Identification and sequence analysis of a novel human leukocyte antigen allele B*51:141.

The newly detected HLA-B*51:141 is distinguished from HLA-B*51:08 by a single-nucleotide exchange at codon 30 where D is replaced by Y.

Quality of life, lifestyle behavior and employment experience: a comparison between young and midlife survivors of gynecology early stage cancers.

GOALS: To evaluate differences and changes in quality of life (QoL), lifestyle behavior and employment experience of young in comparison to midlife adults in response to early stage gynecologic cancer diagnoses. METHODS: 263 patients, divided into two age groups (Group A: ≤ 45 and Group B: >45 years), were interviewed on their QoL, lifestyle behavior (dietary habits, tobacco and alcohol use, physical activity) and employment experience (employment status and working time) at diagnosis and within 4 years from the treatment. The QoL was evaluated by European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire C30 (QLQ-C30) and its specific modules for each cancer type (in particular endometrium, cervix, ovarian and breast). RESULTS: Global health status was significantly different between the two groups. In the younger age group a more relevant cancer interference on family life and social activities and a greater impact on perception of health status have been observed. Young women were more affected by fatigue, constipation, gastrointestinal symptoms, lymphedema, poor body image and impaired sexuality. Cancer diagnosis had a major negative impact on employment of younger patients. Conversely, younger patients had overall better health behavior. They reported a higher daily intake of fruits and vegetables, along with lower alcohol consumption, furthermore they were a little more physically active than midlife adults. CONCLUSIONS: To enhance quality of life and to promote healthy lifestyle behavior of female cancer patients, particularly in younger age, it is essential to assure multidisciplinary approaches with specific medical intervention and psychosocial supports. Indeed, midlife adults seem to have a more rapid adaptive tendency to return towards levels of well-being, following cancer diagnosis and treatment, than younger patients.

Human papillomavirus (HPV) genotypes and HPV16 variants and risk of adenocarcinoma and squamous cell carcinoma of the cervix.

OBJECTIVE: Human papillomavirus (HPV) genotypes have been extensively studied in uterine cervix squamous cell carcinoma and HPV16 variants have been found to be associated with increased cancer risk, but few reports have been published on genotype distribution and HPV16 variant prevalence in adenocarcinoma tumors. The objective of this study was to analyze viral genotypes and HPV16 intratypic variants in cervical adenocarcinoma and squamous cell carcinoma of Italian women. METHODS: A total of 39 invasive adenocarcinoma and 132 squamous cell carcinoma were reviewed and classified according to the modified WHO classification. HPV sequences were detected by nested PCR, using the broad spectrum consensus-primer pairs MY09/MY11 and the GP5+/GP6+ system, and genotyped by nucleotide sequence analysis. The HPV16-positive cases were amplified with E6-specific oligonucleotides and amplimers subjected to direct nucleotide sequence for variant identification. RESULTS: The prevalence rate of any HPV infection was 72% in adenocarcinoma, and 85% in cervical squamous cell carcinoma. Among the 140 HPV-positive cancer cases, a total of nine mucosal HPV genotypes (HPV16, 18, 31, 33, 35, 39, 45, 58, 82) epidemiologically classified as carcinogenic or probably carcinogenic viruses were identified. The HPV type 16 was the most common viral type representing 64% and 73% of all infections in adenocarcinoma and squamous cell carcinoma, respectively. The E6 nucleotide sequence analysis of HPV16 isolates allowed the identification of Asian American (AA) variants in 33% of adenocarcinoma and in 20% of squamous cell carcinoma suggesting their stronger association with cancer of glandular origin. CONCLUSION: These results suggest that HPV16 has a high prevalence in both invasive adenocarcinoma and squamous cell carcinoma from Italian patients. Moreover this study confirms previous observations, summarized in a systematic review of the literature, on the increased cancer risk of HPV16 AA class in adenoglandular cancer, possibly related to their more oncogenic behavior compared to HPV16 European variants.

Th2 polarization in peripheral blood mononuclear cells from human immunodeficiency virus (HIV)-infected subjects, as activated by HIV virus-like particles.

We have recently shown that human immunodeficiency virus type 1 (HIV-1) Pr55(gag) virus-like particles (HIV-VLPs), produced in a baculovirus expression system and presenting a gp120 molecule from a Ugandan HIV-1 isolate of clade A, induce maturation and activation of monocyte-derived dendritic cells (MDDCs) with a production of Th1- and Th2-specific cytokines. Furthermore, HIV-VLP-loaded MDDCs are able to induce a primary and secondary response in autologous human CD4(+) T cells in an ex vivo immunization assay. In the present study, we show that similar data can be obtained directly with fresh peripheral blood mononuclear cells (PBMCs), and the HIV-1 seropositivity status, with either low or high viremia, does not significantly impair the immune activation status and the responsiveness of circulating monocyte CD14(+) cell populations to an immunogenic stimulus. Some HIV-1-seropositive subjects, however, show a complete lack of maturation induced by HIV-VLPs in CD14(+) circulating cells, which does not consistently correlate with an advanced status of HIV-1 infection. The established Th2 polarization in both HIV-seropositive groups is efficiently boosted by HIV-VLP induction and does not switch into a Th1 pattern, strongly suggesting that specific Th1 adjuvants would be required for therapeutic effectiveness in HIV-1-infected subjects. These results indicate the possibility of screening PBMCs for donor susceptibility to an immunogen treatment, which would greatly simplify the identification of "responsive" vaccinees as well as the understanding of eventual failures in individuals enrolled in clinical trials.

Cellular prognostic markers in hepatitis-related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and accounts for about 6% of all new cancers diagnosed worldwide. Moreover, it is the third and the fifth leading cause of death from cancer in men and women, respectively. HBV and HCV chronic infection is the main risk factor for HCC. A range of therapies are used in the management of HCC according to the extent and severity of liver disease. In this perspective, evaluation of prognosis represents a crucial step for proper management of HCC patients. However, the clinical outcome can be significantly different in HCC patients within the same stage of disease. Therefore, many efforts have been made to define new parameters with more precise prognostic value, and the search for HCC prognostic markers is gaining momentum. The present review aims at providing an update on cellular prognostic markers for HCC.

HIV type 1 subtype A epidemic in injecting drug user (IDU) communities in Iran.

HIV-1 C2-V3 subgenomic regions of the env gene from Iranian seropositive injecting drug users (IDUs) living in Mashhad have been analyzed to evaluate molecular and phylogenetic relationships with IDUs living in Tehran and identify possible common founder virus isolates. The results show that the viral sequences of the Iranian IDUs are strongly related and form a single cluster within the A subtype related to African Ugandan/Kenyan sub-Saharan isolates. Pairwise nucleotide alignment shows higher average divergence values within the Mashhad group than the Tehran group. Furthermore, the Mashhad sequences show much less conserved amino acid residues in the V3 loop than the Tehran sequences. These data represent the first macro-analysis of the HIV-1 molecular evolution in the Iran and Middle East epidemics and may be extremely relevant to guide the development and implementation of diagnostic as well as preventive/therapeutic approaches in this region.

Genetic and phylogenetic characterization of structural genes from non-B HIV-1 subtypes in Italy.

A molecular and phylogenetic characterization on env and gag subgenomic regions has been performed in our laboratory on HIV-1 variants identified in seropositive individuals residing in Italy, infected in the 1999-2001 period, and five non-B-subtype HIV-1 isolates have been described. To confirm the phylogenetic characterization and to determine the genomic organization of three non-B HIV-1 isolates (A, G, and CRF02- AG), the complete gag, pol, and gp120 ORFs (approx. 6900 bp) have been sequenced for each of them. The phylogenetic tree analyses performed on the whole sequence or on individual genes suggested, for the A and G isolates, the identification of divergent strains that do not cluster into any of the known subsubtypes. This has been further validated by pairwise distance analysis. On the contrary, the phylogenetic classification of the CRF02-AG isolate has been confirmed and an overall typical pattern of intragenomic breakpoints has been observed by a Simplot analysis. These results confirm the constant HIV-1 molecular evolution and indicate the relevance of a continuous molecular monitoring of HIV-1 isolates for the development of appropriate vaccine candidates.

Functional characterization of biodegradable nanoparticles as antigen delivery system.

BACKGROUND: Peptide based vaccines may suffer from limited stability and inefficient delivery to professional antigen-presenting cells (APCs), such as dendritic cells (DCs). In order to overcome such limitations, several types of biodegradable nanoparticles (NPs) have been developed as carrier system for antigens. The present study describes for the first time the extensive biological characterization of cationic NPs made of poly (D,L-lactide-co-glycolide) (PLGA) and polyethylenimine (PLGA/PEI) as delivery system for protein/peptide antigens, with potential in therapeutic cancer vaccine development. RESULTS: Flow cytometry as well as confocal laser scanning microscopy (CLSM) showed that PLGA/PEI NPs are more readily taken up than PLGA NPs by both human CD14(+) monocytes and mouse Hepa 1-6 hepatoma cell line. No signs of toxicity were observed in either cellular setting. Sequential image acquisition by TEM showed an intracellular apical localization for PLGA NPs and a perinuclear localization for PLGA/PEI NPs. Both NPs showed a clathrin-dependent as well as a caveolin-dependent internalization pathway and, once in the cells, they formed multivesicular endosomes (MVE). Finally, an ex vivo priming experiment showed that PLGA/PEI NPs are comparable to PLGA NPs in delivering a non-self antigen (i.e., ovalbumin - OVA) to immature dendritic cells (imDCs), which matured and induced autologous naïve CD4(+) T cells to differentiate to memory (i.e., central memory and effector memory) cells. Such a differentiation was associated with a Th1 phenotype suggesting a downstream activation and amplification of a CD8(+) T cell cytotoxic response. The same OVA antigen in a soluble form was unable to induce maturation of DCs, indicating that both NP formulations provided an intrinsic adjuvanting effect combined to efficient antigen delivery. CONCLUSIONS: Our study represents the first report on side-by-side comparison of PLGA and PLGA/PEI NPs as strategy for protein antigen delivery. PLGA/PEI NPs are superior for cellular uptake and antigen delivery as compared to PLGA NPs. Such an evidence suggests their great potential value for vaccine development, including therapeutic cancer vaccines.

The uganda study on HPV variants and genital cancers.

BACKGROUND: Genital cancers in Uganda have been the most frequently diagnosed cancer in men as well as in women since the 1950s. Genetic studies have detected HPV-16 variants of Af1 class and identified a new sub-class designated Af1-u. OBJECTIVES: The main goal of this study is to analyze the prevalence of HPV strains and HPV variants in anogenital lesions of Ugandan male and female subjects in order to possibly determine their role in the pathogenesis of such lesions and to develop an Ugandan preventive HPV vaccine program. STUDY DESIGN: The study is planning to enroll male and female subjects affected by genital lesions, in particular to collect 200 scrapes/biopsies from women with normal ectocervical epithelium as well as with all different degrees of ectocervical lesions (from CIN 1/LSIL to cervical carcinoma). All samples are analyzed by PCR amplification of the L1 conserved region (nt 6584-7035) and the E6/E7 genes (nt 34-880), nucleotide sequence analysis, homology and phylogenetic studies. Variant distribution studies will be followed by serological studies of prevalence and incidence in 1000 women. PRELIMINARY RESULTS AND CONCLUSIONS: Penile cancers from the Kyadondo County have been analyzed for the presence of HPV sequences. More recently 16 ectocervical scrapes and three biopsies have been received from women attending the Nsambya Hospital and analyzed for the presence and type of HPVs. Our results, obtained by PCR and sequencing analysis, allowed the identification of HPV-16 Af1 sequences in 100% of tumor tissue and in 6.25% of scrapes. HPV 45 was identified only in one tumor together with HPV 16 infection. HPV 33 and HPV 58 were present in 20% and 40%, respectively of HPV positive benign samples. The results are showing a narrowing of the HPV pattern in more advanced lesions, suggesting that mainly HPV-16 Af1 patients are progressing to cancer.

High efficient production of Pr55(gag) virus-like particles expressing multiple HIV-1 epitopes, including a gp120 protein derived from an Ugandan HIV-1 isolate of subtype A.

The main goal of this study was to investigate a novel approach for an efficient and reproducible production of Virus-Like Particles (VLPs) expressing multiple HIV-1 epitopes. The HIV-1 Pr55(gag)-based VLPs have been produced in a Baculovirus expression system, using a transfer vector able to support the independent expression of different open reading frames (ORFs). In this regard, the gp120 derived from 94UG018 HIV-1(A) isolate, previously studied in our laboratory, has been packaged into the VLPs together with nef and pol ORFs. In particular, the gp120(UG) sequence shows a 90% homology in the V3 region compared to African HIV-1 strains of the A-clade. This novel approach is extremely effective for the production of VLPs expressing all the epitopes, as confirmed by Western Blot characterization. Furthermore, the resulting HIV-VLP(A)s show the expected density (1.14--1.18 g/ml) on a 10--60% sucrose gradient and the morphology of an immature virion at standard transmission electron microscopy. Our results demonstrate that this strategy is highly efficient for expressing a balanced amount of multiple epitopes and their packaging in VLP structures, without affecting the Pr55(gag) autoassembling capacities. Furthermore, the genetic transposition performed in a modified E. coli represents a methodological improvement, allowing a faster and more reproducible identification of recombinant Baculovirus DNA molecules.

Induction of neutralizing antibodies and cytotoxic T lymphocytes in Balb/c mice immunized with virus-like particles presenting a gp120 molecule from a HIV-1 isolate of clade A.

We have recently developed a candidate HIV-1 vaccine based on virus-like particles (VLPs) expressing a gp120 from an Ugandan HIV-1 isolate of the clade A (HIV-VLP(A)s). In vivo immunogenicity experiments were performed in Balb/c mice, with an immunization schedule based on a multiple-dose regimen of HIV-VLP(A)s without adjuvants, showing a significant induction of both humoral and cellular immunity. The Env-specific cellular response was investigated in vitro, scoring for both the proliferative response of T helper cells and the cytolytic activity of cytotoxic T lymphocytes (CTLs). Furthermore, immune sera showed >50% neutralization activity against both the autologous field isolate and the heterologous T cell adapted B-clade HIV-1(IIIB) viral strain. This is one of the first examples of HIV-1 vaccines based on antigens derived from the A clade, which represents >25% of all isolates identified world wide. In particular, the A clade is predominant in sub-Saharan countries, where 70% of the global HIV-1 infections occur, and where vaccination is the only rational strategy for an affordable prevention against HIV-1 infection.

Molecular and phylogenetic characterization of HIV variants in Italian primary HIV infections (PHI): identification of non-B subtype variants.

The distribution of Human Immunodeficiency Virus type 1 (HIV-1) clades is evaluated in primary HIV-1 infections (PHIs) occurring through sexual transmission in Lombardia, the Italian region with the highest prevalence/incidence of HIV-1 infections. The two primary inclusion parameters for enrollment were sexual transmission and < 1 year seroconversion. Thirty-four enrolled patients have been analysed so far at the molecular level, to characterize their infecting HIV-1 population. Two HIV-1 genomic regions with different rates of genetic variability, the hypervariable C2-V3 fragment of the env gene and the conserved 5' end of the gag p17, were amplified by Polymerase Chain Reaction (PCR) in peripheral blood mononuclear cells (PBMCs) and characterized by direct DNA sequence analysis. Pairwise nucleotide alignment and phylogenetic analyses show that, although with a high range of nucleotide variability, 32 out of the 34 HIV-1 isolates identified in this PHI cohort fall under the clade B genotype. The two remaining isolates, detected in a couple formed by a Nigerian woman and her Italian partner, consistently cluster with clade G standards in both sub-genomic regions. The amino acid sequences confirm this classification, showing clade-specific residues both in the V3 and p17 regions. These data suggest that the B clade is still prevalently associated with acute primary HIV-1 infections occurring in Italy through sexual transmission. However, the significant intra-clade variability and the identification of non-B clades strongly indicate the relevance of continuous molecular monitoring of the HIV-1 isolates circulating in Italy, for prognostic evaluations as well as preventive and therapeutic strategies.

Electrochemotherapy as "new standard of care" treatment for cutaneous Kaposi's sarcoma.

BACKGROUND: Electrochemotherapy (ECT) is a novel modality for the treatment of skin nodules and cutaneous or subcutaneous tumors that allows delivery of low and non-permeant drug into cells. The aim of this prospective single-center study was to evaluate ECT efficacy in the local treatment of Classic Kaposi's sarcoma (CKS) skin localization stage I-II sec. Brambilla et al. METHODS: Nineteen consecutive patients affected by classic KS were included in this study. All patients underwent blood sampling and concurrent incisional biopsy for histological diagnosis and Kaposi's sarcoma related herpes virus 8 (HHV-8) molecular analysis. ECT treatment of KS cutaneous lesions were performed according to the European Standard Operating Procedures of Electrochemotherapy (ESOPE). The primary endpoint of the study was the evaluation of ECT efficacy in the treatment of KS skin nodules and the assessment of HHV-8 viral load in the peripheral blood following the ECT therapy. RESULTS: Complete response (CR) was observed in 14 (73.6%) patients after first ECT session, while 3 (15.7%) and 2 (10.5%) out of 19 patients received a second and a third ECT treatment, respectively. Clinical response dragged out the whole follow-up period that ranged between 6 and 31 months with a median of 16 months. CONCLUSIONS: Clinical management of CKS skin localizations still represents a challenging task for surgeons and oncologists. Therefore, according to this and other author's recent experiences, ECT is claimed to become the "new standard of care" as first line treatment strategy for stage I-II CKS patients.

Generation of HIV-1 Virus-Like Particles expressing different HIV-1 glycoproteins.

Elicitation of a potent and broadly neutralizing antibody response is the main goal of an effective preventive HIV-1 vaccine. It has been shown by us and others that the expression of Env glycoproteins on the surface of particulate structures, such as Virus-Like Particles (VLPs), could be a more efficient strategy to deliver conformational epitopes to the immune system. To this aim, VLPs expressing native HIV Env gp140 or gp41 glycoproteins have been produced in insect cells using a baculovirus expression system and characterized for appropriate protein expression. VLP-bound HIV gp140 glycoprotein showed the appropriate expression and trimeric conformation. Immunogenicity studies have been performed in BALB/C mice by intra-peritoneal administration and sera from immunized mice have been tested in ELISA assays, for their reactivity with HIV specific antigens, as well as in ex vivo neutralization assay. Sera from immunized animals showed a high reactivity with individual HIV proteins expressed in VLPs. Results of TZM-bl based neutralization assay show that combined sera from animals independently immunized with gp140- or full-length-gp41-expressing VLPs have an additive/synergistic effect in the neutralization activity of HIV pseudoviruses. In conclusion, novel VLPs expressing different HIV Env glycoproteins with native trimeric conformation have been generated, showing the induction of effective antibody response with neutralization activity in TZM-bl neutralization assay. These results confirm the effectiveness of VLPs as presentation and delivery system for conformational proteins and show the improved neutralization activity upon the combination of anti-sera elicited by different HIV envelope antigens, suggesting the possibility of broadening the spectrum of viral epitopes targeted by immune response.

HIV-Gag VLPs presenting trimeric HIV-1 gp140 spikes constitutively expressed in stable double transfected insect cell line.

We have previously described the establishment and characterization of a stably transfected insect cell line for the constitutive and efficient expression of Pr55 HIV Gag proteins, which auto-assemble into enveloped Virus-Like Particles (VLPs) released into the cell culture supernatant. Such HIV-Gag VLPs have been shown to elicit a specific systemic humoral response in vivo, proving the appropriate antigenic presentation of the HIV Gag protein to the immune system. Here we describe the establishment of a stable double transfected insect cell line for the constitutive and reproducible production of Pr55Gag-VLPs expressing on their surface trimeric forms of HIV-1 envelope glycoproteins. The persistence of HIV coding genes has been verified in clonal resistant insect cells, the protein expression and conformation has been verified by Western blot analysis. The resulting HIV-VLPs have been visualized by standard transmission electron microscopy and their immunogenicity has been evaluated in vivo. This represents, to our knowledge, the first example of stable double transfected insect cell line for the constitutive production of enveloped HIV-Gag VLPs presenting trimeric HIV-gp140 on their surface.

Constitutive expression of HIV-VLPs in stably transfected insect cell line for efficient delivery system.

We have previously developed HIV-1 Pr55gag-based virus-like particles (HIV-VLPs) as presentation and delivery model using a transient Baculovirus expression system. Here we describe the establishment and characterization of stably transfected insect cell line for the constitutive and reproducible production of HIV-VLPs. The persistence of HIV gag coding gene has been verified in clonal resistant insect cells and the protein expression has been confirmed by Western blot analysis. The resulting HIV-VLPs have been evaluated by standard transmission electron microscopy and their immunogenicity has been evaluated in vivo. Our results demonstrate that this strategy is highly efficient for constitutive expression of conformational enveloped VLPs which can be employed as presentation and delivery system for pathogen as well as tumor-associated antigens. This represents, to our knowledge, the first example of stably transfected insect cell line for the constitutive production of VLPs.

Short communication: limited induction of IL-10 in PBMCs from HIV-infected subjects treated with HIV-VLPs.

We have recently shown that HIV-1 Pr55gag virus-like particles (HIV-VLPs), produced in a baculovirus expression system and presenting a gp120 molecule from a Ugandan HIV-1 isolate of clade A (HIV-VLP(A)s), induce maturation and activation of antigen-presenting cells (APCs) in fresh peripheral blood mononuclear cells (PBMCs) from seronegative as well as seropositive, with either low or high viremia, HIV-1 subjects. A Th2 polarization has been observed in both HIV seropositive groups, which is efficiently boosted by HIV-VLP induction and does not switch into a Th1 pattern. Here we show that the production of the known immune-suppressive IL-10 is induced in both HIV-seropositive groups at a significantly lower level by HIV-VLPs compared to LPS. These levels, however, appear to still negatively interfere with the innate as well as adaptive Th1-polarized response observed in HIV-seropositive groups. These results indicate that vaccines and novel adjuvants (i.e., TLR agonists, such as LPS) must be evaluated not only for their immunogenicity but also for their potential immune-suppressive effects. In this perspective, fresh ex vivo PBMCs can be of high value for screening the responses as well as eventual failures of vaccinees enrolled in clinical trials.