Carlo Parravicini

Differential Viral Protein Expression in Kaposi’s Sarcoma-Associated Herpesvirus-Infected Diseases : Kaposi’s Sarcoma, Primary Effusion Lymphoma, and Multicentric Castleman’s Disease

Kaposis sarcoma (KS)-associated herpesvirus (KSHV) is linked to KS, primary effusion lymphomas (PEL), and a subset of multicentric Castlemans disease (MCD). Transcript mapping studies using PEL cell lines have allowed preliminary classification of viral gene expression into constitutive (class I) and inducible (class II/III) categories. To determine whether viral gene expression differs in vivo, we examined tissue sections of KSHV-infected disorders, using specific antibodies against proteins that are representative of the different expression classes of KSHV genes. ORF73/LANA appears to be a surrogate marker for KSHV infection because it is constitutively expressed in vitro and in vivo in all KSHV-infected cells. Expression of vIRF1, vIL6, and PF-8 proteins in the infected B cells of MCD lymph nodes reproduces the expression pattern observed in TPA-stimulated KSHV-infected B-cell lines. In contrast, the protein expression of the inducible viral genes that we tested in KS and PEL biopsies is restricted to PF-8 and vIL6, respectively. The tightly restricted expression of KSHV proteins in vivo differs from the dysregulated expression of inducible KSHV genes in vitro and suggests that viral gene expression in KSHV-infected cell lines does not accurately reflect what occurs in diseased tissues. These differences may be related to either cell-specific or immune restriction of viral replication.

Epstein-Barr virus DNA in cerebrospinal fluid from patients with AIDS-related primary lymphoma of the central nervous system

Epstein-Barr virus (EBV) is constantly associated with AIDS-related primary lymphomas of the central nervous system (CNS). To assess whether EBV DNA in cerebrospinal fluid (CSF) could be used as a tumour marker, CSF samples that had been taken within 180 days before death from 85 patients with HIV infection and neurological disorders at necropsy were examined retrospectively by nested polymerase chain reaction (PCR) for EBV. Histologically evident primary CNS lymphomas were found in 17 patients, and EBV was shown in tissue by in-situ hybridisation in 16 of the 16 cases examined. All 17 patients with primary CNS lymphoma had EBV DNA in CSF. EBV DNA was found in CSF from 1 of 68 HIV-infected patients without histologically detectable lymphoma at necropsy. PCR for EBV DNA in CSF was 100% sensitive and 98.5% specific for AIDS-associated primary CNS lymphoma, and may be useful as a diagnostic tumour marker.

Kaposi's Sarcoma-Associated Herpesvirus LANA2 Is a B-Cell-Specific Latent Viral Protein That Inhibits p53

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is associated with three proliferative diseases ranging from viral cytokine-induced hyperplasia to monoclonal neoplasia: multicentric Castlemans disease (CD), Kaposis sarcoma (KS), and primary effusion lymphoma (PEL). Here we report a new latency-associated 1,704-bp KSHV spliced gene belonging to a cluster of KSHV sequences having homology to the interferon regulatory factor (IRF) family of transcription factors. ORFK10.5 encodes a protein, latency-associated nuclear antigen 2 (LANA2), which is expressed in KSHV-infected hematopoietic tissues, including PEL and CD but not KS lesions. LANA2 is abundantly expressed in the nuclei of cultured KSHV-infected B cells. Transcription of K10.5 in PEL cell cultures is not inhibited by DNA polymerase inhibitors nor significantly induced by phorbol ester treatment. Unlike LANA1, LANA2 does not elicit a serologic response from patients with KS, PEL, or CD as measured by Western blot hybridization. Both KSHV vIRF1 (ORFK9) and LANA2 (ORFK10.5) appear to have arisen through gene duplication of a captured cellular IRF gene. LANA2 is a potent inhibitor of p53-induced transcription in reporter assays. LANA2 antagonizes apoptosis due to p53 overexpression in p53-null SAOS-2 cells and apoptosis due to doxorubicin treatment of wild-type p53 U2OS cells. While LANA2 specifically interacts with amino acids 290 to 393 of p53 in glutathione S-transferase pull-down assays, we were unable to demonstrate LANA2-p53 interaction in vivo by immunoprecipitation. These findings show that KSHV has tissue-specific latent gene expression programs and identify a new latent protein which may contribute to KSHV tumorigenesis in hematopoietic tissues via p53 inhibition.

Leishmaniasis among organ transplant recipients

Leishmaniasis is a rarely reported disease among transplant recipients; however, the number of published cases has quadrupled since the beginning of the 1990s. Most cases have been observed in patients living in countries of the Mediterranean basin. Leishmaniasis is most commonly associated with kidney transplantation (77%), and cases are also recorded among patients undergoing liver, heart, lung, pancreas, and bone marrow transplantation. Visceral leishmaniasis (VL) is the most frequently observed clinical presentation, followed by mucosal leishmaniasis and more rarely cutaneous leishmaniasis. Transplant recipients with VL develop the classic clinical form of the disease, which is a febrile hepatosplenic and pancytopenic syndrome. Immunodepression seems to predispose to development of mucosal leishmaniasis caused by viscerotropic strains. Early diagnosis of VL is crucial for patient therapy and outcome; however, this is frequently overlooked or delayed in transplant patients. Pentavalent antimonials are the most commom form of treatment for VL, but have a high incidence of toxicity (34%). Although used in fewer patients, liposomal amphotericin B seems to be better tolerated and should be considered as first-line therapy in transplant recipients.

Clinical Use of Polymerase Chain Reaction Performed on Peripheral Blood and Bone Marrow Samples for the Diagnosis and Monitoring of Visceral Leishmaniasis in HIV-Infected and HIV-Uninfected Patients : A Single-Center, 8-Year Experience in Italy and Review of the Literature

BACKGROUND To overcome some of the limitations of conventional microbiologic techniques, polymerase chain reaction (PCR)-based assays are proposed as useful tools for the diagnosis of visceral leishmaniasis. PATIENTS AND METHODS A comparative study using conventional microbiologic techniques (i.e., serologic testing, microscopic examination, and culture) and a Leishmania species-specific PCR assay, using peripheral blood and bone marrow aspirate samples as templates, was conducted during an 8-year period. The study cohort consisted of 594 Italian immunocompetent (adult and pediatric) and immunocompromised (adult) patients experiencing febrile syndromes associated with hematologic alterations and/or hepatosplenomegaly. Identification of the infecting protozoa at the species level was directly obtained by PCR of peripheral blood samples, followed by restriction fragment-length polymorphism analysis of the amplified products, and the results were compared with those of isoenzyme typing of Leishmania species strains from patients, which were isolated in vitro. RESULTS Sixty-eight patients (11.4%) had a confirmed diagnosis of visceral leishmaniasis. Eleven cases were observed in human immunodeficiency virus (HIV)-uninfected adults, 20 cases were observed in HIV-infected adults, and the remaining 37 cases were diagnosed in HIV-uninfected children. In the diagnosis of primary visceral leishmaniasis, the sensitivities of the Leishmania species-specific PCR were 95.7% for bone marrow aspirate samples and 98.5% for peripheral blood samples versus sensitivities of 76.2%, 85.5%, and 90.2% for bone marrow aspirate isolation, serologic testing, and microscopic examination of bone marrow biopsy specimens, respectively. None of 229 healthy blood donors or 25 patients with imported malaria who were used as negative control subjects had PCR results positive for Leishmania species in peripheral blood samples (i.e., specificity of Leishmania species-specific PCR, 100%). PCR and restriction fragment-length polymorphism analysis for Leishmania species identification revealed 100% concordance with isoenzyme typing in the 19 patients for whom the latter data were available. CONCLUSIONS PCR assay is a highly sensitive and specific tool for the diagnosis of visceral leishmaniasis in both immunocompetent and immunocompromised patients and can be reliably used for rapid parasite identification at the species level.

Activation of latent Kaposi's sarcoma-associated herpesvirus by demethylation of the promoter of the lytic transactivator

Kaposis sarcoma-associated herpesvirus (KSHV) is strongly linked to Kaposis sarcoma, primary effusion lymphomas, and a subset of multicentric Castlemans disease. The mechanism by which this virus establishes latency and reactivation is unknown. KSHV Lyta (lytic transactivator, also named KSHV/Rta), mainly encoded by the ORF 50 gene, is a lytic switch gene for viral reactivation from latency, inasmuch as it is both essential and sufficient to drive the entire viral lytic cycle. Here we show that the Lyta promoter region was heavily methylated in latently infected cells. Treatment of primary effusion lymphoma-delivered cell lines with tetradecanoylphorbol acetate caused demethylation of the Lyta promoter and induced KSHV lytic phase in vitro. Methylation cassette assay shows demethylation of the Lyta promoter region was essential for the expression of Lyta. In vivo, biopsy samples obtained from patients with KSHV-related diseases show the most demethylation in the Lyta promoter region, whereas samples from a latently infected KSHV carrier remained in a methylated status. These results suggest a relationship among a demethylation status in the Lyta promoter, the reactivation of KSHV, and the development of KSHV-associated diseases.

Heterogeneity of spindle cells in Kaposi's sarcoma: comparison of cells in lesions and in culture

The immunophenotype of spindle cells in epidemic, endemic, and classic (sporadic) Kaposis sarcoma (KS) lesions was defined by the demonstration of various cell markers and compared with that of KS-derived cell lines. No significant histological or immunophenotypic differences were observed between the three clinical types of KS at comparable stages. The spindle-cell compartment of the different KS types was composed predominantly of a mixture of proliferating CD45+/CD68+ bone-marrow-derived monocytes and TE7+/collagen+ fibroblastic cells with varying expression of EN4/PAL-E/CD31/CD34/CD36 endothelial-associated antigens and/or smooth-muscle-specific alpha-actin (alpha-actin). The latter cells appeared to represent transitional forms of fibroendothelial and fibromyocytic cells. The in vitro cultured KS-derived cell lines (KS-3, KS-6, and KS-8) expressed the fibroblastic antigen TE7 and smooth-muscle-specific alpha-actin but not leukocytic or endothelial-associated antigens consistent with the phenotype of fibromyoid spindle cells of primary lesions. Neither HIV antigen nor provirus DNA was demonstrable in the epidemic KS lesions. The observed heterogeneity of the spindle-cell compartment further substantiates the view that Kaposis sarcoma, irrespective of clinical setting, expresses salient features more compatible with reactive, tumor-like lesion than clonal sarcoma.

Latent BK virus infection and Kaposi's sarcoma pathogenesis

We have analyzed by PCR skin lesions from classic, endemic and AIDS‐related Kaposis sarcoma (KS), as well as from KS‐derived cell lines, the presence of ubiquitous transforming viruses. BK virus (BKV), a transforming human papovavirus which has been associated with human tumors, was detected in 100% of KS skin lesions and 75% of KS cell lines. KS specimens contained a full‐length, intact BKV early region, but minor rearrangements were observed in some tumors. BKV was also detected with a high prevalence (57–67%) in genital tissues and sperm, thus fulfilling the role of a sexually transmitted agent in KS. The closely related JC virus (JCV), which has never been associated with human malignancies, was present in 11–20% of KS specimens and was detected with a low prevalence (0–21%) in genital tissues and sperm. Simian virus 40 (SV40) was not detected in any KS lesions. Herpes simplex virus (HSV) DNA sequences were detected in 20–25% of KS lesions. Malignant human papillomavirus (HPV) types 16 and 11 were detected in KS specimens with a similar prevalence of 11–83%, suggesting that the presence of HPV‐transforming sequences is not a specific trait of HPV interaction with KS tissue. Furthermore, JCV, SV40, HSV and HPV DNA sequences were not detected in KS cell lines, suggesting that these viruses are not associated to KS neoplastic cells in KS tissue. KS cell lines were also negative for DNA sequences of KS‐HV, the novel herpesvirus detected in primary KS lesions. The constant association of BKV DNA with KS lesions and KS cell lines suggests that BKV‐transforming functions may participate in the development of KS.

Thrombotic Microangiopathy in Patients with Acquired Immunodeficiency Syndrome Before and During the Era of Introduction of Highly Active Antiretroviral Therapy

The incidence of thrombotic microangiopathy (TMA) was retrospectively evaluated in a cohort of 1223 patients with acquired immunodeficiency syndrome (AIDS) who were observed from January 1985 through December 1996 (before the era of highly active antiretroviral therapy [HAART]), and the incidence was prospectively assessed for 347 patients with AIDS during the period of January 1997 through December 2000 (during the HAART era). Seventeen cases were reported in the former cohort (1.4%). The increased risk of developing TMA was statistically significant in patients with cryptosporidiosis or AIDS-related cancer but not in those with other diseases. In the 1997-2000 cohort, no cases were observed during follow-up. TMA is associated with conditions observed in the advanced phases of human immunodeficiency virus infection. The disappearance of TMA during the HAART era may be explained by the lower percentage of patients with long-lasting CD4+ T cell depletion, advanced AIDS, or cryptosporidiosis or who have undergone multiple courses of chemotherapy for treatment of cancer.

Major histocompatibility complex class I molecules are down-regulated at the cell surface by the K5 protein encoded by Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8

The expression of major histocompatibility complex class I (MHC-I) molecules at the cell surface was down-regulated in BC-3 cells infected with Kaposis sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 at early times after treatment with 12-O-tetradecanoylphorbol acetate (TPA), and in HeLa cells transfected with the K5 gene of KSHV. However, an immunoprecipitation study on these cells with anti-MHC-I monoclonal antibody revealed that there was no significant reduction in the synthesis of MHC-I molecules. A pulse-chase analysis followed by endoglycosidase H digestion also demonstrated the stability and transport of MHC-I molecules from the endoplasmic reticulum to at least the medial-GOLGI: K5 antigen was clearly detected by immunohistological examination of samples from Kaposis sarcoma, primary effusion lymphoma and Castlemans disease. These results suggest that the down-regulation of MHC-I molecules by K5 gene expression during reactivation may be important for evading immunological surveillance in the host.