High throughput testing of drug library substances and monoclonal antibodies for capacity to reduce formation of cystatin C dimers to identify candidates for treatment of hereditary cystatin C amyloid angiopathy.

OBJECTIVE: To establish a high-throughput system for testing the ability of drugs or monoclonal antibodies to reduce the in vitro formation of cystatin C dimers to identify substances potentially useful for treatment of patients with hereditary cystatin C amyloid angiopathy (HCCAA). METHODS: Various combinations of incubation temperature, time period, guanidinium chloride concentration and concentration of cystatin C monomers were tested in low-volume formats to induce dimer formation of recombinant cystatin C. The extent of dimerization was analysed by gel filtration chromatography and agarose gel electrophoresis. RESULTS: A high-throughput system based upon agarose gel electrophoresis was developed and used to test 1040 drugs in a clinical drug library for their capacity to reduce cystatin C dimer formation in vitro. Seventeen substances reducing dimer formation by more than 30% were identified. A similar system for testing the capacity of monoclonal antibodies against cystatin C to reduce the in vitro formation of cystatin C dimers was also developed and used to test a panel of 12 monoclonal antibodies. Seven antibodies reducing dimer formation by more than 30% were identified and the two most potent, Cyst28 and HCC3, reduced dimerization by 75 and 60%, respectively. CONCLUSION: We constructed a simple high-throughput system for testing the capacity of drugs and monoclonal antibodies to reduce the in vitro formation of cystatin C dimers and several candidates for treatment of HCCAA could be identified.

Two Independent HIV Epidemics in Saint Petersburg, Russia Revealed by Molecular Epidemiology.

The HIV epidemic in Russia, one of the world's fastest growing, has been concentrated mostly among people who inject drugs (PWID). We sought to explore the epidemiology of the epidemic in St. Petersburg by sampling from the highest risk groups of PWID and men who have sex with men (MSM) and use viral sequencing data to better understand the nature of the city's epidemic. Serological testing confirmed an HIV prevalence among PWID in excess of 40%. All but 1 of 110 PWID whose blood samples were tested for genetic diversity were infected by subtype A virus, specifically by the AFSU strain. The remaining person was infected with a CRF-06cpx recombinant. Analysis of pairwise genetic distance among all PWID studied revealed an average of 3.1% sequence divergence, suggesting clonal introduction of the AFSU strain and/or constraints on sequence divergence. The HIV prevalence was less than 10% among MSM. All 17 sequences from HIV-infected MSM were found to be a clade B virus with a much higher average sequence diversity of 15.7%. These findings suggest two independent epidemics with little overlap between the two highest at-risk populations, which will require different HIV prevention approaches.