative to cyclosporine A monotherapy, CMPD 167 with cyclosporine A delayed alloantibody production, suppressed cardiac allograft vasculopathy, and tended to further prolong graft survival. CCR5 therefore represents an attractive therapeutic target for attenuating postsurgical stress responses and favorably modulating pathogenic alloimmunity in primates, including man.

Automated agar plate streaker: a linear plater on Society for Biomolecular Sciences standard plates.

Several protocols for bacterial isolation and techniques for aerobic plate counting rely on the use of a spiral plater to deposit concentration gradients of microbial suspensions onto a circular agar plate to isolate colony growth. The advantage of applying a gradient of concentrations across the agar surface is that the original microbiological sample can be applied at a single concentration rather than as multiple serial dilutions. The spiral plater gradually dilutes the sample across a compact area and therefore saves time preparing dilutions and multiple agar plates. Commercial spiral platers are not automated and require manual sample loading. Dispensing of the sample volume and rate of gradients are often very limited in range. Furthermore, the spiral sample application cannot be used with rectangular microplates. Another limitation of commercial spiral platers is that they are useful only for dilute, filtered suspensions and cannot plate suspensions of coarse organic particles therefore precluding the use of many kinds of microorganism-containing substrata. An automated agar plate spreader capable of processing 99 rectangular microplates in unattended mode is described. This novel instrument is capable of dispensing discrete volumes of sample in a linear pattern. It can be programmed to dispense a sample suspense at a uniform application rate or across a decreasing concentration gradient.

Interpretation of high-throughput liquid chromatography mass spectrometry data for quality control analysis and analytical method development.

An approach to rapidly process and interpret high-throughput liquid chromatography mass spectrometry data is presented. This approach applies an in-house developed computer application to process LC-MS report files containing spectral and chromatographic data from four different detectors (i.e. electrospray positive ionization, electrospray negative ionization mass spectrometry, UV absorption, and evaporative light scattering detection). Properties characteristic of detection and chromatographic retention are extracted and populated into a database. Approaches to applying this analytical information database for quality control analysis of ca. 400,000 samples are presented. Compound quality assessment methods employing average purity and detection data fields are compared to methods employing multiple quality control criteria (e.g. detection, purity, retention, and signal to noise). Structural similarity searches were applied with the analytical information database to identify compounds that may be undetectable by electrospray mass spectrometry. In addition, an approach to applying the database to aid in the selection of analytical detection and chromatography conditions for rapid analytical method development is also discussed.

Isolation and structure of antagonists of chemokine receptor (CCR5).

Human CCR5 is a G-coupled receptor that binds to the envelope protein gp120 and CD4 and mediates the HIV-1 viral entry into the cells. The blockade of this binding by a small molecule receptor antagonist could lead to a new mode of action agent for HIV-1 and AIDS. Screening of natural product extracts led to the identification of anibamine (1), a novel pyridine quaternary alkaloid as a TFA salt, from Aniba sp.; ophiobolin C from fermentation extracts of fungi Mollisia sp.; and 19,20-epoxycytochalasin Q from Xylaria sp. Formation of the TFA salt of anibamine is plausibly an artifact of the isolation. The identity of the natural counterion is unknown. Anibamine.TFA competed for the binding of 125I-gp120 to human CCR5 with an IC50 of 1 microM. Ophiobolin C and 19,20-epoxycytochalasin Q exhibited binding IC50) values of 40 and 60 microM, respectively.