Reduced cytotoxicity by mutation of lysine 590 of Pseudomonas exotoxin can be restored in an optimized, lysine-free immunotoxin.

Immunotoxins, which are fusion proteins of an antibody fragment and a fragment of a bacterial or a plant toxin, induce apoptosis in target cells by inhibition of protein synthesis. ADP-ribosylating toxins often have few lysine residues in their catalytic domain. As they are the target for ubiquitination, the low number of lysines possibly prevents ubiquitin-dependent degradation of the toxin in the cytosol. To reduce this potential degradation, we aimed to generate a lysine-free (noK), Pseudomonas exotoxin (PE)-based immunotoxin. The new generation 24 kDa PE, which lacks all but the furin-cleavage site of domain II, was mutated at lysine 590 (K590) and at K606 in a CD22-targeting immunotoxin and activity was determined against various B cell malignancies in vitro and in vivo. On average, K590 mutated to arginine (R) reduced cytotoxicity by 1.3-fold and K606R enhanced cytotoxicity by 1.3-fold compared to wild type (wt). Mutating K590 to histidine or deleting K590 did not prevent this loss in cytotoxicity. Neither stability nor internalization rate of K590R could explain reduced cytotoxicity. These results highlight the relevance of lysine 590 for PE intoxication. In line with in vitro results, the K606R mutant was more than 1.8-fold more active than the other variants in vivo suggesting that this single mutation may be beneficial when targeting CD22-positive malignancies. Finally, reduced cytotoxicity by K590R was compensated for by K606R and the resulting lysine-free variant achieved wt-like activity in vitro and in vivo. Thus, PE24-noK may represent a promising candidate for down-stream applications that would interfere with lysines.

Usability and Suitability of the Omics-Integrating Analysis Platform tranSMART for Translational Research and Education.

BACKGROUND: Platforms like tranSMART assist researchers in analyzing clinical and corresponding omics data. Usability is an important, yet often overlooked, factor affecting the adoption and meaningful use. Analyses on the specific needs of translational researchers and considerations about the application of such platforms for education are rare. OBJECTIVES: The aim of this study was to test whether tranSMART can be used in education and how well medical students and professional researchers can handle it; to identify which kind of translational researchers-in terms of skills, experienced limitations, and available data-can take advantage of tranSMART; and to evaluate the usability and to generate recommendations for improvements. METHODS: An online-based test has been done by medical students (N = 109) and researchers (N = 26). The test comprised 13 tasks in the context of four typical research scenarios based on experimental and clinical data. A web questionnaire was provided to identify both the needs and the conditions of research as well as to evaluate the system's usability based on the "System Usability Scale" (SUS). RESULTS: Students and researchers were able to handle tranSMART well and coped with most scenarios: cohort identification, data exploration, hypothesis generation, and hypothesis validation were answered with a rate of correctness between 82 and 100%. Of the total, 72.2% of the teaching researchers considered tranSMART suitable for their lessons and 84.6% of the researchers considered the platform useful for their daily work; 65.4% of the researchers named the nonavailability of a platform like tranSMART as a restriction on their research. The usability was rated "acceptable" with a SUS of 70.8. CONCLUSION: tranSMART is potentially suitable for education purposes and fits most of the needs of translational researchers. Improvements are needed on the presentation of analysis results and on the guidance of users through the analysis, especially to ensure the compliance of the analysis with the requirements of statistical testing.

Predictive value of PD-L1 based on mRNA level in the treatment of stage IV melanoma with ipilimumab.

INTRODUCTION: PD-L1 is established as a predictive marker for therapy of non-small cell lung cancer with pembrolizumab. Furthermore, PD-L1 positive melanoma has shown more favorable outcomes when treated with anti-PD1 antibodies and dacarbazine compared to PD-L1 negative melanoma. However, the role of PD-L1 expression with regard to response to checkpoint inhibition with anti-CTLA-4 is not clear, yet. In addition, the lack of standardization in the immunohistochemical assessment of PD-L1 makes the comparison of results difficult. In this study, we investigated the PD-L1 gene expression with a new fully automated technique via RT-PCR and correlated the findings with the response to the anti-CTLA-4 antibody ipilimumab. MATERIALS AND METHODS: Within a retrospective multi-center trial, PD-L1 gene expression was evaluated in 78 melanoma patients in a total of 111 pre-treatment tumor samples from 6 skin cancer centers and analyzed with regard to response to ipilimumab. For meaningful statistical analysis, the cohort was enriched for responders with 30 responders and 48 non-responders. Gene expression was assessed by quantitative RT-PCR after extracting mRNA from formalin-fixed paraffin embedded tumor tissue and correlated with results from immunohistochemical (IHC) stainings. RESULTS AND DISCUSSION: The evaluation of PD-L1 expression based on mRNA level is feasible. Correlation between PD-L1 expression as assessed by IHC and RT-PCR showed varying levels of concordance depending on the antibody employed. RT-PCR should be further investigated to measure PD-L1 expression, since it is a semi-quantitative method with observer-independent evaluation. With this approach, there was no statistical significant difference in the PD-L1 expression between responders and non-responders to the therapy with ipilimumab. The evaluation of PD-L1 expression based on mRNA level is feasible. Correlation between PD-L1 expression as assessed by IHC and RT-PCR showed varying levels of concordance depending on the antibody employed. RT-PCR should be further investigated to measure PD-L1 expression, since it is a semi-quantitative method with observer-independent evaluation. With this approach, there was no statistical significant difference in the PD-L1 expression between responders and non-responders to the therapy with ipilimumab.

A role for MALT1 activity in Kaposi's sarcoma-associated herpes virus latency and growth of primary effusion lymphoma.

Primary effusion lymphoma (PEL) is an incurable malignancy that develops in immunodeficient patients as a consequence of latent infection of B-cells with Kaposi's sarcoma-associated herpes virus (KSHV). Malignant growth of KSHV-infected B cells requires the activity of the transcription factor nuclear factor (NF)-κB, which controls maintenance of viral latency and suppression of the viral lytic program. Here we show that the KSHV proteins K13 and K15 promote NF-κB activation via the protease mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1), a key driver of NF-κB activation in lymphocytes. Inhibition of the MALT1 protease activity induced a switch from the latent to the lytic stage of viral infection, and led to reduced growth and survival of PEL cell lines in vitro and in a xenograft model. These results demonstrate a key role for the proteolytic activity of MALT1 in PEL, and provide a rationale for the pharmacological targeting of MALT1 in PEL therapy.

Structural proteins of Kaposi's sarcoma-associated herpesvirus antagonize p53-mediated apoptosis.

The tumor suppressor p53 is a central regulatory molecule of apoptosis and is commonly mutated in tumors. Kaposi's sarcoma-associated herpesvirus (KSHV)-related malignancies express wild-type p53. Accordingly, KSHV encodes proteins that counteract the cell death-inducing effects of p53. Here, the effects of all KSHV genes on the p53 signaling pathway were systematically analyzed using the reversely transfected cell microarray technology. With this approach we detected eight KSHV-encoded genes with potent p53 inhibiting activity in addition to the previously described inhibitory effects of KSHV genes ORF50, K10 and K10.5. Interestingly, the three most potent newly identified inhibitors were KSHV structural proteins, namely ORF22 (glycoprotein H), ORF25 (major capsid protein) and ORF64 (tegument protein). Validation of these results with a classical transfection approach showed that these proteins inhibited p53 signaling in a dose-dependent manner and that this effect could be reversed by small interfering RNA-mediated knockdown of the respective viral gene. All three genes inhibited p53-mediated apoptosis in response to Nutlin-3 treatment in non-infected and KSHV-infected cells. Addressing putative mechanisms, we could show that these proteins could also inhibit the transactivation of the promoters of apoptotic mediators of p53 such as BAX and PIG3. Altogether, we demonstrate for the first time that structural proteins of KSHV can counteract p53-induced apoptosis. These proteins are expressed in the late lytic phase of the viral life cycle and are incorporated into the KSHV virion. Accordingly, these genes may inhibit cell death in the productive and in the early entrance phase of KSHV infection.

Molecular staging of lymph node-negative colon carcinomas by one-step nucleic acid amplification (OSNA) results in upstaging of a quarter of patients in a prospective, European, multicentre study.

BACKGROUND: Current histopathological staging procedures in colon carcinomas depend on midline division of the lymph nodes with one section of haematoxylin & eosin (H&E) staining only. By this method, tumour deposits outside this transection line may be missed and could lead to understaging of a high-risk group of stage UICC II cases, which recurs in ∼20% of cases. A new diagnostic semiautomated system, one-step nucleic acid amplification (OSNA), detects cytokeratin (CK) 19 mRNA in lymph node metastases and enables the investigation of the whole lymph node. The objective of this study was to assess whether histopathological pN0 patients can be upstaged to stage UICC III by OSNA. METHODS: Lymph nodes from patients who were classified as lymph node negative after standard histopathology (single (H&E) slice) were subjected to OSNA. A result revealing a CK19 mRNA copy number >250, which makes sure to detect mainly macrometastases and not isolated tumour cells (ITC) or micrometastases only, was regarded as positive for lymph node metastases based on previous threshold investigations. RESULTS: In total, 1594 pN0 lymph nodes from 103 colon carcinomas (median number of lymph nodes per patient: 14, range: 1-46) were analysed with OSNA. Out of 103 pN0 patients, 26 had OSNA-positive lymph nodes, resulting in an upstaging rate of 25.2%. Among these were 6/37 (16.2%) stage UICC I and 20/66 (30.3%) stage UICC II patients. Overall, 38 lymph nodes were OSNA positive: 19 patients had one, 3 had two, 3 had three, and 1 patient had four OSNA-positive lymph nodes. CONCLUSIONS: OSNA resulted in an upstaging of over 25% of initially histopathologically lymph node-negative patients. OSNA is a standardised, observer-independent technique, allowing the analysis of the whole lymph node. Therefore, sampling bias due to missing investigation of certain lymph node tissue can be avoided, which may lead to a more accurate staging.

IFN-γ and TNF-α-induced GBP-1 inhibits epithelial cell proliferation through suppression of ß-catenin/TCF signaling.

Proinflammatory cytokines induce guanylate-binding protein 1 (GBP-1) protein expression in intestinal epithelial tissues. GBP-1 has been described as influencing a number of cellular processes important for epithelial homeostasis, including cell proliferation. However, many questions remain as to the role of GBP-1 in intestinal mucosal homeostasis. We therefore sought to investigate the function of proinflammatory cytokine-induced GBP-1 during intestinal epithelial cell proliferation. Through the use of complementary GBP-1 overexpression and small interfering RNA-mediated knockdown studies, we now show that GBP-1 acts to inhibit pro-mitogenic ß-catenin/T cell factor (TCF) signaling. Interestingly, proinflammatory cytokine-induced GBP-1 was found to be a potent suppressor of ß-catenin protein levels and ß-catenin serine 552 phosphorylation. Neither glycogen synthase kinase 3ß nor proteasomal inhibition alleviated GBP-1-mediated suppression of cell proliferation or ß-catenin/TCF signaling, indicating a non-canonical mechanism of ß-catenin inhibition. Together, these data show that cytokine-induced GBP-1 retards cell proliferation by forming a negative feedback loop that suppresses ß-catenin/TCF signaling.

Autonomously vascularized cellular constructs in tissue engineering: opening a new perspective for biomedical science.

In tissue engineering cell cultures play a crucial role besides the matrix materials for the end of substituting lost tissue functions. The cell itself is situated at the cross-roads leading to different orders of scale, from molecule to organism and different levels of function, from biochemistry to macrophysiology. Extensive in vitro investigations have dissected a vast amount of cellular phenomena and the role of a number of bioactive substances has been elucidated in the past. Further, recombinant DNA technologies allow modulation of the expression profiles of virtually all kinds of cells. However, issues of vascularization in vivo limit transferability of these observations and restrict upscaling into clinical applications. Novel in vivo models of vascularization have evolved inspired from reconstructive microsurgical concepts and they encompass axial neovascularization by means of vascular induction. This work represents a brief description of latest developments and potential applications of neovascularization and angiogenesis in tissue engineering.

Expression of calcitonin receptor-like receptor in human vascular tumours.

BACKGROUND: Vascular tumours such as Kaposi's sarcoma and capillary haemangioma are characterised by abnormal vascularisation and proliferation of endothelial cells or neoplastic cells. Adrenomedullin, a potent vasodilative peptide, and its receptor, calcitonin receptor-like receptor (CRLR), play an important part in angiogenesis. AIM: To establish whether this system also plays a part in vascular diseases, showing abnormal proliferation such as vascular tumours. METHODS: CRLR expression was investigated in several specimens of Kaposi's sarcoma and other vascular tumours, using immunohistochemical analysis with a previously described CRLR-specific polyclonal antibody and reverse transcriptase-polymerase chain reaction. RESULTS: Intense and specific CRLR-immunoreactive staining of neoplastic cells was observed in all specimens, which was of greater intensity than similar staining of adjacent normal endothelium. CONCLUSIONS: CRLR is expressed in vascular tumours and, with adrenomedullin, may have a role in neoplastic vascular growth.

The helical domain of GBP-1 mediates the inhibition of endothelial cell proliferation by inflammatory cytokines.

Inflammatory cytokines (IC) activate endothelial cell adhesiveness for monocytes and inhibit endothelial cell growth. Here we report the identification of the human guanylate binding protein-1 (GBP-1) as the key and specific mediator of the anti-proliferative effect of IC on endothelial cells. GBP-1 expression was induced by IC, downregulated by angiogenic growth factors, and inversely related to cell proliferation both in vitro in microvascular and macrovascular endothelial cells and in vivo in vessel endothelial cells of Kaposi's sarcoma. Experimental modulation of GBP-1 expression demonstrated that GBP-1 mediates selectively the anti-proliferative effect of IC, without affecting endothelial cell adhesiveness for monocytes. GBP-1 anti-proliferative activity did not affect ERK-1/2 activation, occurred in the absence of apoptosis, was found to be independent of the GTPase activity and isoprenylation of the molecule, but was specifically mediated by the C-terminal helical domain of the protein. These results define GBP-1 as an important tool for dissection of the complex activity of IC on endothelial cells, and detection and specific modulation of the IC-activated non-proliferating phenotype of endothelial cells in vascular diseases.

Clearance of human herpesvirus 8 from blood and regression of leukopenia-associated aggressive classic Kaposi's sarcoma during interferon-alpha therapy: a case report.

A human immunodeficiency virus-negative woman with severe classic Kaposi's sarcoma, idiopathic leukopenia, and massive spread of human herpesvirus 8 (HHV-8) in circulating cells showed stable disease remission in response to systemic interferon-alpha treatment that was accompanied by increased CD3(+) and CD4(+) T cell numbers and complete clearance of HHV-8 from the circulation. These results suggest a direct relationship between HHV-8 clearance from blood and regression of Kaposi's sarcoma and are consistent with the in vitro inhibitory effects of interferon-alpha on HHV-8 infection.

Activation of matrix-metalloproteinase-2 and membrane-type-1-matrix-metalloproteinase in endothelial cells and induction of vascular permeability in vivo by human immunodeficiency virus-1 Tat protein and basic fibroblast growth factor.

Previous studies indicated that the Tat protein of human immunodeficiency virus type-1 (HIV-1) is a progression factor for Kaposi's sarcoma (KS). Specifically, extracellular Tat cooperates with basic fibroblast growth factor (bFGF) in promoting KS and endothelial cell growth and locomotion and in inducing KS-like lesions in vivo. Here we show that Tat and bFGF combined increase matrix-metalloproteinase-2 (MMP-2) secretion and activation in endothelial cells in an additive/synergistic manner. These effects are due to the activation of the membrane-type-1-matrix-metalloproteinase and to the induction of the membrane-bound tissue inhibitor of metalloproteinase-2 (TIMP-2) by Tat and bFGF combined, but also to Tat-mediated inhibition of both basal or bFGF-induced TIMP-1 and -2 secretion. Consistent with this, Tat and bFGF promote vascular permeability and edema in vivo that are blocked by a synthetic MMP inhibitor. Finally, high MMP-2 expression is detected in acquired immunodeficiency virus syndrome (AIDS)-KS lesions, and increased levels of MMP-2 are found in plasma from patients with AIDS-KS compared with HIV-uninfected individuals with classic KS, indicating that these mechanisms are operative in AIDS-KS. This suggests a novel pathway by which Tat can increase KS aggressiveness or induce vasculopathy in the setting of HIV-1 infection.

Inverse relation of Fas-ligand and tumor-infiltrating lymphocytes in angiosarcoma: indications of apoptotic tumor counterattack.

Fas and Fas-L regulate immune responses through the induction of cell death. Fas-L is commonly expressed in activated immune cells and in the endothelium. In the latter it contributes to the inhibition of transvascular cell migration by the induction of apoptosis in Fas-bearing lymphocytes. Here we investigated whether the Fas/Fas-L system may regulate lymphocyte invasion into angiosarcomas. Fas and Fas-L expression was quantitatively determined in different grade angiosarcomas (n = 40) and related to the number of extravasated tumor-infiltrating lymphocytes (TILs). Fas expression was detected in < 50% of the cases. In positive tumors both the number of Fas-positive cells and the staining intensity were highly variable and did not correlate with the number of TILs, the mean time of survival, and the histopathological tumor grade. By contrast, Fas-L expression was detected in >70% of the cases and the relative numbers of Fas-L-positive cells correlated inversely with the numbers of CD3- and CD8-positive TILs (P < or = 0.004). The survival times of patients with high Fas-L-expressing angiosarcomas were significantly reduced as compared to patients with low Fas-L-expressing tumors. Our results show that angiosarcomas with low Fas-L expression are characterized by numerous TILs, whereas sarcomas with high Fas-L expression show significantly reduced numbers of TILs. These results suggest that the Fas/Fas-L system may repress TIL invasion into angiosarcoma and by this may contribute to the evasion of the anti-tumor immune surveillance of angiosarcoma in the course of an apoptotic tumor counterattack mechanism.

Kaposi's sarcoma-associated herpesvirus serology in Europe and Uganda: multicentre study with multiple and novel assays.

A multicentre study was undertaken to define novel assays with increased inter-assay concordance, sensitivity, specificity and predictive value for serological diagnosis of human herpesvirus type 8 (HHV-8) infection. A total of 562 sera from European and Ugandan human immunodeficiency virus (HIV)-infected or uninfected individuals with or without Kaposi's sarcoma (KS) and blood donors were examined under code by 18 different assays in seven European laboratories. Sera from KS patients and all non-KS sera found positive by at least 70%, 80%, or 90% of the assays were considered "true positive." The validity of the assays was then evaluated by univariate logistic regression analysis. Two immunofluorescence assays (IFA) for detection of antibodies against HHV-8 lytic (Rlyt) or latent (LLANA) antigens and two enzyme-linked-immunosorbent assays (ELISA) (M2, EK8.1) for detection of antibodies against HHV-8 structural proteins were found to be highly concordant, specific, and sensitive, with odds ratios that indicated a high predictive value. When used together, the two IFA (Rlyt-LLANA) showed the best combination of sensitivity (89.1%) and specificity (94.9%). The performance of these assays indicate that they may be used for the clinical management of individuals at risk of developing HHV-8 associated tumours such as allograft recipients.

Serum concentrations of fibroblast growth factor 2 are increased in HIV type 1-infected patients and inversely related to survival probability.

HIV-1-infected patients develop a generalized vasculopathy that is clinically most evident as Kaposi's sarcoma (KS), a multifocally appearing endothelial cell-derived tumor. Fibroblast growth factor 2 (FGF-2) is a potent autocrine and paracrine mitogen of endothelial cells and has been implicated in the cell proliferation and angiogenesis observed in KS. Here we determined by ELISA the FGF-2 serum concentrations in different clinical groups of HIV-1-infected patients. AIDS-KS patients (n = 53) and HIV-1-infected patients without KS (n = 39) revealed significantly increased FGF-2 serum concentrations (median, 4.5 and 4.6 pg/ml, respectively), as compared with the healthy control group (n = 22; median, 2.2 pg/ml; p < 0.01). FGF-2 concentrations were highest in untreated HIV-1-infected patients (median, 8.6 pg/ml) and were significantly decreased in patients undergoing antiretroviral therapy (AZT-median, 4.5 pg/ml; HAART-median, 2.5 pg/ml; p < 0.01). In addition, FGF-2 serum concentrations above 5.2 pg/ml were associated with a statistically significant higher risk of death in HIV-1-infected patients. Multivariate analysis showed that this effect is independent of CD4 levels, localization of KS (cutaneous or visceral), AIDS-defining opportunistic diseases, and therapy. Circulating FGF-2 may contribute to AIDS-associated vasculopathy and may be a sensitive and easily accessible surrogate marker to determine the survival time of HIV-1-infected patients and the efficacy of antiretroviral therapy.

Transcription pattern of human herpesvirus 8 open reading frame K3 in primary effusion lymphoma and Kaposi's sarcoma.

Human herpesvirus 8 (HHV-8) is found in immunoblastic B cells of patients with multicentric Castleman's disease (MCD) and, predominantly in a latent form, in primary effusion lymphoma (PEL) cells and Kaposi's sarcoma (KS) spindle cells. Recent studies have shown that upon reactivation, HHV-8 expresses factors that downregulate major histocompatibility class I proteins and coactivation molecules and that may enable productively infected cells to escape cytotoxic T lymphocytes and natural killer cell responses. One of these viral factors is encoded by open reading frame (ORF) K3. Here we show that in PEL cells, ORF K3 is expressed through viral transcripts that are induced very early upon virus reactivation, including bicistronic RNA molecules containing coding sequences from viral ORFs K3 and 70. Specifically, we found that a bicistronic transcript was expressed in the absence of de novo protein synthesis, thereby identifying a novel HHV-8 immediate-early gene product. Several features of the RNA molecules encoding the K3 product, including multiple transcriptional start sites, multiple donor splicing sites, and potential alternative ATG usage, suggest that there exists a finely tuned modulation of ORF K3 expression. By contrast, ORF K3 transcripts are not detected in the majority of cells present in KS lesions that are latently infected by the virus, suggesting that there are other, as-yet-unknown mechanisms of immune evasion for infected KS spindle cells. Nevertheless, because HHV-8 viremia precedes the development of KS lesions and is associated with the recrudescence of MCD symptoms, the prompt expression of ORF K3 in productively infected circulating cells may be important for virus pathogenesis. Thus, molecules targeting host or viral factors that activate ORF K3 expression or inactivate the biological functions of the K3 product should be exploited for the prevention or treatment of HHV-8-associated diseases in at-risk individuals.

Biology of Kaposi's sarcoma.

Kaposi's sarcoma (KS) is an angioproliferative disease occurring in several different clinical-epidemiological forms that, however, share the same histological traits and are all associated with infection by the human herpesvirus 8 (HHV8). KS initiates in a context of immune dysregulation characterised by CD8+ T cell activation and the production of Th1-type cytokines that induce a generalised activation of endothelial cells leading to adhesion and tissue extravasation of lympho-monocytes, spindle cell formation and angiogenesis. These phenomena are triggered or enhanced by infection with HHV8 that, in turn, is reactivated by the same cytokines. Productively-infected circulating cells are recruited into 'activated' tissue sites where HHV8 finds an optimal environment for establishing a persistent, latent infection of KS spindle cells (KSC). HHV8 dissemination is favoured by virus escape mechanisms and immune dysregulation, and leads to immune responses that are not effective against the virus but, paradoxically, exacerbates the reactive process. Although early KS is a reactive process of polyclonal nature that can regress, in time it can progress in to a true sarcoma. The progression of KS appears to be due to the deregulated expression of oncogenes and oncosuppressor genes, to the long-lasting expression of the HHV8 latency genes and, for AIDS-KS, is promoted by the proliferative and angiogenic effects of the HIV-1 Tat protein.

Human herpesvirus-8 and Kaposi's sarcoma: relationship with the multistep concept of tumorigenesis.

Kaposi's sarcoma (KS) develops through discrete inflammatory-angiogenic stages of polyclonal nature (early-stage lesions) to monomorphic nodules of spindle-shaped cells that can be clonal (late-stage lesions) and resemble true sarcomas. Molecular and epidemiological studies indicate that development of KS is tightly associated with infection by the human herpesvirus-8 (HHV-8). However, only individuals with specific conditions of immunodysregulation develop KS. In these individuals the systemic and tissue increase of Th-1-type cytokines (IC) reactivate HHV-8 infection, leading to increased viral load, antibody titers, and an expanded cell tropism that precedes the clinical appearance of KS. Recruitment of the virus into tissues by infected monocytes and other cell types is facilitated by the endothelial cell activation due to IC. In clinical lesions, HHV-8 infection increases with lesion stage and in late-stage lesions most of the spindle cells are latently infected, whereas only few lyrically infected cells are present, suggesting that latent genes may have a role in the transformation of the early inflammatory-hyperplastic lesion into a real sarcoma. The development of tumors, however, is regulated through a multistep process based on the acquisition by cells of several different capabilities leading to malignant growth. Here we review the available data on the expression of HHV-8-encoded genes in primary KS lesions and, in view of their biological activity, analyze their potential function in different steps of tumorigenesis. By this pragmatic approach interesting insights into potential key functions of HHV-8-encoded genes are found and steps of potential cooperativity with other viral factors (HIV-1-Tat) in the pathogenesis of KS are identified.

Reactivation and role of HHV-8 in Kaposi's sarcoma initiation.

Kaposi's sarcoma (KS) is an angioproliferative disease occurring in several clinical-epidemio-logic forms but all associated with infection by the human herpesvirus-8 (HHV-8). At least in early stages, KS is a reactive disease associated with a state of immune dysregulation characterized by CD8+ T-cell activation and production of Th1-type inflammatory cytokines (IC) that precedes lesion development. In fact, evidence indicates that IC can trigger lesion formation by inducing the activation of endothelial cells that leads to adhesion and tissue extravasation of lymphomonocytes, spindle cell formation, and angiogenesis, and HHV-8 reactivation that, in turn, leads to virus spread to all circulating cell types and virus dissemination into tissues. Due to virus escape mechanisms and deficient immune responses toward HHV-8, virus reactivation and spread are not controlled by the immune system but induce immune responses that may paradoxically exacerbate the reactive process. The virus is recruited into "activated" tissue sites where it finds an optimal environment for growth. In fact, viral load is very low in early lesions, whereas almost all spindle cells are infected in late-stage lesions. Although early KS is a reactive process of polyclonal nature that can regress, in time and in the presence of immunodeficiency, it can progress to a true sarcoma. This is likely due to the long-lasting expression of HHV-8 latency genes in spindle cells associated with the deregulated expression of oncogenes and oncosuppressor genes and, for AIDS-KS, with the effects of the HIV-1 Tat protein.

Cytokine-mediated growth promotion of Kaposi's sarcoma and primary effusion lymphoma.

Kaposi's sarcoma (KS) is an angioproliferative disease particularly frequent and aggressive in patients with AIDS but occurring also in post-transplant patients or in immunocompetent individuals of certain geographic areas. At least in its early stages, KS behaves as a reactive hyperplastic process mediated by inflammatory cytokines and angiogenic factors triggered or exacerbated by human herpesvirus-8 (HHV-8) infection. The HIV Tat protein appears to be responsible for the highly aggressive nature of AIDS-KS. Over time, however, KS may evolve into a true sarcoma in association with the expression of oncogenes and/or HHV-8 latency genes endowed with growth and anti-apoptotic properties. HHV-8 infection is also associated with primary effusion lymphoma (PEL), a rare tumor that similarly develops more frequently in the setting of HIV infection. HHV-8 latency genes are likely to contribute to the neoplastic phenotype of PEL cells, whose growth in vivo may require cytokines and factors from the host, or encoded by the virus.