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Objective: To investigate whether the upregulatory effect of interleukin (IL)-10 on HIV expression in a model of latent HIV infection is mediated by induction of endogenous tumour necrosis factor (TNF)-alpha and TNF receptors (TNFR). Design: The latently HIV-infected promonocytic cell line U1 was examined, because in this in vitro model IL-10 has been shown to synergize with multiple cytokines, including TNF-alpha, in enhancing HIV production. Methods: Membrane-bound TNF-alpha, TNFR-1 and TNFR-2 surface expression were determined by flow cytometry. TNF-alpha mRNA was estimated by competitive polymerase chain reaction (PCR), and TNF-alpha, soluble TNFR-1 and soluble TNFR-2 super natant content by enzyme-linked immunosorbent assay. HIV-1 expression was quantitated by reverse transcriptase assay and p24 antigen release. Results: We demonstrated that IL-10 induces a time and cell-concentration dependent upregulation of HIV expression in U1 cells. This effect is mediated through the endogenous production of TNF-alpha as demonstrated by blocking experiments with anti-TNF-alpha antibodies and by detection of IL-10-induced increase of TNF-alpha mRNA by competitive PCR. More importantly, IL-10 is able to upregulate membrane-bound TNF-alpha and TNFR-1, along with a consistent increase in the shedding of soluble TNFR-1 without inducing detectable TNF-alpha secretion. Conclusions: IL-10 activates HIV expression through the membrane-bound TNF-alpha/TNFR-1 pathway, suggesting an amplification mechanism of HIV expression that might occur during cell-to-cell interaction. This positive regulatory effect of IL-10 in an in vitro model of chronic HIV infection is consistent with the inexorable progression of disease seen in advanced patients when both IL-10 and TNF-alpha are elevated.

The long-term kinetics of T cell production following highly active antiretroviral therapy (HAART) were investigated in blood and lymph node in a group of HIV-infected subjects at early stage of established infection and prospectively studied for 72 wk. Before HAART, CD4 and CD8 T cell turnover was increased. However, the total number of proliferating CD4+T lymphocytes, i.e., CD4+Ki67+T lymphocytes, was not significantly different in HIV-infected (n= 73) and HIV-negative (n= 15) subjects, whereas proliferating CD8+Ki67+T lymphocytes were significantly higher in HIV-infected subjects. After HAART, the total body number of proliferating CD4+Ki67+T lymphocytes increased over time and was associated with an increase of both naive and memory CD4+T cells. The maximal increase (2-fold) was observed at week 36, whereas at week 72 the number of proliferating CD4+T cells dropped to baseline levels, i.e., before HAART. The kinetics of the fraction of proliferating CD4 and CD8 T cells were significantly correlated with the changes in the total body number of these T cell subsets. These results demonstrate a direct relationship betweenex vivomeasures of T cell production and quantitative changes in total body T lymphocyte populations. This study provides advances in the delineation of the kinetics of T cell production in HIV infection in the presence and/or in the absence of HAART.

Evolutionary patterns of virus replication and distribution in lymphoid tissue during the early phases of HIV infection have not been delineated. Lymph node (LN) biopsies were excised from patients at different times after the estimated time of primary infection. Within 3 months of the acute viral syndrome, HIV was mostly present in individual virus-expressing cells in LNs; trapping of virions in the follicular dendritic cell (FDC) network was minimal or absent, but was the predominant form of HIV detected in LNs of subjects with chronic infection, either recent (4–20 months after primary infection) or long-term (>2–3 years after primary infection). Plasma viremia was significantly higher in patients during the first 3 months than in those recently infected; however, there were no significant differences in the number of virus-expressing cells per square millimeter of LN tissue in these two groups. Numbers of virus-expressing cells in lymphoid tissue were significantly lower in the subjects with long-term infection than in the other two groups. Therefore, during the transition from primary to chronic HIV infection, the level of HIV replication in lymphoid tissue remains elevated despite the fact that viremia is significantly downregulated. These findings have implications for therapeutic strategies in primary HIV infection and in recent seroconvertors.

Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.

Poor penetration of antitumor drugs into the extravascular tumor tissue is often a major factor limiting the efficacy of cancer treatments. Our group has recently described a strategy to enhance tumor penetration of chemotherapeutic drugs through use of iRGD peptide (CRGDK/RGPDC). This peptide comprises two sequence motifs: RGD, which binds to αvβ3/5 integrins on tumor endothelia and tumor cells, and a cryptic CendR motif (R/KXXR/K-OH). Once integrin binding has brought iRGD to the tumor, the peptide is proteolytically cleaved to expose the cryptic CendR motif. The truncated peptide loses affinity for its primary receptor and binds to neuropilin-1, activating a tissue penetration pathway that delivers the peptide along with attached or co-administered payload into the tumor mass. Here, we describe the design of a new tumor-penetrating peptide based on the current knowledge of homing sequences and internalizing receptors. The tumor-homing motif in the new peptide is the NGR sequence, which binds to endothelial CD13. The NGR sequence was placed in the context of a CendR motif (RNGR), and this sequence was embedded in the iRGD framework. The resulting peptide (CRNGRGPDC, iNGR) homed to tumor vessels and penetrated into tumor tissue more effectively than the standard NGR peptide. iNGR induced greater tumor penetration of coupled nanoparticles and co-administered compounds than NGR. Doxorubicin given together with iNGR was significantly more efficacious than the drug alone. These results show that a tumor-specific, tissue-penetrating peptide can be constructed from known sequence elements. This principle may be useful in designing tissue-penetrating peptides for other diseases. Cancer Res; 73(2); 804–12. ©2012 AACR.

Various NGR-containing peptides have been exploited for targeted delivery of drugs to CD13-positive tumor neovasculature. Recent studies have shown that compounds containing this motif can rapidly deamidate and generate isoaspartate-glycinearginine (isoDGR), a ligand of alpha v beta 3-integrin that can be also exploited for drug delivery to tumors. We have investigated the role of NGR and isoDGR peptide scaffolds on their biochemical and biological properties. Peptides containing the cyclic CNGRC sequence could bind CD13-positive endothelial cells more efficiently than those containing linear GNGRG. Peptide degradation studies showed that cyclic peptides mostly undergo NGR-to-isoDGR transition and CD13/integrin switching, whereas linear peptides mainly undergo degradation reactions involving the alpha-amino group, which generate non-functional six/seven-membered ring compounds, unable to bind alpha v beta 3, and small amount of isoDGR. Structure-activity studies showed that cyclic isoDGR could bind alpha v beta 3 with an affinity >100-fold higher than that of linear isoDGR and inhibited endothelial cell adhesion and tumor growth more efficiently. Cyclic isoDGR could also bind other integrins (alpha v beta 5, alpha v beta 6, alpha v beta 8, and alpha 5 beta 1), although with 10-100-fold lower affinity. Peptide linearization caused loss of affinity for all integrins and loss of specificity, whereas alpha-amino group acetylation increased the affinity for all tested integrins, but caused loss of specificity. These results highlight the critical role of molecular scaffold on the biological properties of NGR/isoDGR peptides. These findings may have important implications for the design and development of anticancer drugs or tumor neovasculature-imaging compounds, and for the potential function of different NGR/isoDGR sites in natural proteins.

Asparagine deamidation in peptides or in fibronectin fragments containing the asparagine-glycine-arginine sequence generates isoaspartate-glycine-arginine (isoDGR), a new αvβ3 integrin-binding motif. Because αvβ3 is expressed in angiogenic vessels, we hypothesized that isoDGR-containing peptides could be exploited as ligands for targeted delivery of drugs to tumor neovasculature. We found that a cyclic CisoDGRC peptide coupled to fluorescent nanoparticles (quantum dots) could bind αvβ3 integrin and colocalize with anti-CD31, anti-αvβ3, and anti-α5β1 antibodies in human renal cell carcinoma tissue sections, indicating that this peptide could efficiently recognize endothelial cells of angiogenic vessels. Using CisoDGRC fused to tumor necrosis factor α (TNF) we observed that ultralow doses (1–10 pg) of this product (called isoDGR-TNF), but not of TNF or CDGRC-TNF fusion protein, were sufficient to induce antitumor effects when administered alone or in combination with chemotherapy to tumor-bearing mice. The antitumor activity of isoDGR-TNF was efficiently inhibited by coadministration with an excess of free CisoDGRC, as expected for ligand-directed targeting mechanisms. These results suggest that isoDGR is a novel tumor vasculature–targeting motif. Peptides containing isoDGR could be exploited as ligands for targeted delivery of drugs, imaging agents, or other compounds to tumor vasculature. [Cancer Res 2008;68(17):7073–82]

Aminopeptidase-N (CD13) is an important target of tumor vasculature-targeting drugs. The authors investigated its expression by immunohistochemistry with three anti-CD13 monoclonal antibodies (WM15, 3D8, and BF10) in normal and pathological human tissues, including 58 normal, 32 inflammatory, and 149 tumor tissue specimens. The three antibodies stained vessels in most neoplastic tissues, interestingly with different patterns. As a matter of fact, WM15 stained almost all intratumor and peritumor capillaries and only partially large vessels, whereas BF10 and 3D8 reacted with arteries and venules and to a lesser extent with capillaries. These antibodies also stained the stroma in about half of neoplastic tissues. In inflammatory lesions, the three antibodies stained vessels and stroma, whereas in normal tissues, they stained a small percentage of blood vessels. Finally, the three antibodies failed to stain endothelial cells of normal colon, whereas they reacted with activated human umbilical vein endothelial cells and with endothelial cells of colon adenocarcinoma vessels. Overall, WM15 was the most specific antibody for angiogenic tumor vessels, suggesting that it may be a good tool for detecting the CD13 form associated with the tumor vasculature. This finding may be relevant for CD13-mediated vascular targeting therapies. (J Histochem Cytochem 59:47-59, 2011)

Asparagine deamidation at the NGR sequence in the 5th type I repeat of fibronectin (FN-I(5)) generates isoDGR, an alpha v beta 3 integrin-binding motif regulating endothelial cell adhesion and proliferation. By NMR and molecular dynamics studies, we analyzed the structure of CisoDGRC (isoDGR-2C), a cyclic beta-peptide mimicking the FN-I(5) site, and compared it with NGR, RGD, or DGR-containing cyclopeptides. Docking experiments show that isoDGR, exploiting an inverted orientation as compared with RGD, favorably interacts with the RGD-binding site of alpha v beta 3, both recapitulating canonical RGD-alpha v beta 3 contacts and establishing additional polar interactions. Conversely, NGR and DGR motifs lack the fundamental pharmacophoric requirements for high receptor affinity. Therefore, unlike NGR and DGR, isoDGR is a new natural recognition motif of the RGD-binding pocket of alpha v beta 3. These findings contribute to explain the different functional properties of FN-I(5) before and after deamidation, and provide support for the hypothesis that NGR 3 isoDGR transition can work as a molecular timer for activating latent integrin-binding sites in proteins, thus regulating protein function.

Homozygous (Delta ccr5/Delta ccr5) and heterozygous (CCR5/Delta ccr5) deletions in the beta-chemokine receptor 5 (CCR5) gene, which encodes for the major co-receptor for macrophage-tropic HIV-1 entry, have been implicated in resistance to HIV infection and in protection against disease progression, respectively. The CCR5/Delta ccr5 genotype was found more frequently in long-term nonprogressors (LTNP) (31.0%) than in progressors (10.6% p < 0.0001), in agreement with previous studies. Kaplan-Meier survival analyses showed that a slower progression of disease, i.e. higher proportion of subjects with CD4(+) T cell counts > 500/mu l (p = 0.0006) and a trend toward a slower progression to AIDS (p = 0.077), was associated with the CCR5/Delta ccr5 genotype. However, when LTNP were analyzed separetely, no significant differences in CD4(+) T cell counts (p = 0.12) and viremia levels (p = 0.65) were observed between the wild-type (69% of LTNP) and the heterozygous (31.0%) genotypes. Therefore, there are other factors which play a major role in determining the status of nonprogression in the majority of LTNP. Furthermore, there was no evidence that the CCR5/Delta ccr5 genotype was associated with different rates of disease progression in the group of progressors. Taken together, these results indicate that the CCR5/Delta ccr5 genotype is neither essential nor sufficient for protection against the progression of HIV disease.

We studied determinants of chronic inflammation and/or immune activation in plasma from patients in the transition from primary to early chronic HIV-1 infection. The following parameters were estimated in seven patients over time: plasma concentrations of soluble CD8 (sCD8), tumor necrosis factor alpha (TNF-alpha), soluble TNF receptor type II (sTNFRII), interleukin 6 (IL-6), soluble IL-6 receptor (slL6R). IL-10, transforming growth factor beta 1 (TGF-beta 1), along with CD4- and CD8-positive T cell counts, p24 antigenemia, and clinical evaluation. Results showed that concentrations of sCD8, TNF-alpha, and sTNFRII, and peripheral CD8-positive lymphocyte counts, were significantly increased in patients, compared to HIV-negative controls, and showed a trend toward normal values over time. Levels of IL6, sIL6R, IL-10, and TGF-beta 1 did not differ from those of controls and did not change over time, Heterogeneity was observed among the patients in terms of CD4-positive T cell depletion, levels of sCD8, concentrations of TNF-alpha/sTNFRII, and clinical outcome, These data indicate that in the transition phase from primary acute to chronic and asymptomatic infection the host immune activation in response to the virus is highly heterogeneous and that the sustained rise in TNF-alpha and its receptor may represent an important therapeutic target in early disease. The persistence of a state of chronic inflammation and/or immune activation could influence the progression of disease independently from CD4-positive T cell counts.