The loss of CCR6+ and CD161+ CD4+ T-cell homeostasis contributes to disease progression in SIV-infected rhesus macaques.

Although previous studies have shown that CD4+ T cells expressing CCR6 and CD161 are depleted from blood during HIV infection, the mechanisms underlying their loss remain unclear. In this study, we investigated how the homeostasis of CCR6+ and CD161+ CD4+ T cells contributes to SIV disease progression and the mechanisms responsible for their loss from circulation. By comparing SIV infection in rhesus macaques (RMs) and natural host sooty mangabeys (SMs), we found that the loss of CCR6+ and CD161+ CD4+ T cells from circulation is a distinguishing feature of progressive SIV infection in RMs. Furthermore, while viral infection critically contributes to the loss of CD161+CCR6-CD4+ T cells, a redistribution of CCR6+CD161- and CCR6+CD161+CD4+ T cells from the blood to the rectal mucosa is a chief mechanism for their loss during SIV infection. Finally, we provide evidence that the accumulation of CCR6+CD4+ T cells in the mucosa is damaging to the host by demonstrating their reduction from this site following initiation of antiretroviral therapy in SIV-infected RMs and their lack of accumulation in SIV-infected SMs. These data emphasize the importance of maintaining CCR6+ and CD161+ CD4+ T-cell homeostasis, particularly in the mucosa, to prevent disease progression during pathogenic HIV/SIV infection.

IL-21 and probiotic therapy improve Th17 frequencies, microbial translocation, and microbiome in ARV-treated, SIV-infected macaques.

Increased mortality in antiretroviral (ARV)-treated, HIV-infected individuals has been attributed to persistent immune dysfunction, in part due to abnormalities at the gastrointestinal barrier. In particular, the poor reconstitution of gastrointestinal Th17 cells correlates with residual translocation of dysbiotic, immunostimulatory microflora across a compromised intestinal epithelial barrier. We have previously demonstrated that oral probiotics promote increased intestinal CD4(+) T-cell reconstitution during ARV treatment in a non-human primate model of HIV infection; however, essential mucosal T-cell subsets, such as Th17 cells, had limited recovery. Here, we sought to promote Th17 cell recovery by administering interleukin (IL)-21 to a limited number of ARV-treated, probiotic-supplemented, Simian Immunodeficiency Virus (SIV)-infected pigtailed macaques. We demonstrate that probiotic and IL-21 supplementation of ARVs are associated with enhanced polyfunctional Th17 expansion and reduced markers of microbial translocation and dysbiosis as compared with infected controls receiving ARVs alone. Importantly, treatment resulted in fewer morbidities compared with controls, and was independent of increased immune activation or loss of viral suppression. We propose that combining ARVs with therapeutics aimed at restoring intestinal stasis may significantly improve disease prognosis of ARV-treated, HIV-infected individuals.

Loss of mucosal CD103+ DCs and IL-17+ and IL-22+ lymphocytes is associated with mucosal damage in SIV infection.

Human immunodeficiency virus (HIV) and Simian immunodeficiency virus (SIV) disease progression is associated with multifocal damage to the gastrointestinal tract epithelial barrier that correlates with microbial translocation and persistent pathological immune activation, but the underlying mechanisms remain unclear. Investigating alterations in mucosal immunity during SIV infection, we found that damage to the colonic epithelial barrier was associated with loss of multiple lineages of interleukin (IL)-17-producing lymphocytes, cells that microarray analysis showed expressed genes important for enterocyte homeostasis, including IL-22. IL-22-producing lymphocytes were also lost after SIV infection. Potentially explaining coordinate loss of these distinct populations, we also observed loss of CD103+ dendritic cells (DCs) after SIV infection, which associated with the loss of IL-17- and IL-22-producing lymphocytes. CD103+ DCs expressed genes associated with promotion of IL-17/IL-22+ cells, and coculture of CD103+ DCs and naïve T cells led to increased IL17A and RORc expression in differentiating T cells. These results reveal complex interactions between mucosal immune cell subsets providing potential mechanistic insights into mechanisms of mucosal immune dysregulation during HIV/SIV infection, and offer hints for development of novel therapeutic strategies to address this aspect of AIDS virus pathogenesis.

Administration of fludarabine-loaded autologous red blood cells in simian immunodeficiency virus-infected sooty mangabeys depletes pSTAT-1-expressing macrophages and delays the rebound of viremia after suspension of antiretroviral therapy.

A major limitation of highly active antiretroviral therapy is that it fails to eradicate human immunodeficiency virus (HIV) infection due to its limited effects on viral reservoirs carrying replication-competent HIV, including monocytes/macrophages (M/M). Therefore, therapeutic approaches aimed at targeting HIV-infected M/M may prove useful in the clinical management of HIV-infected patients. In previous studies, we have shown that administration of fludarabine-loaded red blood cells (RBC) in vitro selectively induces cell death in HIV-infected M/M via a pSTAT1-dependent pathway. To determine the in vivo efficacy of this novel therapeutic strategy, we treated six naturally simian immunodeficiency virus (SIV)-infected sooty mangabeys (SMs) with either 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) only, fludarabine-loaded RBC only, or PMPA in association with fludarabine-loaded RBC. The rationale of this treatment was to target infected M/M with fludarabine-loaded RBC at a time when PMPA is suppressing viral replication taking place in activated CD4+ T cells. In vivo administration of fludarabine-loaded RBC was well tolerated and did not induce any discernible side effect. Importantly, addition of fludarabine-loaded RBC to PMPA delayed the rebound of viral replication after suspension of therapy, thus suggesting a reduction in the size of SIV reservoirs. While administrations of fludarabine-loaded RBC did not induce any change in the CD4+ or CD8+ T-cell compartments, we observed, in chronically SIV-infected SMs, a selective depletion of M/M expressing pSTAT1. This study suggests that therapeutic strategies based on the administration of fludarabine-loaded RBC may be further explored as interventions aimed at reducing the size of the M/M reservoirs during chronic HIV infection.

Perturbations of cell cycle control in T cells contribute to the different outcomes of simian immunodeficiency virus infection in rhesus macaques and sooty mangabeys.

In contrast to human immunodeficiency virus (HIV) infection of humans and experimental simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs), SIV infection of sooty mangabeys (SMs), a natural host African monkey species, is typically nonpathogenic and associated with preservation of CD4+ T-cell counts despite chronic high levels of viral replication. In previous studies, we have shown that the lack of SIV disease progression in SMs is related to lower levels of immune activation and bystander T-cell apoptosis compared to those of pathogenic HIV/SIV infection (G. Silvestri, D. Sodora, R. Koup, M. Paiardini, S. O'Neil, H. M. McClure, S. I. Staprans, and M. B. Feinberg, Immunity 18:441-452, 2003; G. Silvestri, A. Fedanov, S. Germon, N. Kozyr, W. J. Kaiser, D. A. Garber, H. M. McClure, M. B. Feinberg, and S. I. Staprans, J. Virol. 79:4043-4054, 2005). In HIV-infected patients, increased T-cell susceptibility to apoptosis is associated with a complex cell cycle dysregulation (CCD) that involves increased activation of the cyclin B/p34-cdc2 complex and abnormal nucleolar structure with dysregulation of nucleolin turnover. Here we report that CCD is also present during pathogenic SIV infection of RMs, and its extent correlates with the level of immune activation and T-cell apoptosis. In marked contrast, naturally SIV-infected SMs show normal regulation of cell cycle control (i.e., normal intracellular levels of cyclin B and preserved nucleolin turnover) and a low propensity to apoptosis in both peripheral blood- and lymph node-derived T cells. The absence of significant CCD in the AIDS-free, non-immune-activated SMs despite high levels of viral replication indicates that CCD is a marker of disease progression during lentiviral infection and supports the hypothesis that the preservation of cell cycle control may help to confer the disease-resistant phenotype of SIV-infected SMs.

Cell-cycle dysregulation in the immunopathogenesis of AIDS.

For a number of years the pathogenesis of AIDS was thought to be essentially related to direct human immunodeficiency virus (HIV)-mediated killing of CD4+ T cells. More recently, attention has shifted to pathogenic models that emphasize the role of generalized immune system activation and the excess apoptosis of uninfected T cells in inducing HIV-associated CD4+ T-cell depletion. The main focus of our research is to better define the determinants and the consequences of these "indirect" mechanisms of immunodeficiency by studying both HIV-infected patients and nonhuman primates infected with simian immunodeficiency virus (SIV). We have discovered that pathogenic models of retroviral infections of primates (i.e., HIV infection in humans and SIV infection in rhesus macaques) are associated with the presence of a set of perturbations of normal cell-cycle control in T lymphocytes. These perturbations, to which we collectively refer to as cell-cycle dysregulation, or CCD, may represent an important biological link between chronic immune activation and excess apoptosis and therefore may play a significant role in the pathogenesis of AIDS. A better understanding of the determinants and consequences of CCD may pave the way for the introduction of new therapeutic strategies to be used in addition to standard antiretroviral therapy in HIV-infected patients.

Cyclin D1 expression in pre-cancerous and cancerous lesions of the canine mammary gland.

Overexpression of cyclin D1, the regulatory subunit of cyclin-dependent kinases (cdk4 and cdk6) involved in cell cycle control, has often been found in breast cancer and other types of human cancer. Increased expression, or stability, of cyclin D1 molecules may cause sufficient cdk4 activation to produce retinoblastoma protein phosphorylation independently of mitogenic signals; this results in commitment of cells to the G1 phase at mitosis. In the present study, cyclin D1 expression was investigated in pre-cancerous and cancerous lesions of the canine mammary gland by a complex experimental approach, which included Western blot and immunohistochemical analysis of cyclin D1 and the related molecular system. Furthermore, to define relationships between cell growth and expression of cyclin D1, proliferative activity was studied by the AgNOR technique. The study provided the following information. Cyclin D1 overexpression was largely independent of the type of proliferative anomaly. Indeed, cyclin D1 was expressed in 60% of the pre-cancerous lesions and in 44% of cancerous lesions. Mitotic activity and cyclin D1 expression were related: mammary lesions that expressed cyclin D1 showed a high proliferative ratio, the opposite being true of cyclin D1-negative cell populations. This study may contribute to the establishment of an animal model for anti-cancer research based on cyclin D1 suppression or cdk inactivation, or both.

Exogenous interleukin-2 administration corrects the cell cycle perturbation of lymphocytes from human immunodeficiency virus-infected individuals.

Human immunodeficiency virus (HIV)-induced immunodeficiency is characterized by progressive loss of CD4(+) T cells associated with functional abnormalities of the surviving lymphocytes. Increased susceptibility to apoptosis and loss of proper cell cycle control can be observed in lymphocytes from HIV-infected individuals and may contribute to the lymphocyte dysfunction of AIDS patients. To better understand the relation between T-cell activation, apoptosis, and cell cycle perturbation, we studied the effect of exogenous interleukin-2 (IL-2) administration on the intracellular turnover of phase-dependent proteins. Circulating T cells from HIV-infected patients display a marked discrepancy between a metabolic profile typical of G(0) and a pattern of expression of phase-dependent proteins that indicates a more-advanced position within the cell cycle. This discrepancy is enhanced by in vitro activation with ConA and ultimately results in a marked increase of apoptotic events. Conversely, treatment of lymphocytes with IL-2 alone restores the phase-specific pattern of expression of cell cycle-dependent proteins and is associated with low levels of apoptosis. Interestingly, exogenous IL-2 administration normalizes the overall intracellular protein turnover, as measured by protein synthesis, half-life of newly synthesised proteins, and total protein ubiquitination, thus providing a possible explanation for the effect of IL-2 on the intracellular kinetics of cell cycle-dependent proteins. The beneficial effect of IL-2 administration is consistent with the possibility of defective IL-2 function in vivo, which is confirmed by the observation that lymphocytes from HIV-infected patients show abnormal endogenous IL-2 paracrine/autocrine function upon in vitro mitogen stimulation. Overall these results confirm that perturbation of cell cycle control contributes to HIV-related lymphocyte dysfunction and, by showing that IL-2 administration can revert this perturbation, suggest a new mechanism of action of IL-2 therapy in HIV-infected patients.

Identification of blood erythroid markers useful in revealing erythropoietin abuse in athletes.

Recombinant human erythropoietin (rEpo) is being used with increasing frequency by endurance athletes to improve aerobic potential. Although rEpo administration has been banned by the International Olympic Committee, no methods are available to unequivocally detect its abuse in sports. Prompted by these considerations, we evaluated the main hematological and biochemical modifications measured in the blood of 18 volunteers upon rEpo administration. Different rEpo regimens, iron, folic acid, and vitamin B12 administration did not significantly modify the percentage increase in hematocrit. However, a significant decrease in circulating ferritin (fr) and an increase in the soluble transferrin receptor (sTfr) were not found in athletes receiving low (30 IU/kg) doses of rEpo. Thus, an increase in the sTfr/fr ratio cannot be used as an indicator of rEpo abuse, at least when the hormone is administered at low concentrations. In contrast, the amounts of beta-globin mRNA detected by quantitative competitive (RT)-PCR in whole blood samples significantly increased above the threshold levels in all of the treatments investigated. Taken together, these data suggest that hematocrit value, reticulocyte count, soluble transferrin receptor content, and concentration of beta-globin mRNA, when included in a new multiparametric formula, can detect rEpo abuse in 57.5% of the samples examined with a confidence interval of 99.99%. Thus, the method reported in this paper could significantly improve the tests currently available, which in similar experiments allowed the detection of rEpo abuse in only 7.6% of the samples examined.

Abnormal intracellular kinetics of cell-cycle-dependent proteins in lymphocytes from patients infected with human immunodeficiency virus: a novel biologic link between immune activation, accelerated T-cell turnover, and high levels of apoptosis.

Human immunodeficiency virus (HIV)-infection is characterized by loss of CD4+ T cells associated with high levels of immune activation, T-cell proliferation, and lymphocyte apoptosis. To investigate the role of intrinsic perturbations of cell-cycle control in the immunopathogenesis of acquired immunodeficiency syndrome (AIDS), we studied the expression of cell-cycle-dependent proteins in lymphocytes from HIV-infected patients. Cyclin B1 expression, Nucleolar Organizer Regions (NORs) number, and NORs area of distribution were all consistently increased in HIV-infected patients, but returned to normal after effective antiretroviral therapy, suggesting that viral replication is directly implicated in the genesis of the observed changes. Analysis of cyclin B1 intracellular turnover showed that the increased cyclin B1 expression is (1) caused by defective degradation in the presence of normal rates of synthesis, and (2) is temporally associated with decreased levels of ubiquitination. After in vitro activation of lymphocytes from healthy individuals, cyclin B1 and cdc25 expression and ubiquitination, p34 cdc2 activity, NORs morphology, and C23/nucleolin localization showed a 72- to 96-hour cyclic pattern that led to a biologic state similar to baseline. On the contrary, complex but consistent changes of the same indices followed activation of T lymphocytes from HIV-infected patients, resulting in a 5-fold increase in apoptosis. Overall, our data indicate that a profound dysregulation of cell-cycle control is present in lymphocytes from HIV-infected patients. This finding may provide a novel biologic link between immune activation, accelerated lymphocyte turnover, and increased apoptosis during HIV infection.

Identification of the main ubiquitination site in human erythroid alpha-spectrin.

Erythroid spectrin is the main component of the red cell membrane skeleton, which is very important in determining the shape, resistance to mechanical stresses and deformability of red cells. Previously we demonstrated that human erythroid alpha-spectrin is ubiquitinated in vitro and in vivo, and using recombinant peptides we identified on repeat 17 the main ubiquitination site of alpha-spectrin. In order to identify the lysine(s) involved in the ubiquitination process, in the present study we mutated the lysines by site-directed mutagenesis. We found that ubiquitination was dramatically inhibited in peptides carrying the mutation of lysine 27 on repeat 17 (mutants K25,27R and K27R). We also demonstrated that the correct folding of this protein is fundamental for its recognition by the ubiquitin conjugating system. Furthermore, the region flanking lysine 27 showed a 75% similarity with the leucine zipper pattern present in many regulatory proteins. Thus, a new potential ubiquitin recognition motif was identified in alpha-spectrin and may be present in several other proteins.

[Update on animal models for testing innovative antineoplastic strategies: the mammary tumor in dogs]. / Messa a punto di modelli animali per la sperimentazione di strategie antineoplastiche innovative: il tumore mammario canino.

In this study, the authors describe a new possible animal model to test new anticancer therapies. The selected animals are domestic animals such as dogs, which develop spontaneous tumours very similar in morphology and biology to human ones, also in relation to similar environmental oncogenic pressures. Cycline D1 overexpression, which has both a prognostic and pathogenetic value, is usually detected in human tumours. Thus, the use of cycline-dependent kinases inhibitors could be of value in anticancer therapy. We studied spontaneous canine mammary tumours in order to test the above hypothesis. Immunohistochemistry, AgNOR and western blotting analysis were performed, and the results revealed that cycline D1 is associated with metabolic, morphological and protein expression patterns typical of proliferating cells. The same protein expression pattern, the use of human antibodies for detecting canine proteins and the availability of neoplastic tissue make these spontaneous canine tumours a reliable model.

Identification of ubiquitinated repeats in human erythroid alpha-spectrin.

The spectrin role(s) is (are) very important for the shape and the physical properties of red cells, such as deformability and resistance to mechanical stresses. Moreover a variety of spectrin diseases are known. We have previously demonstrated [Corsi, D., Galluzzi, L., Crinelli, R. & Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935] that human erythroid alpha-spectrin is ubiquitinated in vitro and in vivo. In order to define the ubiquitinated repeats of this long protein and find out a possible function, we have produced recombinant peptides encompassing the alphaIII-, alphaIV-, alphaV- and EF hand domains of alpha-spectrin chain. These peptides were tested in in vitro ubiquitin conjugation assays and two regions susceptibles to ubiquitination were found. The first one, in the alphaIV-domain, includes the repeat 17 and the second one, in the alphaV-domain, includes the repeat 20 and a part of repeat 21. We also demonstrated that the susceptibility to ubiquitination of the alphaV-domain is reduced by interaction with the corresponding portion of beta-spectrin chain (betaIV-domain). Thus, at least ubiquitination of alphaV-domain is susceptible to cytoskeleton assembly and spectrin dimerization.

Unscheduled cyclin B expression and p34 cdc2 activation in T lymphocytes from HIV-infected patients.

OBJECTIVE: To study the role of cell cycle regulation during HIV infection by investigating in vivo and in vitro cyclin B and p34 cdc kinase expression. METHODS: Cyclin B expression was analysed by Western blot in CD4 and CD8 cells from 25 HIV-infected patients and 24 uninfected individuals. In eight patients, a sequential analysis was performed after initiation of antiretroviral therapy (ART), and correlations with CD4 cell count and HIV viremia were studied. Sequential changes in cyclin B expression and p34 cdc kinase expression and activity were also studied in lymphocytes activated in vitro with phytohaemagglutinin (PHA). RESULTS: Lymphocytes from untreated HIV-infected patients demonstrate persistent in vivo overexpression of cyclin B in both CD4 and CD8 cell subpopulations. When cells are stimulated to proliferate in vitro, biochemical events that characterize the entrance into the cell cycle [ornithine decarboxylase (ODC) activity, interleukin 2 production, interleukin 2 alpha-chain receptor (IL-2R, CD25) expression, total protein synthesis, total DNA synthesis] show similar timing and sequence in lymphocytes from HIV-infected and uninfected individuals. However, in peripheral blood lymphocytes (PBL) from HIV-infected patients, cyclin B and p34 cdc kinase show premature expression during the cell cycle. Both in vivo cyclin B overexpression and in vitro unscheduled cyclin B expression were almost completely reversed 2-4 weeks after initiation of effective ART. CONCLUSION: Increased and unscheduled expression of cyclin B and p34 cdc kinase is consistently observed in CD4 and CD8 cells from HIV-infected patients, both in vivo and after in vitro mitogenic stimulation. These alterations correlate with the level of viremia and may provide a link between the perturbation of lymphocyte proliferative homeostasis and the exaggerated propensity towards apoptosis.

Alteration of alpha-spectrin ubiquitination due to age-dependent changes in the erythrocyte membrane.

Mammalian red blood cell alpha-spectrin is ubiquitinated in vitro and in vivo [Corsi, D., Galluzzi, L., Crinelli, R., Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935]. This process shows a cell age-dependent decrease, with senescent red blood cells having approximately one third of the amount of ubiquitinated alpha-spectrin found in young cells. In-vitro ubiquitination of alpha-spectrin was dependent on the source of the red cell membranes (those from older cells are less susceptible to ubiquitination than those from younger cells), on the source of ubiquitin-conjugating enzymes (those from older cells catalyze the process at a reduced rate compared to those from younger cells) and on the ubiquitin isopeptidase activity (which decreases during red cell ageing). However, once alpha-spectrin has been extracted from the membranes of young or old red blood cells, it is susceptible to ubiquitination to a similar extent regardless of source. This suggests that it is the membrane architecture, and not spectrin itself, that is responsible for the age-dependent decline in ubiquitination. Furthermore, spectrin oligomers, tetramers and dimers are also equally susceptible to ubiquitination. As spectrin ubiquitination occurs on domains alphaIII and alphaV of alpha-spectrin, and domain alphaV contains the nucleation site for the association of the alpha- and beta-spectrin chains, alterations in ubiquitination during red cell ageing could affect the stability and deformability of the erythrocyte membrane.