Growth Factors and COX2 Expression in Canine Perivascular Wall Tumors.

Canine perivascular wall tumors (PWTs) are a group of subcutaneous soft tissue sarcomas developing from vascular mural cells. Mural cells are involved in angiogenesis through a complex crosstalk with endothelial cells mediated by several growth factors and their receptors. The evaluation of their expression may have relevance since they may represent a therapeutic target in the control of canine PWTs. The expression of vascular endothelial growth factor (VEGF) and receptors VEGFR-I/II, basic fibroblast growth factor (bFGF) and receptor Flg, platelet-derived growth factor B (PDGFB) and receptor PDGFRß, transforming growth factor ß1 (TGFß1) and receptors TGFßR-I/II, and cyclooxygenase 2 (COX2) was evaluated on frozen sections of 40 PWTs by immunohistochemistry and semiquantitatively scored to identify their potential role in PWT development. Statistical analysis was performed to analyze possible correlations between Ki67 labeling index and the expression of each molecule. Proteins of the VEGF-, PDGFB-, and bFGF-mediated pathways were highly expressed in 27 (67.5%), 30 (75%), and 19 (47.5%) of 40 PWTs, respectively. Proteins of the TGFß1- and COX2-mediated pathways were highly expressed in 4 (10%) and 14 (35%) of 40 cases. Statistical analysis identified an association between VEGF and VEGFR-I/II (P = .015 and .003, respectively), bFGF and Flg (P = .038), bFGF and PDGFRß (P = .003), and between TGFß1 and COX2 (P = .006). These findings were consistent with the mechanisms that have been reported to play a role in angiogenesis and in tumor development. No association with Ki67 labeling index was found. VEGF-, PDGFB-, and bFGF-mediated pathways seem to have a key role in PWT development and growth. Blockade of tyrosine kinase receptors after surgery could represent a promising therapy with the aim to reduce the PWT relapse rate and prolong the time to relapse.

Clinical characteristics and factors predicting evolution of asymptomatic IgM monoclonal gammopathies and IgM-related disorders.

We evaluated the prognostic features of 384 asymptomatic IgM-monoclonal gammopathies (aIgM-MGs) and 74 IgM-related disorders (IgM-RDs), two clinically distinct groups as proposed by the Second International Workshop on Waldenström's Macroglobulinemia (WM). The cumulative probability of evolution to lymphoid malignancy at 5 and 10 years was 8% (95% CI, 5-13%) and 29% (95% CI, 21-38%), respectively, in aIgM-MGs; it was 9% (95% CI, 4-20%) and 16% (95% CI, 7-31%), respectively, in IgM-RDs (P=0.26). At a median follow-up of 45 months (12-233), 45 aIgM-MGs (11.7%) evolved to symptomatic WM (n=41), non-Hodgkin's lymphoma (NHL) (n=2), IgM multiple myeloma (n=1), and primary amyloidosis (n=1). At a median follow-up of 60 months (13-195), seven IgM-RDs (9.5%) evolved to symptomatic WM (n=6), and B-chronic lymphocytic leukaemia (n=1). At univariate analysis, in aIgM-MGs bone marrow lymphoplasmacytic infiltration, high erythrocyte sedimentation rate (ESR), haemoglobin level, IgM size, and lymphocytosis significantly correlated with evolution probability. At multivariate analysis, the latter two parameters strongly correlated with prognosis, haemoglobin being associated with a trend for a higher progression risk. In IgM-RDs IgM size, neutropenia, lymphocytosis, detectable Bence Jones proteinuria, and high ESR were associated with evolution probability. In conclusion, asymptomatic IgM-MGs and IgM-RDs are distinct clinical entities with similar probability of transformation to lymphoid malignancy.

Efficacy of an early intensification treatment integrating chemotherapy, autologous stem cell transplantation and radiotherapy for poor risk primary mediastinal large B cell lymphoma with sclerosis.

The aim of our study was to evaluate the impact of an early intensification programme including chemotherapy (CHT), autologous stem cell transplantation (ASCT) and radiation therapy (RT) in patients with primary mediastinal large B cell lymphoma (MLCL) with sclerosis presenting with adverse prognostic factors. Between 1993 and 1999, 19 patients with MLCL were referred to our institution. Four patients were classified as low risk according to the age-adjusted International Prognostic Index (AA-IPI). Fifteen (79%) were categorised in the high-intermediate or high risk group and were considered eligible for ASCT. Induction therapy consisted of VACOP-B (etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone and bleomycin) for 12 weeks. After induction therapy the four low risk patients achieved a complete remission (CR) and did not undergo ASCT. Of the 15 poor risk patients, five achieved CR, seven partial remission (PR), and three showed refractory disease (RD). All these patients received mobilising therapy consisting of high-dose cyclophosphamide. After peripheral stem cell (PSC) collection, to obtain a greater tumor mass reduction before transplantation, the seven patients in PR underwent further treatment with high-dose etoposide and those with RD received two cycles of DHAP (dexamethasone, cytarabine and cisplatin). At the time of ASCT, seven patients were in CR, six in PR and two had RD. After transplantation using BEAM as preparative regimen, all patients but one achieved a CR. Seven patients with minimal (<25%) residual mass at computed tomography scan received further mediastinal RT even if they had a negative Ga(67) scan. At a median follow-up of 35 months from transplantation the disease free survival is 93%. The outcome following this programme of early intensification in poor prognosis MLCL results in a high incidence of durable remissions even in patients with refractory disease.

Cell-to-cell contact results in a selective translocation of maternal human immunodeficiency virus type 1 quasispecies across a trophoblastic barrier by both transcytosis and infection.

Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant child's virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies.

An unusual HIV type 1 env sequence embedded in a mosaic virus from Cameroon: identification of a new env clade. European Network on the study of in utero transmission of HIV-1.

An atypical HIV-1 strain (CAM001) was identified in a pregnant Cameroonian woman in 1995. HMA subtyping of the env region was unsuccessful, and sequence analyses were performed. Unique sequence motifs were found at the V3 tip (GAGRALHA and GAGRAWIHA), and phylogenetic studies showed that the env C2-V5 sequence branched within group M but remained distinct from all known HIV-1 subtypes, while p17 gag branched with the subtype F sequences. Four other HIV group M viruses, undetermined by HMA, of African origin were found to cluster with CAM001 in the C2-V5 sequences. With the BLAST method, we found in databases three strains whose V3 sequences also clustered with CAM001. These unusual env sequences from eight HIV-1 strains derived from Cameroon formed a separate cluster in HIV-1 group M, which we designated k.

Nonproductive human immunodeficiency virus type 1 infection of human fetal astrocytes: independence from CD4 and major chemokine receptors.

Human immunodeficiency virus type 1 (HIV-1) infection of the brain is associated with neurological manifestations both in adults and in children. The primary target for HIV-1 infection in the brain is the microglia, but astrocytes can also be infected. We tested 26 primary HIV-1 isolates for their capacity to infect human fetal astrocytes in culture. Eight of these isolates, independent of their biological phenotype and chemokine receptor usage, were able to infect astrocytes. Although no sustained viral replication could be demonstrated, the virus was recovered by coculture with receptive cells such as macrophages or on stimulation with interleukin-1beta. To gain knowledge into the molecular events that regulate attachment and penetration of HIV-1 in astrocytes, we investigated the expression of several chemokine receptors. Fluorocytometry and calcium-mobilization assay did not provide evidence of expression of any of the major HIV-1 coreceptors, including CXCR4, CCR5, CCR3, and CCR2b, as well as the CD4 molecule on the cell surface of human fetal astrocytes. However, mRNA transcripts for CXCR4, CCR5, Bonzo/STRL33/TYMSTR, and APJ were detected by RT-PCR. Furthermore, infection of astrocytes by HIV-1 isolates with different chemokine receptor usage was not inhibited by the chemokines SDF-1beta, RANTES, MIP-1beta, or MCP-1 or by antibodies directed against the third variable region or the CD4 binding site of gp120. These data show that astrocytes can be infected by primary HIV-1 isolates via a mechanism independent of CD4 or major chemokine receptors. Furthermore, astrocytes are potential carriers of latent HIV-1 and on activation may be implicated in spreading the infection to other neighbouring cells, such as microglia or macrophages.

CXCR4 is a functional coreceptor for infection of human macrophages by CXCR4-dependent primary HIV-1 isolates.

The identification of HIV-1 coreceptors has provided a molecular basis for the tropism of different HIV-1 strains. CXC chemokine receptor-4 (CXCR4) mediates the entry of both primary and T cell line-adapted (TCLA) syncytia-inducing strains. Although macrophages (M phi) express CXCR4, this coreceptor is assumed to be nonfunctional for HIV-1 infection. We addressed this apparent paradox by infecting human monocyte-derived M phi with primary and TCLA isolates that were rigorously characterized for coreceptor usage and by adding the natural CXCR4 ligand, stem cell differentiation factor-1, to specifically block CXCR4-mediated entry. Our results show that primary HIV-1 isolates that selectively use CXCR4 productively infected both normal and C-C chemokine receptor-5-null M phi. By contrast, M phi supported the entry of CXCR4-dependent TCLA strains with variable efficiency but were not productively infected. Thus, the tropism of HIV isolates results from complex virus/host cell interactions both at the entry and postentry levels.

The role of virologic and immunologic factors in mother-to-child transmission of HIV-1.

PROBLEM: More than 90% of human immunodeficiency virus type 1 (HIV-1) infection in children is acquired by mother-to-child transmission. However, infection of the child occurs in between 14 and 35% of cases. METHOD OF STUDY: To understand the mechanisms involved in HIV-1 transmission, we have investigated the antigenic, molecular, and phenotypic characteristics of the virus harbored in infected mothers and their children. RESULTS: A clear correlation was observed between the transmission of the virus and the isolation of viral variants with a rapidly replicating and syncytium-inducing phenotype from the mother. Furthermore, non-transmitting mothers were able to neutralize several primary isolates more frequently than transmitting mothers. The comparison of the viral phenotype and genotype of mother-child pairs showed that the transmitted virus did not have common features, suggesting that transmission is usually not a selective process. CONCLUSIONS: This study suggests that transmission is governed by an interaction of both viral and immunological factors. The results obtained indicate that different strategies can be applied for the prevention of transmission.

In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression.

Following the identification of the C-C chemokines RANTES, MIP-1alpha and MIP-1beta as major human immunodeficiency virus (HIV)-suppressive factors produced by CD8+ T cells, several chemokine receptors were found to serve as membrane co-receptors for primate immunodeficiency lentiretroviruses. The two most widely used co-receptors thus far recognized, CCR5 and CXCR4, are expressed by both activated T lymphocytes and mononuclear phagocytes. CCR5, a specific RANTES, MIP-1alpha and MIP-1 receptor, is used preferentially by non-MT2-tropic HIV-1 and HIV-2 strains and by simian immunodeficiency virus (SIV), whereas CXCR4, a receptor for the C-X-C chemokine SDF-1, is used by MT2-tropic HIV-1 and HIV-2, but not by SIV. Other receptors with a more restricted cellular distribution, such as CCR2b, CCR3 and STRL33, can also function as co-receptors for selected viral isolates. The third variable region (V3) of the gp120 envelope glycoprotein of HIV-1 has been fingered as a critical determinant of the co-receptor choice. Here, we document a consistent pattern of evolution of viral co-receptor usage and sensitivity to chemokine-mediated suppression in a longitudinal follow-up of children with progressive HIV-1 infection. Viral isolates obtained during the asymptomatic stages generally used only CCR5 as a co-receptor and were inhibited by RANTES, MIP-1alpha and MIP-1beta, but not by SDF-1. By contrast, the majority of the isolates derived after the progression of the disease were resistant to C-C chemokines, having acquired the ability to use CXCR4 and, in some cases, CCR3, while gradually losing CCR5 usage. Surprisingly, most of these isolates were also insensitive to SDF-1, even when used in combination with RANTES. An early acquisition of CXCR4 usage predicted a poor prognosis. In children who progressed to AIDS without a shift to CXCR4 usage, all the sequential isolates were CCR5-dependent but showed a reduced sensitivity to C-C chemokines. Discrete changes in the V3 domain of gp120 were associated with the loss of sensitivity to C-C chemokines and the shift in co-receptor usage. These results suggest an adaptive evolution of HIV-1 in vivo, leading to escape from the control of the antiviral C-C chemokines.

C-C chemokines released by lipopolysaccharide (LPS)-stimulated human macrophages suppress HIV-1 infection in both macrophages and T cells.

Human immunodeficiency virus-1 (HIV-1) expression in monocyte-derived macrophages (MDM) infected in vitro is known to be inhibited by lipopolysaccharide (LPS). However, the mechanisms are incompletely understood. We show here that HIV-1 suppression is mediated by soluble factors released by MDM stimulated with physiologically significant concentrations of LPS. LPS-conditioned supernatants from MDM inhibited HIV-1 replication in both MDM and T cells. Depletion of C-C chemokines (RANTES, MIP-1 alpha, and MIP-1 beta) neutralized the ability of LPS-conditioned supernatants to inhibit HIV-1 replication in MDM. A combination of recombinant C-C chemokines blocked HIV-1 infection as effectively as LPS. Here, we report an inhibitory effect of C-C chemokines on HIV replication in primary macrophages. Our results raise the possibility that monocytes may play a dual role in HIV infection: while representing a reservoir for the virus, they may contribute to the containment of the infection by releasing factors that suppress HIV replication not only in monocytes but also in T lymphocytes.

Human recombinant antibody fragments specific for a rye-grass pollen allergen: characterization and potential applications.

One of the major allergens from the pollen of perennial rye grass (Lolium perenne), Lol pII, was used to isolate specific antibody fragments from a random combinatorial library displaying a large repertoire of human Fab on filamentous phages. After five panning cycles on recombinant Lol pII immunotubes, phage binders were isolated and the antibody fragments expressed as soluble Fab molecules in the Escherichia coli periplasm. The DNA sequencing of the clones producing antibodies with the highest binding activity showed three of them to be identical, while one differed by two amino acid substitutions in the heavy chain. The antibody fragments were produced in milligram amounts, affinity-purified and further characterized. They bound the natural allergen as well as the recombinant one, with no cross-reactivity with other allergens contained in the pollen extract of L. perenne. One antibody bound the allergen with Kd = 2.63 x 10(-9) M, as demonstrated by the surface plasmon resonance technique, and was able to compete with a fraction of serum IgE. Epitope mapping using synthetic peptides revealed that antigenic domains, located between amino acids 39 and 51 of Lol pII, are recognized by Fab and polyclonal IgE from sera of allergic donors. The Fab fragments inhibited the binding of serum IgE to the allergen. In vitro experiments on whole blood from allergic subjects showed that recombinant Fab fragments had a blocking activity on histamine release from cells challenged with recombinant Lol pII allergen. Thus, serum IgE and recombinant Fab fragments recognize common epitopes, although they represent the outcome of different maturation and/or selection processes. Our molecular and functional findings altogether indicate that allergen-specific human antibodies may be useful for the characterization of the antigenic structure of allergens. We conclude that a phage library is a powerful source of anti-allergen human antibodies with high affinity and high specificity. Moreover, these molecules may be potentially innovative reagents for the treatment of atopic allergy.

Production, purification and characterization of an anti-(carcinoembryonic antigen) recombinant single-chain Fv antibody fragment.

Specific targeting of radioactive agents to tumour cells has been the main objective of the in vivo use of monoclonal antibodies and their fragments. In particular, specific antibodies to carcinoembryonic antigen (CEA)-expressing tumours can be used either for diagnosis or therapy, if targeting could be improved. The expression of antibody fragments in bacteria allows the preparation of engineered molecules with antigen-binding properties and a better penetration into the tumour. A specific anti-CEA single-chain Fv fragment was produced in bacteria and purified. Its binding activity has been demonstrated in ELISA, immunocytochemistry, immunohistochemistry, fluorescence-activated cell sorting and the kinetic parameters determined by the plasmon surface resonance.