Immunology and Pathology Core

Core DirectorsJoan W. Berman, PhD and Sunhee C Lee, MD

Departments of Pathology and Microbiology and Immunology

Staff MembersLillie Lopez and Namjong Choi

Core AimsAim 1. To provide investigators with the research infrastructure and

optimization of technologies and protocols to perform state-of-the art immunologic and biochemical assays. These assays will quantify responses of cells infected with or stimulated by HIV and AIDS-associated pathogens.

Aim 2. To provide cell isolation and tissue culture service Aim 3. To provide investigators with a facility using appropriate biohazard

containment that is capable of examining the effects of infection with HIV and AIDS-associated pathogens on tissues by characterizing morphological, molecular and phenotypical changes using histological, immunohistochemical, in situ hybridization analyses and live cell imaging.

Aim 4. To train CFAR investigators in the performance of techniques that are extant in the Core and to assist investigators in the development of new immunological assays and pathological techniques. To provide extensive mentoring in the immunology and pathogenesis of HIV/AIDS and OI.

Core Training programs

• Provide training in microscopy, RNA and protein techniques

• Provide training in immunopathology• Provide training in data analysis and presentation• Provide mentoring for project development relevant

to the immunopathology of HIV/AIDS and OI • Provide grant writing assistance for protocols and

experimental approaches

Core services• Histopathology

– customize fixation and tissue processing and sectioning, frozen and fixed• Immunohistochemistry

– optimize primary and secondary antibodies and provide control tissues• Cytokine and Chemokine and other soluble mediator measurements (ELISA, multiplex assays, FACS, MSD)

– Optimize and customize assay protocols• RNA analysis (Q-RT-PCR)

– Identify and test primers and optimize assay conditions• Protein extraction and analysis

– Interface with proteomic core (imaging MS)• Culture of human and rodent CNS cells (endothelial cells, astrocytes, microglia and neurons)• Isolation of PBMCs and subsets (monocytes, T cells, B cells and progenitors)• Live cell imaging

– available for HIV-infected tissue and cells– calcium imaging– time-lapse photography

• Fluorescence and confocal microscopy– Customize and optimize appropriate reagents– Provide control tissues and reagents

Expansion & Innovation ofCore Activities

• Addition of new microscope and live cell imaging• Addition of human PBMC and subset isolation service

and training• Imaging mass spectroscopy with Proteomics Facility• Addition of assistance with grant writing:

experimental design and methods

Microglial reactivity in human cerebral malaria

Sarah Hochman, MD, “Malaria/HIV co-infection”

Recent Improvements to Live Cell imaging Facility

• Now have Cy2, Cy3, Cy5 and Cy7 filters• A new objective for low magnification • Incubation system with CO2, O2 and humidity control and heating stages• Longer times for live cell imaging• Anti-vibration table with compressor• New software for calcium imaging and time lapse microscopy

Microscope setupUpgrade, including incubation system, CO2,

and O2 control plus software

Use of Live cell imaging system

Eliseo Eugenin et al., submitted

Imaging mass spectrometry (iMS)

Nature Reviews 2010

•Greater sensitivity and a linear dynamic range (at least 5 logs)•Ability to perform instantaneous detection of binding with minimal or no need for washing steps (advantage for low affinity interactions and kinetically sensitive measurements)•Can perform up to 10 analyses per well

MSD Electrochemiluminescence

Figure. Multiplex cytokine analysis with the MSD system. Capture mAbs are printed on the MSD plate well surface in defined positional arrays. Up to 10 spots can be printed by the manufacturer per well. The MSD Sector Imager plate scanner detects and quantitates light emitted from each spot separately, allowing multiple analytes to be quantitated using a mixture of detection mAbs. (Figure adapted from MSD website, http://www.meso-scale.com).

Conclusion

Immunology and Pathology Core provides state-of-the art services, consultation, and

mentoring to facilitate outstanding HIV/OI research programs