A collaborative study on larval excretory/secretory antigens of Toxocara canis for the immunodiagnosis of human toxocariasis with ELISA.

Two batches of excretory/secretory (E/S) antigens from second stage larvae of Toxocara canis maintained in vitro were prepared independently in two different laboratories (Zürich and Basel) and analysed in order to obtain information for future efforts to standardize the enzyme-linked immunosorbent assay (ELISA) used for the serodiagnosis of human toxocariasis. SDS-PAGE and "Western-blotting" revealed at least 10 different antigenic components common to the two antigen preparations. However, distinct qualitative and quantitative differences among the two E/S-antigens were observed, since one antigen had a more complex composition than the other. Despite these differences, an accordance of serodiagnosis was obtained in 80% of 25 sera from patients with suspected Toxocara infection tested independently in two different ELISA systems (Basel and Zürich) with the corresponding E/S-antigens. The specificity was 93% as determined (BS-antigen, BS-ELISA) by testing 46 out of 3396 sera from patients with parasitologically proven extra-intestinal helminthic infections. Cross-reactions occurred mainly with sera from patients infected with filariae (5 from 13 cases) exhibiting very high extinction values in their homologous ELISA-system. The reproducibility (intra- and inter-test variations) of two ELISA systems using the corresponding E/S-antigens varied from 5-15%. The results demonstrate that T. canis E/S-antigens may well be applicable for standardization of the ELISA used for the serodiagnosis of human toxocariasis.

The human IgG anti-carbohydrate repertoire exhibits a universal architecture and contains specificity for microbial attachment sites.

Despite the paradigm that carbohydrates are T cell-independent antigens, isotype-switched glycan-specific immunoglobulin G (IgG) antibodies and polysaccharide-specific T cells are found in humans. We used a systems-level approach combined with glycan array technology to decipher the repertoire of carbohydrate-specific IgG antibodies in intravenous and subcutaneous immunoglobulin preparations. A strikingly universal architecture of this repertoire with modular organization among different donor populations revealed an association between immunogenicity or tolerance and particular structural features of glycans. Antibodies were identified with specificity not only for microbial antigens but also for a broad spectrum of host glycans that serve as attachment sites for viral and bacterial pathogens and/or exotoxins. Tumor-associated carbohydrate antigens were differentially detected by IgG antibodies, whereas non-IgG2 reactivity was predominantly absent. Our study highlights the power of systems biology approaches to analyze immune responses and reveals potential glycan antigen determinants that are relevant to vaccine design, diagnostic assays, and antibody-based therapies.

Human DMTF1β antagonizes DMTF1α regulation of the p14(ARF) tumor suppressor and promotes cellular proliferation

The human DMTF1 (DMP1) transcription factor, a DNA binding protein that interacts with cyclin D, is a positive regulator of the p14ARF (ARF) tumor suppressor. Our earlier studies have shown that three differentially spliced human DMP1 mRNAs, α, β and γ, arise from the human gene. We now show that DMP1α, β and γ isoforms differentially regulate ARF expression and promote distinct cellular functions. In contrast to DMP1α, DMP1β and γ did not activate the ARF promoter, whereas only β resulted in a dose-dependent inhibition of DMP1α-induced transactivation of the ARF promoter. Ectopic expression of DMP1β reduced endogenous ARF mRNA levels in human fibroblasts. The DMP1β- and γ-isoforms share domains necessary for the inhibitory function of the β-isoform. That DMP1β may interact with DMP1α to antagonize its function was shown in DNA binding assays and in cells by the close proximity of DMP1α/β in the nucleus. Cells stably expressing DMP1β, as well as shRNA targeting all DMP1 isoforms, disrupted cellular growth arrest induced by serum deprivation or in PMA-derived macrophages in the presence or absence of cellular p53. DMP1 mRNA levels in acute myeloid leukemia samples, as compared to granulocytes, were reduced. Treatment of acute promyelocytic leukemia patient samples with all-trans retinoic acid promoted differentiation to granulocytes and restored DMP1 transcripts to normal granulocyte levels. Our findings imply that DMP1α- and β-ratios are tightly regulated in hematopoietic cells and DMP1β antagonizes DMP1α transcriptional regulation of ARF resulting in the alteration of cellular control with a gain in proliferation.

Human 3β-hydroxysteroid dehydrogenase deficiency seems to affect fertility but may not harbor a tumor risk: lesson from an experiment of nature.

CONTEXT 3β-hydroxysteroid dehydrogenase deficiency (3βHSD) is a rare disorder of sexual development and steroidogenesis. There are two isozymes of 3βHSD, HSD3B1 and HSD3B2. Human mutations are known for the HSD3B2 gene which is expressed in the gonads and the adrenals. Little is known about testis histology, fertility and malignancy risk. OBJECTIVE To describe the molecular genetics, the steroid biochemistry, the (immuno-)histochemistry and the clinical implications of a loss-of-function HSD3B2 mutation. METHODS Biochemical, genetic and immunohistochemical investigations on human biomaterials. RESULTS A 46,XY boy presented at birth with severe undervirilization of the external genitalia. Steroid profiling showed low steroid production for mineralocorticoids, glucocorticoids and sex steroids with typical precursor metabolites for HSD3B2 deficiency. The genetic analysis of the HSD3B2 gene revealed a homozygous c.687del27 deletion. At pubertal age, he showed some virilization of the external genitalia and some sex steroid metabolites appeared likely through conversion of precursors secreted by the testis and converted by unaffected HSD3B1 in peripheral tissues. However, he also developed enlarged breasts through production of estrogens in the periphery. Testis histology in late puberty revealed primarily a Sertoli-cell-only pattern and only few tubules with arrested spermatogenesis, presence of few Leydig cells in stroma, but no neoplastic changes. CONCLUSIONS The testis with HSD3B2 deficiency due to the c.687del27 deletion does not express the defective protein. This patient is unlikely to be fertile and his risk for gonadal malignancy is low. Further studies are needed to obtain firm knowledge on malignancy risk for gonads harboring defects of androgen biosynthesis.

BIOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF MURINE-TUMOR SPECIFIC TRANSPLANTATION ANTIGENS (ISOTACHOPHORESIS, IMMUNOGENICITY)

Tumor-specific transplantation antigens (TSTA) are individually distinct neoantigens expressed on the cells of chemically-induced neoplasms. TSTA are operationally defined by immunization of syngeneic mice against challenge with viable tumor cells. Immunization with cell surface or extracted TSTA induces specific resistance to transplanted tumor cells. The biological and biochemical nature of TSTA was investigated in the 3-methylcholanthrene-induced fibrosarcomas of female C3H/HeJ mice, MCA-F and MCA-D. Tumor cell suspensions were extracted by treatment with 3M KCl or 2.5% butanol solutions and the TSTA was partially purified by preparative isoelectric focusing. The isoelectric pH of TSTA purified from 3M KCl extracts was 5.8-6.0, and from butanol extracts was 6.4-6.6. Whereas immunization with 10('5) and 10('6) irradiated tumor cells induces complete rejection of tumor cell challenge over a two-fold-log dose range, immunization with ug quantities within a one-fold-log dose range of extracted TSTA induces only partial resistance to tumor challenge. Reduced immunogenicity of extracted TSTA is hypothesized to result from immunization of mice with insufficiently purified TSTA preparations. The hypothesis predicts that immunization with highly purified TSTA, free from interfering substances, induces complete rejection of tumor challenge over a broad dose range. To test the hypothesis preparative isotachophoresis (pITP) was used to purify TSTA from electrofocused TSTA fractions. Significant purification was achieved, as immunization with 15 pg to 1.5 ug (5 logs) of pITP-purified TSTA extracted from the MCA-F, or with 1 pg to 10 ng (4 logs) of TSTA from the MCA-D tumor induced specific resistance to tumor challenge. Despite 50,000 fold purification of TSTA, immunization induced partial, not complete, rejection of transplanted tumor cells. This suggests a clear dissociation of the immunogenicity and purification of extracted TSTA, indicating that the induction of partial immunity to tumor challenge is an intrinsic property of extracted TSTA.^

The role of functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) in normal human prostate epithelial cells and prostate cancer development

15-Lipoxygenase 2 (15-LOX2) is a recently cloned human lipoxygenase that shows tissue-restricted expression in prostate, lung, skin, and cornea. The protein level and enzymatic activity of 15-LOX2 have been shown to be down-regulated in prostate cancers compared with normal and benign prostate tissues. We report the cloning and functional characterization of 15-LOX2 and its three splice variants (termed 15-LOX2sv-a, 15-LOX2sv-b, and 15-LOX2sv-c) from primary prostate epithelial (NHP) cells. Western blotting with multiple NHP cell strains and prostate cancer (PCa) cell lines reveals that the expression of 15-LOX2 is lost in all PCa cell lines, accompanied by decreased enzymatic activity. 15-LOX2 is expressed at multiple subcellular locations, including cytoplasm, cytoskeleton, cell-cell border, and nucleus. Surprisingly, the three splice variants of 15-LOX2 are mostly excluded from the nucleus. To elucidate the relationship between nuclear localization, enzymatic activity, and tumor suppressive functions, we established PCa cell clones stably expressing 15-LOX2 or 15-LOX2sv-b. The 15-LOX2 clones express 15-LOX2 in the nuclei and possess robust enzymatic activity, whereas 15-LOX2sv-b clones show neither nuclear protein localization nor arachidonic acid-metabolizing activity. Interestingly, both 15-LOX2- and 15-LOX2sv-b-stable clones proliferate much slower in vitro when compared with control clones. When orthotopically implanted in nude mouse prostate, both 15-LOX2 and 15-LOX2sv-b suppress PC3 tumor growth in vivo. Finally, cultured NHP cells lose the expression of putative stem/progenitor cell markers, slow down in proliferation, and enter senescence. Several pieces of evidence implicate 15-LOX2 plays a role in replicative senescence of NHP cells: (1) promoter activity and the mRNA and protein levels of 15-LOX2 and its splice variants are upregulated in serially passaged NHP cells, which precede replicative senescence and occur in a cell-autonomous manner; (2) PCa cells stably expressing 15-LOX2 or 15-LOX2sv-b show a passage-related senescence-like phenotype; (3) enforced expression of 15-LOX2 or 15-LOX2sv-b in young NHP cells induce partial cell-cycle arrest and senescence-like phenotypes. Together, these results suggest that 15-LOX2 suppress prostate tumor development and do not necessarily depend on arachidonic acid-metabolizing activity and nuclear localization. Also, 15-LOX2 may serve as an endogenous prostate senescence gene and its tumor-suppressing functions might be associated with its ability to induce cell senescence. ^

EVIDENCE OF HUMAN ENDOGENOUS RETROVIRUS K INVOLVEMENT IN HUMAN CANCER

Many lines of clinical and experimental evidence indicate a viral role in carcinogenesis (1-6). Our access to patient plasma, serum, and tissue samples from invasive breast cancer (N=19), ductal carcinoma in situ (N=13), malignant ovarian cancer (N=12), and benign ovarian tumors (N=9), via IRB-approved and informed consent protocols through M.D. Anderson Cancer Center, as well as normal donor plasmas purchased from Gulf Coast Regional Blood Center (N=6), has allowed us to survey primary patient blood and tissue samples, healthy donor blood from the general population, as well as commercially available human cell lines for the presence of human endogenous retrovirus K (HERV-K) Env viral RNA (vRNA), protein, and viral particles. We hypothesize that HERV-K proteins are tumor-associated antigens and as such can be profiled and targeted in patients for diagnostic and therapeutic purposes. To test this hypothesis, we employed isopycnic ultracentrifugation, a microplate-based reverse transcriptase enzyme activity assay, reverse transcription – polymerase chain reaction (RT-PCR), cDNA sequencing, SDS-PAGE and western blotting, immunofluorescent staining, confocal microscopy, and transmission electron microscopy to evaluate v HERV-K activation in cancer. Data from large numbers of patients tested by reverse transcriptase activity assay were analyzed statistically by t-test to determine the potential use of this assay as a diagnostic tool for cancer. Significant reverse transcriptase enzyme activity was detected in 75% of ovarian cancer patients, 53.8% of ductal carcinoma in situ patient, and 42.1% of invasive breast cancer patient samples. Only 11.1% of benign ovarian patient and 16.7% of normal donor samples tested positive. HERV-K Env vRNA, or Env SU were detected in the majority of cancer types screened, as demonstrated by the results shown herein, and were largely absent in normal controls. These findings support our hypothesis that the presence of HERV-K in patient blood circulation is an indicator of cancer or pre-malignancy in vivo, that the presence of HERV-K Env on tumor cell surfaces is indicative of malignant phenotype, and that HERV-K Env is a tumor-associated antigen useful not only as a diagnostic screening tool to predict patient disease status, but also as an exploitable therapeutic target for various novel antibody-based immunotherapies.

HUMAN ENDOGENOUS RETROVIRUS K AS A NOVEL TUMOR-ASSOCIATED ANTIGEN FOR DEVELOPMENT OF AN OVARIAN CANCER VACCINE

HUMAN ENDOGENOUS RETROVIRUS K AS A NOVEL TUMOR-ASSOCIATED ANTIGEN FOR DEVELOPMENT OF AN OVARIAN CANCER VACCINE Publication No.________Kiera Rycaj, B.S.Supervisory Professor: Feng Wang-Johanning, Ph.D., M.D. Ovarian cancer (OC) is the fourth most common cancer in women, and the most lethal gynecologic malignancy in the United States. Adequate screening methodologies are currently lacking and most women first present with either stage III or IV disease. To date, there has been no substantial decrease in death rates and the majorities of patients relapse and die from their disease despite response to first-line therapy. Several proteins, such as CA-125, are elevated in OC, but none has proven specific and sensitive enough to serve as a screening tool or for tumor cell recognition and lysis. It has been proposed that human endogenous retrovirus sequences (HERVs) may play a role in the etiology of certain cancers. In a previous study, we showed that HERV-K envelope (env) proteins are widely expressed in human invasive breast cancer (BC) and ductal carcinoma in situ (DCIS), and elicit both serologic and cell-mediated immune responses in BC patients. We also reported the expression of multiple HERV genes and proteins in OC cell lines and tissues. In this study, we strengthened our previous data by determining that HERV-K env mRNAs are expressed in 69% of primary OC tissues (n=29), but in only 24% of benign tissues (N=17). Immmunohistochemistry (IHC) staining revealed HERV-Kpositivecancer cells detected in endometrioid adenocarcinoma and serous adenocarcinoma but not in benign cyst or normal epithelium biopsies. Immunofluorescence staining (IFS) showed greater cell surface expression of HERV-K in OC samples compared to adjacent uninvolved samples. Enzyme-linked immunosorbent assay (ELISA) data confirmed that a humoral immune response is elicited against HERV-K in OC patients. T-cell responses against HERV-K in lymphocytes from OC patients stimulated with autologous HERV-K pulsed dendritic cells included induction of T-cell proliferation and IFN-γ production. HERV-K–specific cytolytic T cells induced greater specific lysis of OC target cells compared to benign and adjacent uninvolved target cells. Finally, upon T regulatory cell (T-reg) depletion, 64% of OC patients displayed an increase in the specific lysis of target cells expressing HERV-K env protein. These findings suggest that HERV-K env protein is a tumor-associated antigen capable of activating both T-cell and B-cell responses in OC patients, and has great potential in the development of immunotherapy regimens against OC.

Role and regulation of c-KIT gene expression in tumor growth and metastasis of human melanoma

The purpose of this study was to investigate the role of the c-KIT receptor in the progression of human melanoma and the mechanism(s) for the regulation of c-KIT gene expression in human melanoma.^ The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP) (metastatic phenotype) are not well-defined. Expression of the tyrosine-kinase receptor c-KIT progressively decreases during local tumor growth and invasion of human melanomas. To provide direct evidence that the metastasis of human melanoma is associated with the loss of c-KIT expression, highly metastatic A375SM cells, which express very low or undetectable levels of c-KIT, were tranduced with the human c-KIT gene. We demonstrated that enforced c-KIT expression in highly metastatic human melanoma cells significantly suppressed their tumorigenicity and metastatic propensity in nude mice. In addition, we showed that the ligand for c-KIT, SCF, induces apoptosis in human melanoma cells expressing c-KIT under both in vitro and in vivo conditions. These results suggest that loss of c-KIT receptor may allow malignant melanoma cells to escape SCF/c-KIT-mediated apoptosis, thus contributing to tumor growth and eventually metastasis.^ Furthermore, we investigated the possible mechanism(s) for the down-regulation of c-KIT gene expression in malignant melanoma. Sequence analysis of the c-KIT promoter indicated that this promoter contains several consensus binding-site sequences including three putative AP2 and two Myb sites. Although Myb was shown to be associated with c-KIT expression in human hemotopoietic cells, we found no correlation between c-KIT expression and Myb expression in human melanoma cell lines. In contrast, we showed that c-KIT expression directly correlates with expression of AP2 in human melanoma cells. We found that highly metastatic cells do not express the transcription factor AP2. Expression of AP2 in A375SM cells (c-KIT-negative and AP2-negative) was enough to restore luciferase activity driven by the c-KIT promoter in a dose-dependent manner. On the other hand, co-expression of the dominant-negative form of AP2 (AP2B) in Mel-501 cells (c-KIT-positive and AP2-positive) resulted in two-fold reduction in luciferase activity. Electrophoretic mobility shift assays revealed that the c-KIT promoter contains functional AP2 binding sites which could associate with AP2 protein. Endogenous c-KIT gene expression levels were elevated in AP2 stably-transfected human melanoma A375SM cells. Expression of exogenous AP2 in A375SM cells inhibited their tumorigenicity and metastatic potential in nude mice. The c-KIT ligand, SCF, also induced apoptosis in the AP2 stably-transfected A375SM cells. The identification of AP2 as an important regulator for c-KIT expression suggests that AP2 may have tumor growth and metastasis inhibitory properties, possibly mediated through c-KIT/SCF effects on apoptosis of human melanoma cells. Since AP2 binding sites were found in the promoters of other genes involved in the progression of human melanoma, such as MMP2 (72 kDa collagenase), MCAM/MUC18 and P21/WAF-1, our findings suggest that loss of AP2 expression might be a crucial event in the development of malignant melanoma. ^

The role of p53 in skin cancer induction by UV radiation and as a potential tumor antigen in UV-induced murine skin tumors

Carcinoma of the skin is the most common type of human cancer in the United States. Ultraviolet radiation (UVR) present in the sunlight is thought to be the major carcinogen responsible for induction of skin cancer. In UV-associated skin carcinogenesis, mutations in p53 are not only present with very high frequency, but occur early in the course of tumor development. In addition, UV-induced skin tumors in mice exhibit unique immunological characteristics. They are highly antigenic and express both individually-specific tumor transplantation antigens recognized by effector T cells and the UV-associated common antigen recognized by UV-induced suppressor T cells. ^ To examine the hypothesis that p53 plays a critical role in preventing skin cancer induction by UVR, mice constitutively lacking one or two functional p53 alleles were compared to wild-type mice for their susceptibility to UV carcinogenesis. Both p53 +/– and –/– mice showed greater susceptibility to skin cancer induction than wild-type mice, and –/– mice were the most susceptible, Accelerated tumor development in the p53 +/– mice was not associated with loss of the remaining wild-type allele of p53 , but in many cases was associated with UV-induced mutations in p53. Our studies clearly demonstrate the essential role of p53 in protection against UV carcinogenesis, particularly in the eye and epidermis. ^ The role of p53 in the antigenicity of UV-induced murine skin tumors was also addressed. Primary UV-induced tumors from p53 –/–, +/– and +/+ mice were transplanted into both normal and immunosuppressed mice, and rates of tumor rejection were compared. Tumors from mice with only one or no functional p53 alleles were less antigenic than those from mice with two functional p53 alleles. Moreover, tumors with no functional p53 also failed to grow well in chronically UV-irradiated mice. These results indicate that p53 contributes to the strong antigenicity of UV-induced murine skin tumors, and suggest that it may play a critical role in expression of the UV-associated common antigen recognized by suppressor T cells. ^ In this study we also monitored the effect of UVR on the development of lymphoid malignancies in p53 deficient mice. The incidence of lymphoid malignancies in UV-irradiated p53 +/– mice was drastically enhanced compared to that in unirradiated counterparts. The immune responses of the mice were identical and were suppressed to the same extent by UV irradiation regardless of the p53 genotype. These data provide the first experimental evidence that exposure to UVR can contribute to the development of lymphoid neoplasms in genetically susceptible hosts. ^

P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase

Protein tyrosine phosphatases (PTPs) have long been thought to play a role in tumor suppression due to their ability to antagonize the growth promoting protein tyrosine kinases. Recently, a candidate tumor suppressor from 10q23, termed P-TEN, was isolated, and sequence homology was demonstrated with members of the PTP family, as well as the cytoskeletal protein tensin. Here we show that recombinant P-TEN dephosphorylated protein and peptide substrates phosphorylated on serine, threonine, and tyrosine residues, indicating that P-TEN is a dual-specificity phosphatase. In addition, P-TEN exhibited a high degree of substrate specificity, showing selectivity for extremely acidic substrates in vitro. Furthermore, we demonstrate that mutations in P-TEN, identified from primary tumors, tumor cells lines, and a patient with Bannayan–Zonana syndrome, resulted in the ablation of phosphatase activity, demonstrating that enzymatic activity of P-TEN is necessary for its ability to function as a tumor suppressor.

Binding of high-risk human papillomavirus E6 oncoproteins to the human homologue of the Drosophila discs large tumor suppressor protein

In the majority of cervical cancers, DNAs of high-risk mucosotpropic human papillomaviruses (HPVs), such as type 16, are maintained so as to express two viral proteins, E6 and E7, suggesting an essential importance to carcinogenesis. The high-risk HPV E6 proteins are known to inactivate p53 tumor suppressor protein but appear to have an additional, molecularly unknown function(s). In this study, we demonstrate that these E6 proteins can bind to the second PDZ domain of the human homologue of the Drosophila discs large tumor suppressor protein (hDLG) through their C-terminal XS/TXV/L (where X represents any amino acid, S/T serine or threonine, and V/L valine or leucine) motif. This finding is similar to the interaction between the adenomatous polyposis coli gene product and hDLG. E6 mutants losing the ability to bind to hDLG are no longer able to induce E6-dependent transformation of rodent cells. These results suggest an intriguing possibility that interaction between the E6 protein and hDLG or other PDZ domain-containing proteins could be an underlying mechanism in the development of HPV-associated cancers.

HLA class I and II antigens are partially co-clustered in the plasma membrane of human lymphoblastoid cells

Major histocompatibility complex (MHC) class II molecules displayed clustered patterns at the surfaces of T (HUT-102B2) and B (JY) lymphoma cells characterized by interreceptor distances in the micrometer range as detected by scanning force microscopy of immunogold-labeled antigens. Electron microscopy revealed that a fraction of the MHC class II molecules was also heteroclustered with MHC class I antigens at the same hierarchical level as described by the scanning force microscopy data, after specifically and sequentially labeling the antigens with 30- and 15-nm immunogold beads. On JY cells the estimated fraction of co-clustered HLA II was 0.61, whereas that of the HLA I was 0.24. Clusterization of the antigens was detected by the deviation of their spatial distribution from the Poissonian distribution representing the random case. Fluorescence resonance energy transfer measurements also confirmed partial co-clustering of the HLA class I and II molecules at another hierarchical level characterized by the 2- to 10-nm Förster distance range and providing fine details of the molecular organization of receptors. The larger-scale topological organization of the MHC class I and II antigens may reflect underlying membrane lipid domains and may fulfill significant functions in cell-to-cell contacts and signal transduction.

CD68+ cells of monocyte/macrophage lineage in the environment of AIDS-associated and classic-sporadic Kaposi sarcoma are singly or doubly infected with human herpesviruses 7 and 6B

Earlier studies have shown that Kaposi sarcomas contain cells infected with human herpesvirus (HHV) 6B, and in current studies we report that both AIDS-associated and classic-sporadic Kaposi sarcoma contain HHV-7 genome sequences detectable by PCR. To determine the distribution of HHV-7-infected cells relative to those infected with HHV-6, sections from paraffin-embedded tissues were allowed to react with antibodies to HHV-7 virion tegument phosphoprotein pp85 and to HHV-6B protein p101. The antibodies are specific for HHV-7 and HHV-6B, respectively, and they retained reactivity for antigens contained in formalin-fixed, paraffin-embedded tissue samples. We report that (i) HHV-7 pp85 was present in 9 of 32 AIDS-associated Kaposi sarcomas, and in 1 of 7 classical-sporadic HIV-negative Kaposi sarcomas; (ii) HHV-7 pp85 was detected primarily in cells bearing the CD68 marker characteristic of the monocyte/macrophage lineage present in or surrounding the Kaposi sarcoma lesions; and (iii) in a number of Kaposi sarcoma specimens, tumor-associated CD68+ monocytes/macrophages expressed simultaneously antigens from both HHV-7 and HHV-6B, and therefore appeared to be doubly infected with the two viruses. CD68+ monocytes/macrophages infected with HHV-7 were readily detectable in Kaposi sarcoma, but virtually absent from other normal or pathological tissues that harbor macrophages. Because all of the available data indicate that HHV-7 infects CD4+ T lymphocytes, these results suggest that the environment of the Kaposi sarcoma (i) attracts circulating peripheral lymphocytes and monocytes, triggers the replication of latent viruses, and thereby increases the local concentration of viruses, (ii) renders CD68+ monocytes/macrophages susceptible to infection with HHV-7, and (iii) the combination of both events enables double infections of cells with both HHV-6B and HHV-7.