Lymphocytes upregulate CD36 in adipose tissue and liver.

CD36 is a multifunctional scavenger receptor and lipid transporter implicated in metabolic and inflammatory pathologies, as well as cancer progression. CD36 is known to be expressed by adipocytes and monocytes/macrophages, but its expression by T cells is not clearly established. We found that CD4 and CD8 T cells in adipose tissue and liver of humans, monkeys, and mice upregulated CD36 expression (ranging from ~5-40% CD36+), whereas little to no CD36 was expressed by T cells in blood, spleen, and lymph nodes. CD36 was expressed predominantly by resting CD38-, HLA.DR-, and PD-1- adipose tissue T cells in monkeys, and increased during high-fat feeding in mice. Adipose tissue and liver promote a distinct phenotype in resident T cells characterized by CD36 upregulation.

PD-1 blocks lytic granule polarization with concomitant impairment of integrin outside-in signaling in the natural killer cell immunological synapse.

BACKGROUND: The inhibitory receptor programmed cell death protein 1 (PD-1) is upregulated on a variety of immune cells, including natural killer (NK) cells, during chronic viral infection and tumorigenesis. Blockade of PD-1 or its ligands produces durable clinical responses with tolerable side effects in patients with a broad spectrum of cancers. However, the underlying molecular mechanisms of how PD-1 regulates NK cell function remain poorly characterized. OBJECTIVE: We sought to determine the effect of PD-1 signaling on NK cells. METHODS: PD-1 was overexpressed in CD16-KHYG-1 (a human NK cell line with both antibody-dependent cellular cytotoxicity through CD16 and natural cytotoxicity through NKG2D) cells and stimulated by exposing the cells to NK-sensitive target cells expressing programmed death ligand 1 (PD-L1). RESULTS: PD-1 engagement by PD-L1 specifically blocked NK cell-mediated cytotoxicity without interfering with the conjugation between NK cells and target cells. Further examination showed that PD-1 signaling blocked lytic granule polarization in NK cells, which was accompanied by failure of integrin-linked kinase, a key molecule in the integrin outside-in signaling pathway, to accumulate in the immunological synapse after NK-target cell conjugation. CONCLUSION: Our results suggest that NK cell cytotoxicity is inhibited by PD-1 engagement, which blocks lytic granule polarization to the NK cell immunological synapse with concomitant impairment of integrin outside-in signaling. This study provides novel mechanistic insights into how PD-1 inhibition disrupts NK cell function.

Sphingosine-1-phosphate receptor 1 mediates elevated IL-6 signaling to promote chronic inflammation and multitissue damage in sickle cell disease.

Sphingosine-1-phosphate (S1P) is a biolipid involved in chronic inflammation in several inflammatory disorders. Recent studies revealed that elevated S1P contributes to sickling in sickle cell disease (SCD), a devastating hemolytic, genetic disorder associated with severe chronic inflammation and tissue damage. We evaluated the effect of elevated S1P in chronic inflammation and tissue damage in SCD and underlying mechanisms. First, we demonstrated that interfering with S1P receptor signaling by FTY720, a U.S. Food and Drug Administration-approved drug, significantly reduced systemic, local inflammation and tissue damage without antisickling effects. These findings led us to discover that S1P receptor activation leads to substantial elevated local and systemic IL-6 levels in SCD mice. Genetic deletion of IL-6 in SCD mice significantly reduced local and systemic inflammation, tissue damage, and kidney dysfunction. At the cellular level, we determined that elevated IL-6 is a key cytokine functioning downstream of elevated S1P, which contributes to increased S1P receptor 1 ( S1pr1) gene expression in the macrophages of several tissues in SCD mice. Mechanistically, we revealed that S1P-S1PR1 signaling reciprocally up-regulated IL-6 gene expression in primary mouse macrophages in a JAK2-dependent manner. Altogether, we revealed that elevated S1P, coupled with macrophage S1PR1 reciprocally inducing IL-6 expression, is a key signaling network functioning as a malicious, positive, feed-forward loop to sustain inflammation and promote tissue damage in SCD. Our findings immediately highlight novel therapeutic possibilities.-Zhao, S., Adebiyi, M. G., Zhang, Y., Couturier, J. P., Fan, X., Zhang, H., Kellems, R. E., Lewis, D. E., Xia, Y. Sphingosine-1-phosphate receptor 1 mediates elevated IL-6 signaling to promote chronic inflammation and multitissue damage in sickle cell disease.

HIV Persistence in Adipose Tissue Reservoirs.

PURPOSE OF REVIEW: The purpose of this review is to examine the evidence describing adipose tissue as a reservoir for HIV-1 and how this often expansive anatomic compartment contributes to HIV persistence. RECENT FINDINGS: Memory CD4 T cells and macrophages, the major host cells for HIV, accumulate in adipose tissue during HIV/SIV infection of humans and rhesus macaques. Whereas HIV and SIV proviral DNA is detectable in CD4 T cells of multiple fat depots in virtually all infected humans and monkeys examined, viral RNA is less frequently detected, and infected macrophages may be less prevalent in adipose tissue. However, based on viral outgrowth assays, adipose-resident CD4 T cells are latently infected with virus that is replication-competent and infectious. Additionally, adipocytes interact with CD4 T cells and macrophages to promote immune cell activation and inflammation which may be supportive for HIV persistence. Antiviral effector cells, such as CD8 T cells and NK/NKT cells, are abundant in adipose tissue during HIV/SIV infection and typically exceed CD4 T cells, whereas B cells are largely absent from adipose tissue of humans and monkeys. Additionally, CD8 T cells in adipose tissue of HIV patients are activated and have a late differentiated phenotype, with unique TCR clonotypes of less diversity relative to blood CD8 T cells. With respect to the distribution of antiretroviral drugs in adipose tissue, data is limited, but there may be class-specific penetration of fat depots. The trafficking of infected immune cells within adipose tissues is a common event during HIV/SIV infection of humans and monkeys, but the virus may be mostly transcriptionally dormant. Viral replication may occur less in adipose tissue compared to other major reservoirs, such as lymphoid tissue, but replication competence and infectiousness of adipose latent virus are comparable to other tissues. Due to the ubiquitous nature of adipose tissue, inflammatory interactions among adipocytes and CD4 T cells and macrophages, and selective distribution of antiretroviral drugs, the sequestration of infected immune cells within fat depots likely represents a major challenge for cure efforts.

Treatment-Naïve HIV-Infected Patients Have Fewer Gut-Homing ß7 Memory CD4 T Cells than Healthy Controls.

OBJECTIVES: The integrin α4ß7 is the gut-homing receptor for lymphocytes. It also is an important co-receptor for human immunodeficiency virus (HIV) via glycoprotein (gp)120 binding. Depletion of gut cluster of differentiation (CD)4 T cells is linked to chronic inflammation in patients with HIV; however, measuring CD4 cells in the gut is invasive and not routine. As such, establishing a peripheral marker for CD4 depletion of the gut is needed. We hypothesized that α4ß7 CD4 T cells are depleted in the peripheral blood of treatment-naïve patients with HIV compared with healthy controls. METHODS: The study groups were treatment-naïve patients with HIV and uninfected controls. Subjects were included if they were 18 years or older with no history of opportunistic infections, active tuberculosis, or cancer. We collected peripheral blood and examined on whole blood using flow cytometry for the following cell surface markers: CD4, CD45RO, chemokine receptor type 5, C-X-C chemokine receptor type 4 (CXCR4), and the integrin ß7. We collected demographic information, including age, sex, and ethnicity, as well as viral load (VL) and CD4 count. Two-sample t tests and Fisher exact tests were used to compare the differences between the two groups. Spearman correlation coefficients were calculated between CD4 count and log10- VL and percentage of CD4+/CD45RO+/ß7+ and log10- VL in patients. RESULTS: Twenty-two subjects were enrolled in the study (12 patients with HIV and 10 controls). There were no differences in age or sex between the two groups. There were more Hispanics and fewer Asians in the group comprising patients with HIV compared with the control group (7 vs 2 and 0 vs 4, P = 0.05, respectively). Patients infected with HIV had significantly lower frequencies of CD4+/CD45RO+/ß7+ cells (median 12%, range 5-18 compared with uninfected controls: median 20%, range 11-26, P = 0.0007). There was a statistically significant difference in the percentage of CD4+/CD45RO+/C-X-C chemokine receptor type 4+ cells between patients (72%, range 60%-91%) compared with controls (79%, range 72%-94%, P = 0.04). The percentage of CD4+/CD45RO+/chemokine receptor type 5+ did not differ between the group of patients with HIV and the control groups (22%, range 11%-57% vs 27%, range 14%-31%; P = 0.8, respectively). There was no correlation between percentage of CD4+/CD45RO+/ß+ cells and log10- VL as measured by the Spearman correlation coefficient (r = 0.05, P = 0.88) in patients infected with HIV. CONCLUSIONS: Memory CD4 ß7+ cells are reduced significantly in the peripheral blood of untreated patients infected with HIV, which could be used as a noninvasive indicator of intestinal CD4 T cell loss and recovery. Further studies are needed to examine whether depletion of these CD4+/CD45RO+/ß7+ cells in the peripheral blood parallels depletion in the gut of treatment-naïve patients with HIV and whether levels return to control levels after treatment.

Postprandial Monocyte Activation in Individuals With Metabolic Syndrome.

CONTEXT: Postprandial hyperlipidemia has been suggested to contribute to atherogenesis by inducing proinflammatory changes in monocytes. Individuals with metabolic syndrome (MS), shown to have higher blood triglyceride concentration and delayed triglyceride clearance, may thus have increased risk for development of atherosclerosis. OBJECTIVE: Our objective was to examine fasting levels and effects of a high-fat meal on phenotypes of monocyte subsets in individuals with obesity and MS and in healthy controls. Design, Setting, Participants, Intervention: Individuals with obesity and MS and gender- and age-matched healthy controls were recruited. Blood was collected from participants after an overnight fast (baseline) and at 3 and 5 hours after ingestion of a high-fat meal. At each time point, monocyte phenotypes were examined by multiparameter flow cytometry. MAIN OUTCOME MEASURES: Baseline levels of activation markers and postprandial inflammatory response in each of the three monocyte subsets were measured. RESULTS: At baseline, individuals with obesity and MS had higher proportions of circulating lipid-laden foamy monocytes than controls, which were positively correlated with fasting triglyceride levels. Additionally, the MS group had increased counts of nonclassical monocytes, higher CD11c, CX3CR1, and human leukocyte antigen-DR levels on intermediate monocytes, and higher CCR5 and tumor necrosis factor-α levels on classical monocytes in the circulation. Postprandial triglyceride increases in both groups were paralleled by upregulation of lipid-laden foamy monocytes. MS, but not control, subjects had significant postprandial increases of CD11c and percentages of IL-1ß+ and tumor necrosis factor-α+ cells in nonclassical monocytes. CONCLUSIONS: Compared to controls, individuals with obesity and MS had increased fasting and postprandial monocyte lipid accumulation and activation.

Human adipose tissue as a reservoir for memory CD4+ T cells and HIV.

OBJECTIVE: The objective of this study is to determine whether adipose tissue functions as a reservoir for HIV-1. DESIGN: We examined memory CD4(+) T cells and HIV DNA in adipose tissue-stromal vascular fraction (AT-SVF) of five patients [four antiretroviral therapy (ART)-treated and one untreated]. To determine whether adipocytes stimulate CD4(+) T cells and regulate HIV production, primary human adipose cells were cocultured with HIV-infected CD4(+) T cells. METHODS: AT-SVF T cells were studied by flow cytometry, and AT-SVF HIV DNA (Gag and Env) was examined by nested PCR and sequence analyses. CD4(+) T-cell activation and HIV production were measured by flow cytometry and ELISA. RESULTS: AT-SVF CD3(+) T cells were activated (>60% CD69(+)) memory CD4(+) and CD8(+) T cells in uninfected and HIV-infected persons, but the AT-SVF CD4(+)/CD8(+) ratio was lower in HIV patients. HIV DNA (Gag and Env) was detected in AT-SVF of all five patients examined by nested PCR, comparably to other tissues [peripheral blood mononuclear cell (PBMC), lymph node or thymus]. In coculture experiments, adipocytes increased CD4(+) T-cell activation and HIV production approximately two to three-fold in synergy with gamma-chain cytokines interleukin (IL)-2, IL7 or IL15. These effects were mitigated by neutralizing antibodies against IL6 and integrin-α1ß1. Adipocytes also enhanced T-cell viability. CONCLUSION: Adipose tissues of ART-treated patients harbour activated memory CD4(+) T cells and HIV DNA. Adipocytes promote CD4(+) T-cell activation and HIV production in concert with intrinsic adipose factors. Adipose tissue may be an important reservoir for HIV.

The effect of extracorporeal photopheresis alone or in combination therapy on circulating CD4(+) Foxp3(+) CD25(-) T cells in patients with leukemic cutaneous T-cell lymphoma.

PURPOSE: Extracorporeal photopheresis (ECP) alone or in combination therapy is effective for treatment of leukemic cutaneous T-cell lymphoma (L-CTCL), but its mechanism(s) of action remain unclear. This study was designed to investigate the effect of ECP on regulatory T cells and CD8(+) T cells in L-CTCL patients. EXPERIMENTAL DESIGN: Peripheral blood from 18 L-CTCL patients at baseline, Day 2, 1 month, 3 month, and 6 month post-ECP therapy was analyzed by flow cytometry for CD4(+) CD25(+/high) , CD4(+) Foxp3(+) CD25(+/-) , CD3(+) CD8(+) , CD3(+) CD8(+) CD69(+) , and CD3(+) CD8(+) IFN-γ(+) T cells. Clinical responses were assessed and correlated with changes in these T-cell subsets. RESULTS: Twelve of 18 patients achieved clinical responses. The average baseline number of CD4(+) CD25(+/high) T cells of PBMCs in L-CTCL patients was normal (2.2%), but increased at 6-month post-therapy (4.3%, P < 0.01). The average baseline number of CD4(+) Foxp3(+) T cells out of CD4(+) T cells in nine evaluable patients was high (66.8 ± 13.7%), mostly CD25 negative. The levels of CD4(+) Foxp3(+) T cells in responders were higher (n = 6, 93.1 ± 5.7%) than nonresponders (n = 3, 14.2 ± 16.0%, P < 0.01), and they declined in parallel with malignant T cells. The numbers of CD3(+) CD8(+) CD69(+) and CD3(+) CD8(+) IFN-γ(+) T cells increased at 3-month post-therapy in five of six patients studied. CONCLUSIONS: Extracorporeal photopheresis alone or in combination therapy might be effective in L-CTCL patients whose malignant T cells have a CD4(+) Foxp3(+) CD25(-) phenotype.

HIV replication in conjunction with granzyme B production by CCR5+ memory CD4 T cells: Implications for bystander cell and tissue pathologies.

Granzyme B (GrzB) is expressed by activated T cells and mediates cellular apoptosis. GrzB also acts as an extracellular protease involved in tissue degradation. We hypothesized that GrzB production from activated memory CD4 T cells may be associated with HIV pathogenesis. We found that stimulated memory CD4 T cells (via costimulation, cytokines, and TLR ligands) concomitantly produced GrzB and HIV. Both GrzB and HIV expression were mainly restricted to CCR5-expressing memory CD4+CD45RO+ T cells, including Th1 and Th17 subsets. Activated memory CD4 T cells also mediated tissue damage, such as disruption of intestinal epithelial monolayers. In non-human primates, CD4 T cells of rhesus macaques (pathogenic SIV hosts) expressed higher GrzB compared to African green monkeys (non-pathogenic SIV hosts). These results suggest that GrzB from CCR5+ memory CD4 T cells may have a role in cellular and tissue pathologies during HIV infection.

PMA induces SnoN proteolysis and CD61 expression through an autocrine mechanism.

Phorbol-12-myristate-13-acetate, also called PMA, is a small molecule that activates protein kinase C and functions to differentiate hematologic lineage cells. However, the mechanism of PMA-induced cellular differentiation is not fully understood. We found that PMA triggers global enhancement of protein ubiquitination in K562, a myelogenous leukemia cell line and one of the enhanced-ubiquitination targets is SnoN, an inhibitor of the Smad signaling pathway. Our data indicated that PMA stimulated the production of Activin A, a cytokine of the TGF-ß family. Activin A then activated the phosphorylation of both Smad2 and Smad3. In consequence, SnoN is ubiquitinated by the APC(Cdh1) ubiquitin ligase with the help of phosphorylated Smad2. Furthermore, we found that SnoN proteolysis is important for the expression of CD61, a marker of megakaryocyte. These results indicate that protein ubiquitination promotes megakaryopoiesis via degrading SnoN, an inhibitor of CD61 expression, strengths the roles of ubiquitination in cellular differentiation.

HIV-1 Vpr induces adipose dysfunction in vivo through reciprocal effects on PPAR/GR co-regulation.

Viral infections, such as HIV, have been linked to obesity, but mechanistic evidence that they cause adipose dysfunction in vivo is lacking. We investigated a pathogenic role for the HIV-1 accessory protein viral protein R (Vpr), which can coactivate the glucocorticoid receptor (GR) and co-repress peroxisome proliferator-activated receptor γ (PPARγ) in vitro, in HIV-associated adipose dysfunction. Vpr circulated in the blood of most HIV-infected patients tested, including those on antiretroviral therapy (ART) with undetectable viral load. Vpr-mediated mechanisms were dissected in vivo using mouse models expressing the Vpr transgene in adipose tissues and liver (Vpr-Tg) or infused with synthetic Vpr. Both models demonstrated accelerated whole-body lipolysis, hyperglycemia and hypertriglyceridemia, and tissue-specific findings. Fat depots in these mice had diminished mass, macrophage infiltration, and blunted PPARγ target gene expression but increased GR target gene expression. In liver, we observed blunted PPARα target gene expression, steatosis with decreased adenosine monophosphate-activated protein kinase activity, and insulin resistance. Similar to human HIV-infected patients, Vpr circulated in the serum of Vpr-Tg mice. Vpr blocked differentiation in preadipocytes through cell cycle arrest, whereas in mature adipocytes, it increased lipolysis with reciprocally altered association of PPARγ and GR with their target promoters. These results delineate a distinct pathogenic sequence: Vpr, released from HIV-1 in tissue reservoirs after ART, can disrupt PPAR/GR co-regulation and cell cycle control to produce adipose dysfunction and hepatosteatosis. Confirmation of these mechanisms in HIV patients could lead to targeted treatment of the metabolic complications with Vpr inhibitors, GR antagonists, or PPARγ/PPARα agonists.

Relationship of ethnicity and CD4 Count with glucose metabolism among HIV patients on Highly-Active Antiretroviral Therapy (HAART).

BACKGROUND: HIV patients on HAART are prone to metabolic abnormalities, including insulin resistance, lipodystrophy and diabetes. This study purports to investigate the relationship of ethnicity and CD4+ T cell count attained after stable highly-active antiretroviral treatment (HAART) with glucose metabolism in hyperrtriglyceridemic HIV patients without a history of diabetes. METHODS: Demographic, anthropometric, clinical, endocrinologic, energy expenditure and metabolic measures were obtained in 199 multiethnic, healthy but hypertriglyceridemic HIV-infected patients [46% Hispanic, 17% African-American, 37% Non-Hispanic White (NHW)] on stable HAART without a history of diabetes. The relationship of glucose and insulin responses to ethnicity, CD4 strata (low (<300/cc) or moderate-to-high (≥ 300/cc)), and their interaction was determined. RESULTS: African-Americans had significantly greater impairment of glucose tolerance (P < 0.05) and HbA1c levels (P < .001) than either Hispanics or NHWs. In multivariate models, after adjusting for confounders (age, sex, HIV/HAART duration, smoking, obesity, glucose, insulin and lipids), African-Americans and Hispanics had significantly higher HbA1c and 2-hour glucose levels than NHW's. Demonstrating a significant interaction between ethnicity and CD4 count (P = 0.023), African Americans with CD4 <300/cc and Hispanics with CD4 ≥300/cc had the most impaired glucose response following oral glucose challenge. CONCLUSIONS: Among hypertriglyceridemic HIV patients on HAART, African-Americans and Hispanics are at increased risk of developing diabetes. Ethnicity also interacts with CD4+ T cell count attained on stable HAART to affect post-challenge glycemic response.

Granzyme B- and Fas ligand-mediated cytotoxic function induced by mitogenic CD28 stimulation of human memory CD4+ T cells.

Some human memory CD4(+) T cells have cytotoxic functions best understood in the context of viral infections; however, their possible role in pathologic processes is understudied. The novel discovery that mitogenic CD28 antibodies induced proliferation and expansion of Tregs offered therapeutic promise for autoimmune disorders. However, the failed TGN1412 trial forced reassessment of this concept. As memory CD4(+) T cells are known to produce toxic molecules, including granzyme B (GrzB) and FasL, we wondered whether mitogenic CD28 was able to induce these cytotoxic molecules. A commercially available mitogenic human CD28 mAb (clone ANC28.1) was used to determine whether mitogenic CD28 induces cytotoxic function from human memory CD4(+) T cells. We found that stimulation of memory CD4(+) T cells by ANC28.1, as well as by conventional costimulation (CD3/CD28 mAb), robustly induced enzymatically active GrzB, along with increased surface expression of FasL. These functional phenotypes were induced in association with increased expression of T cell activation markers CD69 and CD25, and elimination of target cells by ANC28.1-activated memory CD4(+) T cells involved both GrzB and FasL. Additionally, ANC28.1-activated memory CD4(+) T cells caused disruption of epithelial cell monolayer integrity, which was partially mediated by GrzB. These findings reveal functions of memory CD4(+) T cells previously unknown to be induced by mitogenic CD28, and suggest that these pathogenic mechanisms may have been responsible for some of the widespread tissue destruction that occurred in the TGN1412 trial recipients.

Human monocytes accelerate proliferation and blunt differentiation of preadipocytes in association with suppression of C/EBPΑ mRNA.

Obesity, type 2 diabetes, and HIV-associated lipodystrophy are associated with abnormalities in adipocyte growth and differentiation. In persons with these conditions, adipose depots contain increased numbers of macrophages, but the origins of these cells and their specific effects are uncertain. Peripheral blood mononuclear cells (PBMC)-derived monocytes, but not T cells, cocultured via transwells with primary subcutaneous preadipocytes, increased proliferation (approximately twofold) and reduced differentiation (~50%) of preadipocytes. Gene expression analyses in proliferating preadipocytes (i.e., prior to hormonal induction of terminal differentiation) revealed that monocytes down-regulated mRNA levels of CCAAT/enhancer binding protein, alpha (C/EBPα) and up-regulated mRNA levels of G0/G1 switch 2 (G0S2) message, genes important for the regulation of adipogenesis and the cell cycle. These data indicate that circulating peripheral blood monocytes can disrupt adipogenesis by interfering with a critical step in C/EBPα and G0S2 transcription required for preadipocytes to make the transition from proliferation to differentiation. Interactions between preadipocytes and monocytes also increased the inflammatory cytokines IL-6 and IL-8, as well as a novel chemotactic cytokine, CXCL1. Additionally, the levels of both IL-6 and CXCL1 were highest when preadipocytes and monocytes were cultured together, compared to each cell in culture alone. Such cross-talk amplifies the production of mediators of tissue inflammation.

Detrimental effects of adenosine signaling in sickle cell disease.

Hypoxia can act as an initial trigger to induce erythrocyte sickling and eventual end organ damage in sickle cell disease (SCD). Many factors and metabolites are altered in response to hypoxia and may contribute to the pathogenesis of the disease. Using metabolomic profiling, we found that the steady-state concentration of adenosine in the blood was elevated in a transgenic mouse model of SCD. Adenosine concentrations were similarly elevated in the blood of humans with SCD. Increased adenosine levels promoted sickling, hemolysis and damage to multiple tissues in SCD transgenic mice and promoted sickling of human erythrocytes. Using biochemical, genetic and pharmacological approaches, we showed that adenosine A(2B) receptor (A(2B)R)-mediated induction of 2,3-diphosphoglycerate, an erythrocyte-specific metabolite that decreases the oxygen binding affinity of hemoglobin, underlies the induction of erythrocyte sickling by excess adenosine both in cultured human red blood cells and in SCD transgenic mice. Thus, excessive adenosine signaling through the A(2B)R has a pathological role in SCD. These findings may provide new therapeutic possibilities for this disease.

Flow cytometric analysis of circulating microparticles in plasma.

Microparticles, which include exosomes, micro-vesicles, apoptotic bodies and apoptotic microparticles, are small (0.05 - 3 mum in diameter), membranous vesicles that can contain DNA, RNA, miRNA, intracellular proteins and express extracellular surface markers from the parental cells. They can be secreted from intracellular multivesicular bodies or released from the surface of blebbing membranes. Circulating microparticles are abundant in the plasma of normal individuals and can be derived from circulating blood cells such as platelets, red blood cells and leukocytes as well as from tissue sources, such as endothelial and placental tissues. Elevated levels of microparticles are associated with various diseases such as thrombosis (platelet microparticles), congestive heart failure (endothelial microparticles), breast cancer patients (leukocyte microparticles) and women with preeclampsia (syncytiotrophoblast microparticles). Although microparticles can be detected by microscopy, enzyme-linked immunoassays and functional assays, flow cytometry is the preferred method because of the ability to quantitate (fluorescent bead- or flow rate-based method) and because of polychromatic capabilities. However, standardization of pre-analytical and analytical modus operandi for isolating, enumerating and fluorescent labeling of microparticles remains a challenge. The primary focus of this article is to review the preliminary steps required to optimally study circulating in vivo microparticles which include: 1) centrifugation speed used, 2) quantitation of microparticles before antibody labeling, 3) levels of fluorescence intensity of antibody-labeled microparticles, 4) polychromatic flow cytometric analysis of microparticle sub-populations and 5) use of polyclonal antibodies designed for Western blotting for flow cytometry. These studies determine a roadmap to develop microparticles as biomarkers for a variety of conditions.

Human CD8(+) T cells clear Cryptosporidium parvum from infected intestinal epithelial cells.

Intracellular protozoans of the genus Cryptosporidium are a major cause of diarrheal illness worldwide, especially in immunocompromised individuals. CD4(+) T cells and interferon-gamma are key factors in the control of cryptosporidiosis in human and murine models. Previous studies led us to hypothesize that CD8(+) T cells contribute to clearance of intestinal epithelial Cryptosporidium infection in humans. We report here that antigen expanded sensitized CD8(+) T cells reduce the parasite load in infected intestinal epithelial cell cultures and lyse infected intestinal epithelial cells. These effects are most likely mediated by the release of cytotoxic granules. Elimination of parasites seems to require antigen presentation through both human leukocyte antigen (HLA)-A and HLA-B. These data suggest that cytotoxic CD8(+) T cells play a role in clearing Cryptosporidium from the intestine, a previously unrecognized feature of the human immune response against this parasite.

Cryptosporidium infection of human intestinal epithelial cells increases expression of osteoprotegerin: a novel mechanism for evasion of host defenses.

Cryptosporidium parasites are pathogens of human intestinal epithelial cells. To determine which genes are regulated during early infection, human ileal mucosa cultured as explants was infected with C. parvum or C. hominis, and gene expression was analyzed by microarray. The gene for osteoprotegerin (OPG) was up-regulated by both parasites. OPG mRNA was also significantly increased in biopsy specimens obtained from a volunteer experimentally infected with C. meleagridis, compared with levels in a prechallenge biopsy specimen. After in vitro infection of HCT-8 cells, there was an early peak in production of OPG mRNA protein. Treatment of infected cells with the OPG ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced epithelial cell apoptosis and reduced parasite numbers, and recombinant OPG blocked these effects. These results suggest a novel TRAIL-mediated pathway for elimination of Cryptosporidium infection and a role for OPG in modulating this host response.

CD8 apoptosis may be a predictor of T cell number normalization after immune reconstitution in HIV.

BACKGROUND: As part of the Houston Vanguard study, a subset of 10 patients randomized to receive IL-2 therapy were compared to 4 patients randomized to not receive IL-2, for markers of T cell activation and death during the first three cycles of IL-2. All patients were treated with combination antiretroviral therapy (ART) and were virally suppressed. The purpose of the study was to examine the role of CD8(+) T cell death in responses to ART and IL-2 therapy. METHODS: Lymphocytes were examined at Day 0, 5 and 30 days during three cycles of IL-2 therapy. CD25, CD38, HLA-DR expression and annexin (cell death) were examined on CD4 and CD8 subpopulations. Follow up studies examined CD4 levels and CD4:CD8 reconstitution after 6 years using both univariant and multivariate analyses. RESULTS: Human lymphocytes responded to IL-2 therapy by upregulation of CD25 on CD4(+) T cells, leading to an increase in CD4 cell counts. CD8(+) T cells did not increase CD25 expression, but upregulated activation antigens (CD38 and DR) and had increased death. At baseline, 7 of the 14 patients had high CD8+ T cell apoptosis (mean 17.0% +/- 6.0). We did an exploratory analysis of immune status after six years, and found that baseline CD8+ T cell apoptosis was correlated with CD4 cell count gain beginning two years post enrollment. Patients with low levels of CD8(+) T cell apoptosis at baseline (mean 2.2% +/- 2.1) had significantly higher CD4 cell counts and more normalized CD4:CD8 ratios than patients with high CD8(+) T cell apoptosis (mean CD4 cell counts 1,209 +/- 164 vs 754 +/- 320 cells/mm(3); CD4:CD8 ratios 1.55 vs. 0.70, respectively). CONCLUSION: We postulate that CD8(+) T cell apoptosis may reflect inherent activation status, which continues in some patients even though viral replication is suppressed which influences the ability of CD4(+) T cells to rebound. Levels of CD8(+) T cell apoptosis may therefore be an independent predictor of immune status, which should be shown in a prospective study.