The saccharide antigen, Gal beta1-3GalNAc or T antigen, is of biologic importance in many systems. It is a tumor-associated carbohydrate antigen, a temporally expressed antigen in germinal center B cells and cortical T cells, a parasite-associated antigen, a spermatozoa vitality marker and an antigen on aged red blood cells. It may play a role in normal cellular adhesion and in tumor cell metastasis. Well characterized monoclonal antibodies (MAb) to Gal beta1-3GalNAc will be useful for further studies in these areas. We developed an IgG3 MAb to Gal beta1-3GalNAc by immunizing the mice with a synthetic Gal beta1-3GalNAc-BSA conjugate. The MAb was analyzed using inhibition enzyme immunoassays with related synthetically prepared sugars to determine the restrictions involved in the antibody binding. Use of synthetic sugars as competitors enabled us to delineate the epitope restrictions on the binding activity of this monoclonal and will enable use of this MAb in studies concerning the biologic importance of this disaccharide.

Endogenously generated protoporphyrin IX (PpIX) from exogenous ALA can be an effective photosensitizer. PpIX accumulation is inversely dependent on available intracellular iron, which is required for the conversion of PpIX to heme. Iron also is necessary for cell replication. Since iron can be toxic, intracellular iron levels are tightly controlled. Activated and proliferating cells respond to the demand for intracellular iron by upregulating membrane expression of the transferrin receptor (CD71) which is needed for iron uptake. We predicted that activated lymphocytes (CD71+) would preferentially accumulate PpIX because of their lower intracellular iron levels and because of competition for iron between ALA-induced heme production and cellular growth processes. Thus, the CD71+ cells could serve as PDT targets. Stimulation of human peripheral blood lymphocytes (PBL) with the mitogens, phytohemagglutinin A, concanavalin A and pokeweed prior to incubation with ALA results in PpIX accumulation correlating with level of activation. Activated lymphocytes expressing high levels of surface CD71 transferrin receptors generated more PpIX than those with low CD71 expression. Incubating activated cells in transferrin depleted medium (thereby decreasing the iron availability) further increased PpIX levels. Malignant, CD71+ T lymphocytes from a patient with cutaneous T-cell lymphoma (CTCL)/Sezary syndrome also accumulated increased PpIX levels in comparison to normal lymphocytes. PDT of activated lymphocytes and Sezary cells after ALA incubation demonstrated preferential killing compared to normal, unstimulated PBL. These findings suggest a possible mechanism for the selectivity of ALA PDT for activated CD71+ cells. They also indicate a clinical use for ALA-PDT in therapy directed towards the malignant lymphocytes in leukemias and lymphomas, and as animmunomodulatory agent.

PSA is currently being used to detect and monitor quantitatively the development of prostate cancer by serum levels of PSA and has also been found to be present in high concentrations in semen. Elegantly simple, sensitive, and reproducible methods have been developed for analysis of the presence of PSA, including the Tandem-E PSA Immunoenzymetric Assay. The most common procedures for the forensic identification of semen have focused on the microscopic detection of sperm, acid phosphatase activity, and immunoelectrophoretic methods for the detection of PSA. Although these methods have been used for many years, there are problems associated with each method. The Tandem-E PSA Immunoenzymetric Assay detected PSA in 100% of the forensic casework fabric samples, 80% of the forensic casework vaginal swabs and 100% of the vasectomized individuals tested. The cut-off values was determined to be 1.77 ng/mL. These results indicate that this method can be used to identify the presence of semen in forensically significant specimens.

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Lymphocytes treated with delta-aminolevulinic acid (ALA) can accumulate the photoactive, fluorescent heme precursor, protoporphyrin IX (PpIX). With visible light illumination, PpIX can be used in photodynamic therapy (ALA-PDT) to kill or functionally alter cells. The aim of this study was to characterize the effects of ALA and ALA-PDT on resting and activated human peripheral blood T lymphocytes. Accumulation of PpIX depends inversely on the rate of its iron-dependent conversion into heme. Activated replicating lymphocytes have low intracellular iron levels, with corresponding increases in the transferrin receptor (CD71). Thus, we expected activated lymphocytes would preferentially accumulate PpIX. Using four-color flow cytometry, we examined ALA-induced PpIX levels in T-cell subsets of resting and activated human peripheral blood mononuclear cells and the relationship between CD71 and PpIX. Peripheral blood mononuclear cells stimulated by phytohemagglutinin (PHA) were simultaneously phenotyped for PpIX, CD71 and the T-cell markers CD3 and CD4 or CD8. In activated cells treated with 0-6 mM ALA for 4 h, PpIX fluorescence was maximal at 1 mM ALA. On a single cell basis, there was a strong correlation between PpIX accumulation and CD71 expression. The ALA-treated, PHA-stimulated, CD71+ lymphocytes had an eight-fold greater mean PpIX fluorescence than nonactivated, CD71- cells. Approximately 87% of the CD4+ and 85% of the CD8+ T cells accumulated PpIX. The PpIX levels of CD8+ cells were about 5% greater than CD4+ cells. In addition, mixed lymphocyte reaction-stimulated cells treated with ALA accumulated more PpIX than controls. Thus, activated cells preferentially accumulate endogenous PpIX when exogenous ALA is administered. Cytotoxicity studies showed that the majority of the activated cells following ALA-PDT were killed but resting cells were spared. Also, in examining activation markers by flow cytometry the number of cells that were positive for activation markers CD38 or CD71 dramatically decreased after ALA and light treatment in activated populations. The data suggest a role for ALA-PDT as an immunomodulator or photocytotoxic agent targeting activated lymphocytes.

Lymphocytes treated with δ‐aminolevulinic acid (ALA) can accumulate the photoactive, fluorescent heme precursor, protoporphyrin IX (PpIX). With visible light illumination, PpIX can be used in photodynamic therapy (ALA‐PDT) to kill or functionally alter cells. The aim of this study was to characterize the effects of ALA and ALA‐PDT on resting and activated human peripheral blood T lymphocytes. Accumulation of PpIX depends inversely on the rate of its iron‐dependent conversion into heme. Activated, replicating lymphocytes have low intracellular iron levels, with corresponding increases in the transferrin receptor (CD71). Thus, we expected activated lymphocytes would preferentially accumulate PpIX. Using four‐color flow cytometry, we examined ALA‐induced PpIX levels in T‐cell subsets of resting and activated human peripheral blood mononuclear cells and the relationship between CD71 and PpIX. Peripheral blood mononuclear cells stimulated by phytohemagglutinin (PHA) were simultaneously phenotyped for PpIX, CD71 and the T‐cell markers CD3 and CD4 or CDS. In activated cells treated with 0‐6mM ALA for 4 h, PpIX fluorescence was maximal at 1 mM ALA. On a single cell basis, there was a strong correlation between PpIX ac‐cumulation and CD71 expression. The ALA‐treated, PHA‐stimulated, CD71+ lymphocytes had an eight‐fold greater mean PpIX fluorescence than nonactivated, CD71‐ cells. Approximately 87% of the CD4* and 85% of the CD8+ T cells accumulated PpIX. The PpIX levels of CDS+ cells were about 5% greater than CD4+ cells. In addition, mixed lymphocyte reaction‐stimulated cells treated with ALA accumulated more PpIX than controls. Thus, activated cells preferentially accumulate endogenous PpIX when exogenous ALA is administered. Cytotoxicity studies showed that the majority of the activated cells following ALA‐PDT were killed but resting cells were spared. Also, in examining activation markers by flow cytometry the number of cells that were positive for activation markers CD38 or CD71 dramatically decreased after ALA and light treatment in activated populations. The data suggest a role for ALA‐PDT as an immunomodulator or photocytotoxic agent targeting activated lymphocytes.

Protoporphyrin IX (PpIX), an endogenously synthesized photosensitizer, can transiently accumulate in activated lymphocytes following administration of the heme precursor 5-aminolevulinic acid (ALA). One possible mechanism of this in lymphocyte accumulation is that actively dividing cells use intracellular iron stores for cytochrome and DNA synthesis and thus do not inactivate PpIX, the photoactive precursor of heme, by iron incorporation. This selective accumulation in activated cells should allow targeting by photodynamic therapy (PDT). To determine the effect of this accumulation, we studied PDT effects on the in vitro correlate of transplantation rejection: the one-way mixed lymphocyte reaction (MLR). Selective phototoxicity was determined by photoirradiating ALA-treated, MLR-activated cells and measuring subsequent stimulation either in a secondary MLR or with phytohemagglutinin (PHA). We found that proliferation of MLR-activated lymphocytes incubated with ALA and treated with light was only 12-20% of controls (ALA+, no light) after rechallenge with the stimulator cells (P < 0.05), although their response to nonspecific PHA stimulation was similar to controls. Thus alloantigen-specific depletion was shown. The data suggest a role for ALA-PDT in the treatment of diseases that require the selective elimination of activated lymphocytes and possibly as an immunomodulator.

Cytomegalovirus (CMV) infection is ubiquitous and results in a wide spectrum of clinical manifestations ranging from asymptomatic infection to severe life threatening disease. Infection in normal children and adults usually causes no symptoms but in the immunocompromised host, CMV may result in severe opportunistic infections with high morbidity and mortality. Historically, virus detection was dependent on culture of the virus or on a centrifugation culture system referred to as a shell vial assay. The shell vial assay frequently lacked sensitivity and was unable to detect infection in its early phase. Also, as with culture assays, the results were affected by antiviral therapy. The CMV antigenemia assay was developed to provide more rapid results and has gained wide usage. This assay is limited to detection of the virus in white blood cells and is more sensitive than culture or the shell vial assay. Application of the polymerase chain reaction (PCR) to these problems has resulted in the development of assays for CMV which are more sensitive than previously available methods. This method employs liquid hybridization with 32P labeled probes and gel retardation analysis for detection of amplified DNA specific for each virus. A comparison of the detection of CMV by an antigenemia assay or the PCR method in the leukocytes of renal transplant patients revealed that the PCR assay detects cytomegalovirus earlier and more consistently than the antigenemia assay. Finally, the application of a fluorescent dye detection system and image analysis of the acrylamide gel with a laser scanner provides additional sensitivity to the detection of cytomegalovirus, as well as avoiding the use of radioactivity, making the assay more adaptable to the clinical laboratory.