Noninvasive genomic detection of melanoma.

BACKGROUND: Early detection and treatment of melanoma is important for optimal clinical outcome, leading to biopsy of pigmented lesions deemed suspicious for the disease. The vast majority of such lesions are benign. Thus, a more objective and accurate means for detection of melanoma is needed to identify lesions for excision. OBJECTIVES: To provide proof-of-principle that epidermal genetic information retrieval (EGIR™; DermTech International, La Jolla, CA, U.S.A.), a method that noninvasively samples cells from stratum corneum by means of adhesive tape stripping, can be used to discern melanomas from naevi. METHODS: Skin overlying pigmented lesions clinically suspicious for melanoma was harvested using EGIR. RNA isolated from the tapes was amplified and gene expression profiled. All lesions were removed for histopathological evaluation. RESULTS: Supervised analysis of the microarray data identified 312 genes differentially expressed between melanomas, naevi and normal skin specimens (P<0·001, false discovery rate q<0·05). Surprisingly, many of these genes are known to have a role in melanocyte development and physiology, melanoma, cancer, and cell growth control. Subsequent class prediction modelling of a training dataset, consisting of 37 melanomas and 37 naevi, discovered a 17-gene classifier that discriminates these skin lesions. Upon testing with an independent dataset, this classifier discerned in situ and invasive melanomas from naevi with 100% sensitivity and 88% specificity, with an area under the curve for the receiver operating characteristic of 0·955. CONCLUSIONS: These results demonstrate that EGIR-harvested specimens can be used to detect melanoma accurately by means of a 17-gene genomic biomarker.

Multiple fibroblast growth factors support growth of the ureteric bud but have different effects on branching morphogenesis.

Together with glial-derived neurotrophic factor (GDNF), soluble factors present in a metanephric mesenchyme (MM) cell conditioned medium (BSN-CM) are necessary to induce branching morphogenesis of the isolated ureteric bud (UB) in vitro (Proc. Natl. Acad. Sci. USA 96 (1999) 7330). Several lines of evidence are presented here in support of a modulating role for fibroblast growth factors (FGFs) in this process. RT-PCR revealed the expression of two FGF receptors, FGFR1(IIIc) and FGFR2(IIIb), in isolated embryonic day 13 rat UBs, which by indirect immunofluorescence displayed a uniform distribution. Rat kidney organ culture experiments in the presence of a soluble FGFR2(IIIb) chimera or a neutralizing antibody to FGF7 suggested an important contribution of FGFs other than FGF7 to the branching program. Several FGFs, including FGF1, FGF2, FGF7 and FGF10, in combination with GDNF and BSN-CM were found to affect growth and branching of the isolated UB, albeit with very different effects. FGF1 and FGF7 were at extreme ends of the spectrum, with FGF10 (more FGF1-like) and FGF2 (more FGF7-like) falling in between. FGF1 induced the formation of elongated UB branching stalks with distinct proliferative ampullary tips, whereas FGF7 induced amorphous buds displaying nonselective proliferation with little distinction between stalks and ampullae. Electron microscopic examination demonstrated that FGF1 treatment induced cytoskeletal organization, intercellular junctions and lumens along the stalk portion of the developing tubules, while the ampullary regions contained 'less differentiated' cells with an abundant secretory apparatus. In contrast, FGF7-induced UBs displayed this 'less differentiated' morphology regardless of position on the structure and were virtually indistinguishable from FGF1-induced ampullae. Consistent with this, GeneChip array analysis (employing a novel nanogram-scale assay consisting of two rounds of amplification and in vitro transcription for analyzing small quantities of RNA) revealed that FGF7-induced UBs expressed more markers of cell proliferation than FGF1, which caused the UB to express cytoskeletal proteins, extracellular matrix proteins, and at least one integrin, some of which may be important in UB branch elongation. Thus, while the various FGFs examined all support UB growth, FGF1 and FGF10 appear to be more important for branching and branch elongation, and may thus play a role in determination of nephron number and patterning in the developing kidney. These in vitro data may help to explain results from knockout and transgenic studies and suggest how different FGFs may, together with GDNF and other factor(s) secreted by MM cells, regulate branching morphogenesis of the UB by their relative effects on its growth, branching and branch elongation and differentiation, thereby affecting patterning in the developing kidney.

Altered apoptosis pathways in mantle cell lymphoma detected by oligonucleotide microarray.

An imbalance between cellular apoptosis and survival may be critical for the pathogenesis of lymphoma. Therefore, the gene expression pattern in lymph node preparations from patients with mantle cell lymphoma (MCL) was compared to the pattern in nonmalignant hyperplastic lymph nodes (HLs). Oligonucleotide microarray analysis was performed comparing 5 MCLs to 4 HLs using high-density microarrays. The expression data were analyzed using Genespring software. For confirmation, the expression of selected genes was analyzed by real-time polymerase chain reaction using the RNA extracted from 16 MCL and 12 HL samples. The focus was on 42 genes that were at least 3-fold down-regulated in MCL; in addition to the B-cell leukemia 2 (BCL2) system other apoptotic pathways were altered in MCL. The FAS-associated via death domain (FADD) gene that acts downstream of the FAS cascade as a key gene to induce apoptosis was more than 10-fold down-regulated in MCL. Furthermore, the death-associated protein 6 (DAXX) gene, the caspase 2 (CASP2) gene, and the RIPK1 domain containing adapter with death domain (RAIDD) gene, which are key genes in other proapoptotic pathways, were also decreased in the MCL samples. The suggestion is made that in addition to the known overexpression of cyclin D1, which drives entry into the cell cycle, disturbances of pathways associated with apoptosis contribute to the development of MCL. (Blood. 2001;98:787-794)

Repression of IL-2 promoter activity by the novel basic leucine zipper p21SNFT protein.

IL-2 is the major autocrine and paracrine growth factor produced by T cells upon T cell stimulation. The inducible expression of IL-2 is highly regulated by multiple transcription factors, particularly AP-1, which coordinately activate the promoter. Described here is the ability of the novel basic leucine zipper protein p21SNFT to repress AP-1 activity and IL-2 transcription. A detailed analysis of the repression by p21SNFT repression on the IL-2 promoter distal NF-AT/AP-1 site demonstrates that it can bind DNA with NF-AT and Jun, strongly suggesting that it represses NF-AT/AP-1 activity by competing with Fos proteins for Jun dimerization. The importance of this repression is that p21SNFT inhibits the trans-activation potential of protein complexes that contain Jun, thereby demonstrating an additional level of control for the highly regulated, ubiquitous AP-1 transcription factor and the IL-2 gene.

HIV-1 infection despite immediate combination antiviral therapy after infusion of contaminated white cells.

We present a sixth human case in which primary human immunodeficiency virus (HIV-1) infection occurred, despite antiretroviral prophylaxis, after accidental inoculation of infected blood. In the prior five instances, variables such as large virus dose, late administration of antivirals, viral resistance to zidovudine, and pre-existent immunosuppression, may have played a role in the treatment failure. In this case, high-dosage oral zidovudine was given within minutes of the accident and replaced 2 1/2 days later with interferon alpha and dideoxyinosine (ddl). Despite aggressive treatment, HIV-1 infection was demonstrated in blood, spleen, and brain tissue at autopsy 16 days later. Of the tissues studied, detection of HIV-1 was most prominent in the spleen. Double-label immunocytochemistry confirmed the morphologic impression that while some of the infected spleen cells were CD3-positive T cells, the majority were macrophages. Thus, current single or dual (zidovudine, ddl-interferon) therapies for accidental HIV-1 inoculation may not be effective in preventing early infection. Further trials in animals appear warranted to evaluate protection by other strategies, such as passive immunity or combinations of agents that penetrate the brain and attack HIV-1 viral replication at differing sites.

Stimulation of HIV replication in mononuclear phagocytes by leukemia inhibitory factor.

This study examined the effects of leukemia inhibitory factor (LIF) on human immunodeficiency virus (HIV) replication in mononuclear phagocytes (MNP). LIF induced a dose-dependent increase in p24 antigen production in the chronically infected promonocytic cell line U1. The magnitude and time kinetics of the LIF effects were similar to interleukin 1 (IL-1), IL-6, and tumor necrosis factor (TNF), other cytokines known to induce HIV replication in this cell line. To characterize mechanisms responsible for these LIF effects, levels of HIV mRNA, activation of the DNA binding protein nuclear factor (NF)-kB, signal transduction pathways, and potential interactions with other cytokines were analyzed. LIF increased steady-state levels of HIV mRNA at 2.0, 4.3, and 9.2 kB. This was detectable by 24 h and persisted until 72 h. The DNA binding protein NF-kB is a central mediator in cytokine activation of HIV transcription. NF-kB levels were higher in unstimulated U1 cells as compared to the parent cell line U937. In both cell lines LIF increased NF-kB activity. Induction of NF-kB and HIV replication by cytokines are at least in part dependent on reactive oxygen intermediates. The oxygen radical scavenger N-acetyl-L-cysteine, but not an inhibitor of nitric oxide synthase, inhibited LIF-induced HIV replication. LIF induces the production of other cytokines in monocytes but its effects on HIV replication were not inhibited by antibodies to IL-1, TNF, or IL-6. These results identify LIF as a stimulus of HIV replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone-like protein: alternative messenger RNA splicing pathways in human cancer cell lines.

Parathyroid hormone-like protein (PLP) is expressed in a wide variety of cancers and exerts diverse biological effects in addition to hypercalcemia. We studied the expression of the gene for PLP in cancer cell lines derived from different tissues that produce PLP. We used the polymerase chain reaction to evaluate the PLP mRNA species produced in these various cell lines by differential transcription initiation and alternative splicing pathways. A series of exon-specific oligonucleotide primers that hybridize to DNA sequences adjacent to exon-intron splice junctions throughout the PLP gene were designed. These primers were used to evaluate steady state levels of PLP mRNA species. Our analysis with promoter-specific primers demonstrated expression from all three putative transcription start sites of PLP, designated P1, P2, and P3. P2-initiated transcripts were present in all of the cell lines, whereas the presence of P1- and P3-initiated messages were cell line specific. Our analysis with carboxy-terminal coding-specific primers demonstrated the utilization of the alternative splicing pathways that produce all three mature PLP polypeptides, PLP-139, -1-173, and -1-141. The 1-139 mRNA species was found in all of the cell lines, whereas the 1-141 and 1-173 mRNA species were cell line specific. These studies demonstrate the prevalence of the P2-initiated mRNA and the PLP1-139 alternative splicing pathway in the tumor cell lines studied and suggest that other pathways of PLP gene expression may be regulated in a cell line-dependent manner. The particular form of PLP expressed by a given cell can influence its biological effects.

Establishment of human T-cell leukemia virus type I T-cell lymphomas in severe combined immunodeficient mice.

Human T-cell leukemia virus type I (HTLV-I) is recognized as the etiologic agent of adult T-cell leukemia (ATL), a disease endemic in certain regions of southeastern Japan, Africa, and the Caribbean basin. Although HTLV-I can immortalize T lymphocytes in culture, factors leading to tumor progression after HTLV-I infection remain elusive. Previous attempts to propagate the ATL tumor cells in animals have been unsuccessful. Severe combined immunodeficient (SCID) mice have previously been used to support the survival of human lymphoid cell populations when inoculated with human peripheral blood lymphocytes (PBL). SCID mice were injected intraperitoneally with PBL from patients diagnosed with ATL, HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), or from asymptomatic HTLV-I-seropositive patients. Many of these mice become persistently infected with HTLV-I. Furthermore, after human reconstitution was established in these mice, HTLV-I-infected cells displayed a proliferative advantage over uninfected human cells. Lymphoblastic lymphomas of human origin developed in animals injected with PBL from two ATL patients. The tumor cells represented outgrowth of the original ATL leukemic clone in that they had monoclonal or oligoclonal integrations of the HTLV-I provirus identical to the leukemic clone and predominantly expressed the cell surface markers, CD4 and CD25. In contrast, cell lines derived by HTLV immortalization of T cells in vitro did not persist or form tumors when inoculated into SCID mice, indicating differences between in vitro immortalized cells and ATL leukemic cells. This system represents the first small animal model to study HTLV-I tumorigenesis in vivo.

HTLV-1 in acquired adult myopathy.

We report a 53-year-old Jamaican man with 20 years of progressing weakness involving proximal limb muscles and neck flexors. Serum CK was 1100 IU/L. EMG demonstrated spontaneous activity, myopathic motor units, and full recruitment patterns in weak muscles. Muscle biopsy revealed marked myofiber degeneration with extensive fibrosis, suggesting a chronic myopathic process. HTLV-1 antibody was present in serum in high titers by ELISA and Western blot. Immunohistochemistry with rabbit polyclonal antisera to HTLV-1 showed rare staining myocytes. PCR demonstrated HTLV-1 DNA in frozen muscle tissue. This myopathy, associated with HTLV-1 infection, has clinical and pathological features similar to a dystrophy. We recommend serological screening for HTLV-1 in cryptogenic adult myopathies.

Peripheral nerve lesions in HTLV-I associated myelopathy (HAM/TSP).

Peripheral nerve dysfunction (PND) was found in as many as 43% of our patients with human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM/TSP). To evaluate the PND further we biopsied the sural nerve in 6 patients. The histological features were varying degrees of demyelination, remyelination, axonal atrophy and degeneration, and perineurial fibrosis. "Globule" or "sausage" formation was prominent in two of the specimens. Inflammatory infiltrates were absent. No deposits of IgG, IgM, IgA, or complement were detected in the biopsies. No viral antigen or proviral DNA was detected. It is proposed that the PND and the histological findings noted are part of HTLV-I-associated disease and not an unrelated disorder. The pathogenesis of the PND remains unclear. There was no evidence of direct viral infection. The histological findings could represent primary changes induced by viral-triggered release of soluble factors, such as cytokines or secondary changes to more proximal disease, e.g., root involvement.

Early viral brain invasion in iatrogenic human immunodeficiency virus infection.

We report a 68-year-old man who received an IV inoculation of WBCs for an indium radionuclide scan containing 600 to 700 tissue culture infectious doses of human immunodeficiency virus type 1 (HIV-1) from an HIV-1-infected individual. The recipient immediately received zidovudine, then was switched to dideoxyinosine and interferon-alpha, but died of hepatorenal syndrome and hepatic encephalopathy 15 days later. HIV-1 cultures were positive from the recipient's blood on day 14 but not days 0, 1, and 8. At autopsy, cultures of parietal lobe isolated HIV-1. HIV-1 nucleic acid was present in several brain areas, but not in several other organs, by two independent laboratories using the polymerase chain reaction. The brain showed mild perivascular cuffing and a mild lymphocytic meningitis, but there was no evidence of glial nodules, giant cells, or white matter abnormalities. HIV-1 pg41 viral antigen was seen by immunoperoxidase staining in rare infiltrating cells within perivascular and subpial spaces. Thus, HIV-1 was isolated from brain 15 days after mistaken HIV-1 inoculation and 1 day after virus was first recovered from blood.

HTLV-I-associated myelopathy: clinicopathologic correlation with localization of provirus to spinal cord.

A 49-year-old South African man developed a rapidly progressive myelopathy 14 months after blood transfusion and died 1 year after the onset of symptoms. Detailed pathologic examination of the spinal cord was consistent with the diagnosis of HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although no HTLV-I viral particles, antigens, or nucleic acids were detected in situ, polymerase chain reaction assays revealed HTLV-I proviral DNA in cervical, thoracic, and lumbar levels of the spinal cord, with the greatest amount being detected at the thoracic level. These findings suggest that the pathogenesis of HAM/TSP depends on direct infection of neural or immune elements within the spinal cord.

Transforming growth factor-beta and suppression of humoral immune responses in HIV infection.

We reported previously that PBMC from HIV+ patients spontaneously release increased levels of TGF beta 1, contributing to defects in cellular immune responses. This study defines the implications of TGF beta overexpression for humoral immunity in HIV infection. We found that upon Staphylococcus aureus Cowan I (SAC) stimulation of cells from HIV+ donors, B-lymphocyte proliferative responses were decreased. This deficiency correlated closely (r = 0.7, P less than 0.001) with increased TGF beta secretion by PBMC from HIV-infected donors. Conditioned medium from HIV+ PBMC and purified TGF beta 1 had similar inhibitory effects on SAC- or EBV-induced B-cell proliferation, and B cells from HIV-infected donors were as sensitive to inhibition by TGF beta as cells from normal donors. Antibodies to TGF beta 1 neutralized the inhibitory effect of HIV+ culture supernatants on normal B cells and increased low proliferative responses by HIV+ cells. Using PWM as stimulus for B cell differentiation, it was shown that activated TGF beta from HIV+ PBMC is able to significantly reduce the induction of immunoglobulins and this effect was also abrogated by anti-TGF beta. These studies support the concept that in HIV infection, TGF beta is a potent suppressor, not only of the cellular, but of the humoral immune responses as well.

IL-6 production by human T lymphocytes. Expression in HTLV-1-infected but not in normal T cells.

IL-6 is an important regulator of humoral and cellular immunity. Although this cytokine is produced by diverse cell types, it is not known whether it is produced by T lymphocytes under physiologic conditions or which agents can induce T cell expression of IL-6. We analyzed the production of IL-6 by human peripheral blood T cells, human thymocytes, and human T cell lines. In pure populations of these cells, stimulated with different combinations of various mitogens and cytokines, IL-6 activity could not be detected. Analysis of purified T-alpha beta and T-gamma delta cells showed that neither T cell subset produced IL-6. Similarly, IL-6 mRNA was not detected in T cell or thymocyte populations for up to 48 h after stimulation. With the use of a PCR assay, IL-6 mRNA in T cells was found to be virtually negligible, and did not change after T cell activation. By in situ hybridization it was shown that the cells expressing IL-6 mRNA after mitogen activation of PBMC do not belong to the T cell lineage. To analyze whether human T cells express IL-6 in vivo, we examined lymphoid tissues by in situ hybridization. In normal human thymus there was no detectable signal for IL-6. Tonsils showed only few positive cells within the parenchyma, but strong expression of IL-6 by epithelial cells in crypts. In contrast to normal lymph node, which contained only rare cells positive for IL-6, a lymph node from a patient with Castleman's disease showed IL-6 expression in cells occupying the marginal sinus and interfollicular areas. Screening of various human T cell lines showed that all cell lines infected with HTLV-1 secrete IL-6 activity and express IL-6 mRNA. In addition, in vitro infection of peripheral blood T cells with HTLV-1 induced de novo synthesis and secretion of IL-6. Furthermore, IL-6 expression in HTLV-1-infected cells was enhanced by stimulation with IL-1 beta or TNF-alpha. In contrast, IL-6 was not detectable in non-infected T cell lines. These studies indicate that IL-6 may not be a physiologic product of human T lymphocytes and that infection of T cells with HTLV-1 results in aberrant expression of this cytokine.

Transforming growth factor beta and noncytopathic mechanisms of immunodeficiency in human immunodeficiency virus infection.

This study examines the contribution of transforming growth factor beta (TGF beta), one of the most potent endogenous immunosuppressive factors, to the development of immunodeficiency in human immunodeficiency virus (HIV) infection. Increased titers of TGF beta were found in supernatants of peripheral blood mononuclear cells (PBMCs) from HIV-infected donors as compared to uninfected controls (P less than 0.001). This correlated closely with defective responses of CD4+ lymphocytes to the recall antigens tuberculin purified protein derivative or tetanus toxoid. The addition of TGF beta-neutralizing antibody to PBMCs partially restored these defective T-cell responses. Furthermore, purified TGF beta or HIV+ PBMC culture supernatants preferentially inhibited proliferation of CD4+ lymphocytes as compared to CD8+ cells. The increased expression of the TGF beta protein was associated with increased TGF beta mRNA as determined by a polymerase chain reaction assay. This increase in TGF beta protein and mRNA was due to a selective upregulation of the TGF beta 1 isoform. These results indicate that overexpression of TGF beta 1 occurs in HIV-infected individuals and that this cytokine can contribute to impaired immune functions and to depletion of CD4+ T lymphocytes.

Interaction of host cell proteins with the human T-cell leukemia virus type I transcriptional control region. II. A comprehensive map of protein-binding sites facilitates construction of a simple chimeric promoter responsive to the viral tax2 gene product.

We present a map describing the binding of cellular proteins to a 300-base pair (bp) region of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat. The map accounts for nearly all of the DNase I protection reported in a previous study using crude nuclear extracts. Notable features include a complex arrangement of overlapping binding sites encompassing the 21-bp repeat elements (see accompanying paper) as well as binding sites for the transcription factors Sp1 and NF-I that significantly deviate from the previously defined consensus recognition sequences. Based on the binding results, we constructed simple chimeric promoters containing 21-bp repeat elements, Sp1-, and nuclear factor I-binding sites upstream of a TATA box. Transient transfection experiments show that these promoters are expressed in T-cells and are regulated by the viral tax2 gene product. Deletion of the Sp1 and nuclear factor I sites abolishes tax-induction, suggesting that one or both of these proteins play a role in mediating the tax-responsiveness conferred by the 21-bp repeat element.

In vitro mutagenesis of the human T-cell leukemia virus types I and II tax genes.

The tax gene of the human T-cell leukemia virus types I and II (HTLV-I and HTLV-II) is essential for viral replication and acts by increasing the level of RNA transcription. The tax genes of HTLV-I and HTLV-II encode proteins of 40 and 37 kilodaltons, respectively. By in vitro mutagenesis of the tax gene, we have investigated those regions of the protein which are essential for its function. Mutation of either the amino- or carboxy-terminal domain of the protein resulted in loss of trans-activation ability. In addition, specificity of its activity with regard to trans-activation of either the HTLV-I or HTLV-II long terminal repeats was conferred by the first 59 amino acids.

Characterization of the human granulocyte-macrophage colony-stimulating factor promoter region by genetic analysis: correlation with DNase I footprinting.

T-cell activation induces expression of the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). To define the molecular events involved in the induction of GM-CSF gene expression more clearly, we prepared and analyzed deletion mutants of GM-CSF promoter recombinant constructs. The results localized inducible expression to a 90-base-pair region (-53 to +37) which is active in uninfected and human T-cell leukemia virus-infected T-cell lines but not in resting or mitogen-stimulated B cells. DNase I footprinting experiments revealed protection of sequences contained within this region, including a repeated nucleotide sequence, CATT(A/T), which could serve as a core recognition sequence for a cellular transcription factor. Upstream of these GM-CSF promoter sequences is a 15-base-pair region (-193 to -179) which has negative regulatory activity in human T-cell leukemia virus-infected T cells. These studies revealed a complex pattern of regulation of GM-CSF expression in T cells; positive and negative regulatory sequences may play critical roles in controlling the expression of this potent granulopoietin in the bone marrow microenvironment and in localized inflammatory responses.

HTLV-II transactivation is regulated by the overlapping tax/rex nonstructural genes.

The human T-cell leukemia virus (HTLV) types I and II have two nonstructural genes that are encoded in overlapping reading frames. One of these genes, known as tax, has been shown to encode a protein responsible for enhanced transcription (transactivation) from the viral long terminal repeats (LTRs). Genetic evidence indicates that the second nonstructural gene of HTLV-II, here designated rex, acts in trans to modulate tax gene-mediated transactivation in a concentration-dependent fashion. The rex gene may regulate the process of transactivation during the viral life cycle.