OTHER SERVICES

1. Immunology: Human cells
Extended immunophenotyping for cell surface or intracellular markers:

  • Maturation markers of T cells (Naïve, Memory, Effector)
  • Natural Regulatory T cell markers
  • Co-stimulatory molecules (e.g. PD-1, PDL-1, CD28, CTLA-4)
  • Dendritic cell characterization
  • Natural Killer Cells

Functional assays

  • T cell proliferation by CFSE dye dilution or Ki7 staining by flow cytometry
  • Lymphocyte proliferation assay (LPA) in response to mitogens, antigens and recall antigens
  • Intracytoplasmic cytokines by flow cytometry
  • Cytokine & chemokine quantitations by Enzyme Immuno Assay, Multiplex assay by Protein Microarray, Single cell level by ELISPOT

General Virology

  • P24 determination
  • HIV antibody screening & confirmation by WB
  • Hepatitis antibody screening for HAV, HBV & HCV
  • HIV-1 proviral DNA
    HIV viral load quantitation: Roche Amplicore – Standard & ultrasensitive, bioMerieux – bDNA, Siemens Medical Solutions – bDNA
    HIV drug resistance testing: Celera – ViroSeq and Siemens Medical Solutions – TruGene
  • HBV and HCV viral load quantitations: Siemens Medical Solutions – bDNA
  • HBV and HCV drug resistance testing: Siemens Medical Solutions – TruGene

2. Specialized Virology Assays and Procedures
Contacts:  Drs. Rebeca Geffin and Walter Scott

The virology component of the University of Miami D-CFAR has expertise in the following assays and procedures.  In some cases we can offer to carry out virology procedures on a fee-for-service basis (fees to be determined).  For other, more specialized procedures, we are interested in establishing collaborative efforts.

Virus quantitation.
Preparation of HIV-1 stocks from clinical specimens, and evaluating these virus stocks by measuring HIV-1 capsid antigen (HIV-1 p24 Ag), HIV-1 infectious titer by limiting dilution titration in activated PBMC.  These procedures are useful for cell culture studies where virus input must be quantitated.

HIV-1 subtype determination by polymerase chain reaction (PCR) assay.  This may be of interest in clinical studies of patients infected with a HIV virus that may not be clade B and therefore may give aberrant results in standard determinations of viral load by molecular methods. 

Assays to determine HIV-1 coreceptor usage by a clinical isolate.
This can be accomplished by various assays but we use PCR amplification, sequencing and determination of overall charge of the HIV envelope V3 loop, which is predictive of coreceptor usage. We will advise if phenotypic confirmation is needed.

HIV-1 fitness assay.
Drug resistance mutations, antibody and CTL escape mutations, and changes in coreceptor specificity may all alter the ability of HIV-1 to replicate and also may affect HIV-1 pathogenesis.  To some extent these effects are reflected in the rate of HIV-1 replication in cell culture.  We feel that the exploration of these assays in connection with specific clinical studies to be a potential area for collaboration. We are currently developing an assay that can measure the ability of a test isolate to compete with a standard isolate for growth in peripheral blood mononuclear cells. This competition assay is more sensitive than conventional measurements of viral growth kinetics.

HIV-1 immune serum neutralization assay.
Humoral antibody response is an important factor in driving the evolution of virus envelope protein.  Specific questions about host response to the HIV-1 infection and HIV-1 adaptation to the host humoral response can be assayed by cell culture assays that measure the ability of patient's serum to neutralize HIV-1 infectivity.  We would welcome inquiries about potential collaborations in this area.

3. Molecular Biology Procedures

Maintenance and storage of cell lines, plasmids and transformed bacteria; Manipulation of DNA, mutagenesis and plasmid propagation.  These are standard procedures that most molecular biology laboratories carry out for themselves.  These procedures could be provided as a service if there is enough interest to provide salary support for the work.  In addition, we are willing to provide trouble-shooting advice.

Lentivirus construction, lentivirus stock preparation and titration.  Use of lentivirus vectors has recently become widely used for a variety of experiments involving the introduction of genes into differentiated mammalian cells and for studying modifications in individual genes of HIV-1. The procedure basically involves the introduction of plasmid DNAs containing various portions of the HIV genome and the envelope gene from vesicular stomatitis virus to provide the packaging materials for forming virus particles that can infect a wide variety of cells. The gene to be delivered by the lentivirus vector is cloned into a plasmid that contains packaging signals so that lentivirus particles are made that contain almost exclusively the gene to be delivered.  Virus stocks are titered and then used to infect cells or tissues in culture or, in some cases, are injected into animals.

Note:  Preparation and use of lentivirus vectors falls under section III-D of the NIH Guidelines for Recombinant DNA Research, which requires prior submission to and approval by the Institutional Biosafety Committee (IBC).  Most lentivirus vector experiments can be carried out under BL2 containment or BL2-plus (using a BL2 facility with the laboratory procedures specified for BL3 experiments).  We will be happy to provide advice on submission of documents to the IBC and evaluation of biosafety plans.

siRNA and dominant negative mutant gene transfer.  These are procedures often used to shut off the activity of specific genes in eukaryotic cells in order to study their functions.  Gene transfer may be accomplished by various methods including transfection and retrovirus medicated transduction.  Collaborative interactions are welcomed on such studies.

Other assays and services
Sexually transmitted infections by serology & PCR:
Syphilis, Chlamydia, Gonorrohea, Candida, Gardnerella, Trichomonas

Cryopreservation for cell and plasma repository.
(Offered by Pahwa, Asthana, Scott and Fletcher Labs)
Clinical specimen processing, isolation of peripheral blood mononuclear cells (PBMC), storing cryopreserved cells, plasma, serum and other clinical materials, maintenance of specimen records and repository collection.

Humanized mouse facility (under development)
Contact: Dr Eckhard Podack

Microbial translocation assays
Contact: Meenakshi Sachdeva