Alessandra Handisurya

Different Heparan Sulfate Proteoglycans Serve as Cellular Receptors for Human Papillomaviruses

ABSTRACT Papillomaviruses replicate in stratified epithelia of skin and mucosa. Infection with certain human papillomavirus (HPV) types is the main cause of anogenital neoplasia, in particular cervical cancer. Early events of papillomavirus infectivity are poorly understood. While heparan sulfate proteoglycans (HSPGs) mediate initial binding to the cell surface, the class of proteins carrying heparan sulfates has not been defined. Here we examined two processes of papillomavirus infection, attachment of virus-like particles (VLP) to cells and infection with authentic HPV type 11 (HPV11) virions. Of the HSPGs, syndecan-1 is the major epithelial form and is strongly upregulated in wound edge keratinocytes. We employed K562 cells, which lack HSPGs except minor amounts of endogenous betaglycan, and stable clones that express cDNAs of syndecan-1, syndecan-4, or glypican-1. Binding of VLP correlated with levels of heparan sulfate on the cell surface. Parental K562 bound HPV16 VLP weakly, whereas all three K562 transfectants demonstrated enhanced binding, with the highest binding capacity observed for syndecan-1-transfected cells, which also expressed the most HSPG. For HPV11 infectivity assays, a high virion inoculum was required to infect K562 cells, whereas ectopic expression of syndecan-1 increased permissiveness eightfold and expression of syndecan-4 or glypican-1 fourfold. Infection of keratinocytes was eliminated by treatment with heparitinase, but not phospholipase C, further implicating the syndecan family of integral membrane proteins as receptor proteins. Human keratinocytes with a homozygous deletion of α6 integrin are permissive for HPV11 infection. These results indicate that several HSPGs can serve as HPV receptors and support a putative role for syndecan-1, rather than α6 integrin, as a primary receptor protein in natural HPV infection of keratinocytes.

Cytokine expression pattern in benign prostatic hyperplasia infiltrating T cells and impact of lymphocytic infiltration on cytokine mRNA profile in prostatic tissue.

The aim of the study is to characterize the type of immune response in benign prostatic hyperplasia (BPH) tissue. BPH tissue–derived T cells (n = 10) were isolated, activated (PMA + ionomycin), and analyzed for intracellular reactivity with anti–IFN-γ and IL-2, -4, -5, -6, -10, and -13, as well as TNF-α and -β by four-color flow cytometry. Lymphokine release was tested using Th1/Th2 cytokine bead arrays. The amount of IFN-γ and IL-2, -4, -13, and TGF-β mRNA expressed in normal prostate (n = 5) was compared with that in BPH tissue separated into segments with normal histology (n = 5), BPH histology with (n = 10) and without (n = 10) lymphocytic infiltration, and BPH nodules (n = 10). Expression of lymphokine receptors was analyzed by immunohistology, flow cytometry, and RT-PCR. We found that 28 ± 18% of BPH T helper cells were IFN-γ+/IL-4− Th1 cells, 10 ± 2% were IFN-γ−/IL-4+ Th2, and 12 ± 6% were IFN-γ+/IL-4+ Th0 cells. In relation, cytotoxic and double-negative BPH T lymphocytes showed a slight decrease in Th1 and Th0 in favor of Th2. In double-positive BPH T lymphocytes, the trend toward Th2 (35 ± 15%) was significant (Th1: 12 ± 7%; Th0: 5 ± 4%). Lymphokine release upon stimulation was found in the case of IL-2, IL-5, IFN-γ, and TNF-α > 4 μg; of IL-4 > 2 μg; and of IL-10 > 1 μg/ml. Expression of lymphokine mRNA in tissue was increased (2- to 10-fold) in infiltrated BPH specimens with and without BPH histology. The infiltrated BPH specimens with normal histology differed from those with BPH histology, most evident by the significant decrease in IFN-γ and the increase in TGF-β mRNA expression. Infiltrated BPH specimens with BPH histology expressed significantly more IFN-γ (5-fold), IL-2 (10-fold), and IL-13 (2.8-fold) when compared with noninfiltrated BPH specimens. BPH nodules, however, showed the highest level of expression of IL-4 and IL-13, with only intermediate levels of IFN-γ and very low levels of IL-2 mRNA. Immune response in histologically less transformed BPH specimens is primarily of type 1, whereas in chronically infiltrated nodular BPH and especially within BPH nodules, it is predominantly of type 0 or type 2.

Vascular endothelial growth factor receptor 2 (VEGFR2) expression in squamous cell carcinomas of the head and neck

Objectives Vascular endothelial growth factor receptor 2 (VEGFR2; Flk‐1 [fetal liver kinase]/KDR [kinase insert domain containing receptor]) has been identified as a high affinity receptor for vascular endothelial growth factor (VEGF) on vascular endothelium. Head and neck squamous cell carcinomas (HNSCC) have already been shown to produce substantial amounts of VEGF. VEGFR2 is supposed to play a major role in tumor‐neoangiogenesis.

EXPRESSION OF THE VEGF-RECEPTOR Flt-1 IN BENIGN, PREMALIGNANT AND MALIGNANT PROSTATE TISSUES

PURPOSE Vascular endothelial growth factor (VEGF) is one of the most potent regulators of angiogenesis and has been shown to act upon two tyrosine kinase family receptors: c-fms-like tyrosine kinase (Flt-1) and fetal liver kinase. Preliminary reports have emphasized that expression of VEGF receptors is endothelial cell-specific. In this study we verified the localization and distribution of Flt-1 protein and mRNA expression in prostatic adenocarcinoma (CaP) as well as prostate intraepithelial neoplasia (PIN) and benign prostatic hyperplasia (BPH). MATERIALS AND METHODS 30 selected surgical specimens exhibiting areas with CaP, PIN and BPH histology were evaluated for Flt-1 protein expression by immunohistochemistry. Results were compared with tumor differentiation (Gleason-Score), serum-PSA and clinical followup. Flt-1 synthesis by prostatic carcinoma cell lines, freshly isolated BPH epithelial cells (BPH-EC) and stromal cells was investigated using RT-PCR and intron spanning primer. RESULTS VEGF receptor Flt-1 specific anti-sera revealed significant staining of prostatic endothelial cells, but the reactivity was not restricted to endothelial cells. BPH-epithelial cells of all specimens reacted significantly with anti-Flt-1. In contrast, tumor cells failed to react with anti-Flt-1 in 56% of the specimens. BPH-EC revealed a uniform anti-Flt-1 reactivity, which was less pronounced and weaker in PIN. Loss of anti-Flt-1 reactivity of prostatic tumor cells did not correlate with preoperative PSA serum levels but increased with tumor dedifferentiation. Interestingly, tumor cells of all CaP specimens with a Gleason score of >8 exhibit no anti-Flt-1 immunoreactivity. Accordingly while PC3, DU145 and LNCaP cells were negative when tested using RT-PCR all BPH tissue derived BPH-EC revealed Flt-1 coding mRNA expression. CONCLUSIONS Widespread distribution of VEGF receptor Flt-1 in BPH, PIN and prostate cancer specimens suggests that VEGF function in prostate is not restricted to endothelial cells and angiogenesis. However, since the receptor is lost in CaP cells and with tumor dedifferentiation, these yet unknown effects of VEGF on epithelial cells are obviously suppressed with malignant transformation.

Papillomavirus-Like Particles Are an Effective Platform for Amyloid-β Immunization in Rabbits and Transgenic Mice

Immunization with amyloid-β (Aβ) prevents the deposition of Aβ in the brain and memory deficits in transgenic mouse models of Alzheimer’s disease (AD), opening the possibility for immunotherapy of AD in humans. Unfortunately, the first human trial of Aβ vaccination was complicated, in a small number of vaccinees, by cell-mediated meningoencephalitis. To develop an Aβ vaccine that lacks the potential to induce autoimmune encephalitis, we have generated papillomavirus-like particles (VLP) that display 1–9 aa of Aβ protein repetitively on the viral capsid surface (Aβ-VLP). This Aβ peptide was chosen because it contains a functional B cell epitope, but lacks known T cell epitopes. Rabbit and mouse vaccinations with Aβ-VLP were well tolerated and induced high-titer autoAb against Aβ, that inhibited effectively assembly of Aβ1–42 peptides into neurotoxic fibrils in vitro. Following Aβ-VLP immunizations of APP/presenilin 1 transgenic mice, a model for human AD, we observed trends for reduced Aβ deposits in the brain and increased numbers of activated microglia. Furthermore, Aβ-VLP vaccinated mice also showed increased levels of Aβ in plasma, suggesting efflux from the brain into the vascular compartment. These results indicate that the Aβ-VLP vaccine induces an effective humoral immune response to Aβ and may thus form a basis to develop a safe and efficient immunotherapy for human AD.

Local Intratumoral Tumor Necrosis Factor-α and Systemic IFN-α2b in Patients with Locally Advanced Prostate Cancer

To examine tolerability and activity of local, intratumoral tumor necrosis factor-α (TNF-α) and systemic interferon-α2b (IFN-α2b) in locally advanced, hormone-resistant prostate cancer (LA-HRPC), 10 patients with LA-HRPC (T4NxM0, n = 3, T4NxM1, n = 5; T4N1M1, n = 2) were treated with recombinant TNF-α injected locally into prostate tumor tissue at 4-week intervals (maximum of four cycles) combined with intermittent subcutaneous (s.c.) administration of 5 × 106 IU IFN-α2b. Twenty-nine TNF-α cycles were administered. Despite significant TNF-α leakage into the systemic circulation 2 h after intraprostatic application (from a mean of 9 to a mean of 416 pg/ml; p = 0.0034), TNF-α (and IFN-α2b) was well tolerated (WHO grade 1-2 toxicity), possibly because of its rapid neutralization by increasing soluble 55-kDa and 75-kDa TNF receptor levels in the serum (mean increase 268% and 91%, respectively) at the same time. TNF-α induced prostate tumor cell necrosis in all patients, leading to a significant reduction of p...

Diseases caused by human papillomaviruses (HPV)

Human papillomaviruses (HPV) are non‐enveloped tumor viruses with a double stranded DNA approximately 8 kilobases in length. The viral genome is enclosed by a spherical capsid with icosahedral symmetry and a diameter of about 55 nm. More than 100 HPV types have been identified. They infect the squamous epithelia of skin and mucosa and usually cause benign papillomas or warts. Persistent infection with high‐risk oncogenic HPV causes all cervical cancers, most anal cancers, and a subset of vulvar, vaginal, penile and oropha‐ryngeal cancers. In recent years cutaneous beta‐HPV types have been associated with the pathogenesis of non‐melanoma skin cancers. Two prophylactic HPV vaccines based on virus‐like particles (VLP) are licensed. These are up to 100% effective in preventing HPV 16 and HPV 18 infections and associated genital lesions in women, who have not been previously infected with these types. One vaccine also prevents genital warts caused by HPV 6 and HPV 11.

Vaccination with prion peptide-displaying papillomavirus-like particles induces autoantibodies to normal prion protein that interfere with pathologic prion protein production in infected cells.

Prion diseases are fatal neurodegenerative disorders caused by proteinaceous infectious pathogens termed prions (PrPSc). To date, there is no prophylaxis or therapy available for these transmissible encephalopathies. Passive immunization with monclonal antibodies recognizing the normal host‐encoded prion protein (PrPC) has been reported to abolish PrPSc infectivity and to delay onset of disease. Because of established immunologic tolerance against the widely expressed PrPC, active immunization appears to be difficult to achieve. To overcome this limitation, papillomavirus‐like particles were generated that display a nine amino acid B‐cell epitope, DWEDRYYRE, of the murine/rat prion protein in an immunogenic capsid surface loop, by insertion into the L1 major capsid protein of bovine papillomavirus type 1. The PrP peptide was selected on the basis of its previously suggested central role in prion pathogenesis. Immunization with PrP–virus‐like particles induced high‐titer antibodies to PrP in rabbit and in rat, without inducing overt adverse effects. As determined by peptide‐specific ELISA, rabbit immune sera recognized the inserted murine/rat epitope and also cross‐reacted with the homologous rabbit/human epitope differing in one amino acid residue. In contrast, rat immune sera recognized the murine/rat peptide only. Sera of both species reacted with PrPC in its native conformation in mouse brain and on rat pheochromocytoma cells, as determined by immunoprecipitation and fluorescence‐activated cell sorting analysis. Importantly, rabbit anti‐PrP serum contained high‐affinity antibody that inhibited de novo synthesis of PrPSc in prion‐infected cells. If also effective in vivo, PrP–virus‐like particle vaccination opens a unique possibility for immunologic prevention of currently fatal and incurable prion‐mediated diseases.

A quadrivalent HPV vaccine induces humoral and cellular immune responses in WHIM immunodeficiency syndrome.

WHIM-syndrome is an inherited immunodeficiency disorder with abnormal susceptibility to human papillomavirus (HPV) infection and diseases. We determined safety and immunogenicity to a quadrivalent HPV vaccine in WHIM-syndrome by detection of HPV-specific antibodies and lymphoproliferation. In virus-like-particle (VLP)-ELISA, a WHIM patient showed antibody titers up to 400 for HPV-6/11/16/18, whereas immuno-competent controls developed titers of 6400-25,600. In pseudovirion assays, the patients neutralization titers ranged from 20 to 400 to the four HPV vaccine types, while titers of 1600-25,600 were detected in healthy vaccinees. Specific proliferation of PBMC of the WHIM patient to the HPV vaccine was demonstrated. This first report on response to HPV vaccination in WHIM-immunodeficiency highlights that patients with WHIM-syndrome, and probably other immunodeficiencies, may benefit from HPV immunoprophylaxis.

Bovine papillomavirus type 1 (BPV1) and BPV2 are closely related serotypes

Infection with bovine papillomavirus type 1 (BPV1) or BPV2 induces fibropapillomas in cows and skin sarcoids in horses. Prophylactic vaccination targeting BPV1 and BPV2 may reduce the incidence of these economically important diseases. The L1 major capsid proteins of BPV1 and BPV2 were expressed in Sf-9 insect cells and both self-assembled into virus-like particles (VLPs). Using conformation-dependent monoclonal antibodies (mAb) both type-specific and shared epitopes were detected. Antisera were raised against BPV1 or BPV2 VLP using alum adjuvant, and their (cross)neutralization capacity was tested by C127 neutralization assays using native BPV1 and BPV2 virions, or by BPV1 pseudovirion assay. Antisera induced by either VLP vaccine were able to robustly (cross-)neutralize heterologous as well as homologous types, indicating that BPV1 and BPV2 are closely related serotypes. These results suggest that a monovalent BPV1 (or BPV2) VLP vaccine may potentially protect against both BPV1 and BPV2 infections and associated diseases.