CHARACTERIZATION OF DIFFERENTIATION AND PROGNOSTIC BIOMARKERS ON CD8+ TUMOR-INFILTRATING LYMPHOCYTES IN METASTATIC MELANOMA

CD8+ cytotoxic T lymphocytes (CTL) frequently infiltrate tumors, yet most melanoma patients fail to undergo tumor regression. We studied the differentiation of the CD8+ tumor-infiltrating lymphocytes (TIL) from 44 metastatic melanoma patients using known T-cell differentiation markers. We also compared CD8+ TIL against the T cells from matched melanoma patients’ peripheral blood. We discovered a novel subset of CD8+ TIL co-expressing early-differentiation markers, CD27, CD28, and a late/senescent CTL differentiation marker, CD57. This CD8+CD57+ TIL expressed a cytolytic enzyme, granzyme B (GB), yet did not express another cytolytic pore-forming molecule, perforin (Perf). In contrast, the CD8+CD57+ T cells in the periphery were CD27-CD28-, and GBHi and PerfHi. We found this TIL subset was not senescent and could be induced to proliferate and differentiate into CD27-CD57+, perforinHi, mature CTL. This further differentiation was arrested by TGF-β1, an immunosuppressive cytokine known to be produced by many different kinds of tumors. Therefore, we have identified a novel subset of incompletely differentiated CD8+ TIL that resembled those found in patients with uncontrolled chronic viral infections. In a related study, we explored prognostic biomarkers in metastatic melanoma patients treated in a Phase II Adoptive Cell Therapy (ACT) trial, in which autologous TIL were expanded ex vivo with IL-2 and infused into lymphodepleted patients. We unexpectedly found a significant positive clinical association with the infused CD8+ TIL expressing B- and T- lymphocyte attentuator (BTLA), an inhibitory T-cell receptor. We found that CD8+BTLA+ TIL had a superior proliferative response to IL-2, and were more capable of autocrine IL-2 production in response to TCR stimulation compared to the CD8+BTLA- TIL. The CD8+BTLA+ TIL were less differentiated and resembled the incompletely differentiated CD8+ TIL described above. In contrast, CD8+BTLA- TIL were poorly proliferative, expressed CD45RA and killer-cell immunoglobulin-like receptors (KIRs), and exhibited a gene expression signature of T cell deletion. Surprisingly, ligation of BTLA by its cognate receptor, HVEM, enhanced the survival of CD8+BTLA+ TIL by activating Akt/PKB. Our studies provide a comprehensive characterization of CD8+ TIL differentiation in melanoma, and revealed BTLA as a novel T-cell differentiation marker along with its role in promoting T cell survival.

The epidemiology of Hepatitis B and Hepatitis C virus infection among college students who are not US/Canadian born in Houston

Introduction. A vast majority of studies conducted in both developed and developing nations have focused on the epidemiology of HBV (Hepatitis B virus) and HCV (Hepatitis C virus) in high-risk populations; low-risk populations have been neglected. Recently Hwang et al conducted a unique large cross-sectional study in American university students that focused on cosmetic procedures and drug use for acquiring these infections among a low-risk young adult population In Houston. ^ Methods. This study is a secondary data analysis of the cross-sectional study conducted by Hwang et al. Data for this anonymous study were collected from 7,960 college students, among whom were the 2,561 non US/Canadian born students included in this study. All students completed a self-administered questionnaire and provided a blood sample. The epidemiology of HBV/HCV and risk factors for acquiring HBV/HCV infection was studied by comparing those with HBV/HCV infection versus those without. Both univariate and multivariate logistic regression was used to analyze the data. ^ Results. Overall prevalence of HBV and HCV infections were 22% and 0.8% respectively. By multivariable analysis, the factors that were independently associated with increased prevalence of HBV infection were increasing age per year (OR=1.06, 95% C.I=1.04-1.08), Black or Asian race (OR=6.21, 95% C.I=3.14-12.27), history of household contact with hepatitis (OR=1.87, 95% C.I=1.15-3.05), and having sexual partner with hepatitis (OR=5.20, 95% C.I=1.5-18.00). For HCV these factors included increasing age per year (OR= 1.08, 95% C.I=1.03-1.14), history of blood transfusion prior to 1991 (OR=25.45, 95% C.I=7.58-85.40), and Injection drug use. (OR=78.15, 95% C.I=12.19-500.85). Cosmetic procedures like tattooing were not significant risk factors for either HBV or HCV infection. ^ Conclusions. In a low-risk adult foreign born population, cosmetic procedures are not significant risk factors for HBV or HCV infection. The prevention strategies of these infections in this population should focus on safe sexual practices/abstinence and HBV vaccination should be provided to adolescents and sexually active adults. ^

Hepatitis B and C infections in U.S. incarcerated populations: Prevalence and related mortality

Published reports have consistently indicated high prevalence of serologic markers for hepatitis B (HBV) and hepatitis C (HCV) infection in U.S. incarcerated populations. Quantifying the current and projected burden of HBV and HCV infection and hepatitis-related sequelae in correctional healthcare systems with even modest precision remains elusive, however, because the prevalence and sequelae of HBV and HCV in U.S. incarcerated populations are not well-studied. This dissertation contributes to the assessment of the burden of HBV and HCV infections in U.S. incarcerated populations by addressing some of the deficiencies and gaps in previous research. ^ Objectives of the three dissertation studies were: (1) To investigate selected study-level factors as potential sources of heterogeneity in published HBV seroprevalence estimates in U.S. adult incarcerated populations (1975-2005), using meta-regression techniques; (2) To quantify the potential influence of suboptimal sensitivity of screening tests for antibodies to hepatitis C virus (anti-HCV) on previously reported anti-HCV prevalence estimates in U.S. incarcerated populations (1990-2005), by comparing these estimates to error-adjusted anti-HCV prevalence estimates in these populations; (3) To estimate death rates due to HBV, HCV, chronic liver disease (CLD/cirrhosis), and liver cancer from 1984 through 2003 in male prisoners in custody of the Texas Department of Criminal Justice (TDCJ) and to quantify the proportion of CLD/cirrhosis and liver cancer prisoner deaths attributable to HBV and/or HCV. ^ Results were as follows. Although meta-regression analyses were limited by the small body of literature, mean population age and serum collection year appeared to be sources of heterogeneity, respectively, in prevalence estimates of antibodies to HBV antigen (HBsAg+) and any positive HBV marker. Other population characteristics and study methods could not be ruled out as sources of heterogeneity. Anti-HCV prevalence is likely somewhat higher in male and female U.S. incarcerated populations than previously estimated in studies using anti-HCV screening tests alone without the benefit of repeat or additional testing. Death rates due to HBV, HCV, CLD/cirrhosis, and liver cancer from 1984 through 2003 in TDCJ male prisoners exceeded state and national rates. HCV rates appeared to be increasing and disproportionately affecting Hispanics. HCV was implicated in nearly one-third of liver cancer deaths. ^

THE NEW ROLE OF PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9: A CONNECTION OF PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9, APOLIPOPROTEIN B, and AUTOPHAGY

Plasma low-density lipoprotein (LDL) levels are positively correlated with the incidence of coronary artery disease. In the circulation, the plasma LDL clearance is mainly achieved by the uptake via LDL receptor (LDLR). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a newly discovered gene, playing an important role in LDL metabolism. Gain-of-function mutations of PCSK9 lead to hypercholesterolemia and loss-of-function mutations of PCSK9 are associated with decrease of LDL cholesterol. The effects of PCSK9 on cholesterol levels are the consequence of a strong interaction between the catalytic domain of PCSK9 and epidermal growth factor-like repeat A (EGF-A) domain of LDLR on the cell surface of hepatocytes. This PCSK9/LDLR complex enters the cell via endocytosis, where both PCSK9 and LDLR are removed via the lysosome pathway, resulting in decreased levels of LDLR and accumulation of LDL in the plasma. However, whether this is the exclusive function of PCSK9 on LDL metabolism was challenged by us; we observed PCSK9 interacted with apolipoprotein B (apoB) and increased apoB production, irrespective of the LDLR. ApoB is the primary structure protein of LDL particle and it also serves as the ligand for the LDL receptor. There is ample evidence showing that the levels of apoB are a better indicator for heart disease than either total cholesterol or LDL cholesterol levels. We used a second-generation adenoviral vector to overexpress PCSK9 (Ad-PCSK9) in wild-type C57BL/6 and LDLR deficient mice (Ldlr-/- and Ldlr-/-Apobec1-/-). Our study revealed that overexpression of PCSK9 promoted the production and secretion of apoB in the form of very-low density lipoprotein (VLDL), which is the precursor of LDL, in the 3 mouse models studied (C57BL/6J, Ldlr-/-, and Ldlr-/-Apobec1-/-). The increased apoB production in mice was regulated at post-transcriptional levels, since there was no difference in apoB mRNA levels between mice treated with Ad-PCSK9 and control vector Ad-Null. By using pulse-chase experiment on primary hepatocytes, we showed that overexpression of PCSK9 increased the secretion of apoB, independent of LDLR. In the circulation, we showed that PCSK9 was associated with LDL particles. By using 3 different protein–protein interaction assays of co-immunoprecipitation, mammalian two-hybrid system, and in situ proximity ligation assay, we demonstrated a direct protein–protein interaction between PCSK9 and apoB. The impact of this interaction inhibited the physiological removal process of apoB via autophagosome/lysosome pathway in an LDLR-independent fashion, resulting in increased production and secretion of apoB-containing lipoproteins. The significance of this process was shown in the Pcsk9 knockout mice in the background of Ldlr-/-Apobec1-/- mice (triple knockout mice); in the absence of Pcsk9 (triple knockout mice) the levels of cholesterol, triacylglycerol, and apoB decreased significantly in comparison to that of Ldlr-/-Apobec1-/- mice. Taken together, our study demonstrated a direct intracellular interaction of PCSK9 with apoB, resulting in the inhibition of apoB degradation via the autophagosome/lysosome pathway independent of LDLR. This discovery provides a new concept of the importance of PCSK9 and suggests new approaches for the therapeutic intervention of hyperlipidemia.

MUTATIONS IN STAT3 ASSOCIATED WITH HUMAN HYPER IgE SYNDROME ENHANCE NFκB AND MAPK-MEDIATED GENE EXPRESSION

Hyper IgE syndrome (HIES) is a multisystem disorder resulting in bone and immune system abnormalities. It is associated with mutations in STAT3, which disrupt protein domains responsible for transcriptional function. Patients with HIES display osteoporosis and enhanced inflammatory cytokine production similar to hematopoietic Stat3-deficient mice. Since osteoclast and inflammatory cytokine genes are NFκB targets, these observations indicate a possible deregulation of NFκB signaling in both mice and humans with STAT3-deficiency. Here, we sought to examine the role of STAT3 in the regulation of NFκB-mediated gene expression through analysis of three HIES STAT3 point mutations in both hematopoietic and non- hematopoietic cells. We found that IL-6-induced tyrosine phosphorylation of STAT3 was partially or completely abrogated by HIES mutations in the transactivation domain (V713L) or SH2 domain (V637M), respectively, in both hematopoietic and non- hematopoietic cells. By contrast, IL-6-induced tyrosine phosphorylation of an HIES mutant in the STAT3 DNA-binding domain (R382W) was intact. The R382W and V713L mutants significantly reduced IL-6-dependent STAT3 transcriptional activity in reporter gene assays. Moreover, the R382W and V637M mutants significantly diminished IL-6-responsive expression of the endogenous STAT3 target gene, Socs3, as assessed by quantitative real-time PCR (qPCR) in the RAW macrophage cell line. These observations indicate the HIES mutants dominantly suppress the transcriptional activity of wild type STAT3, albeit to varying degrees. All three HIES mutants enhanced LPS-induced expression of the NFκB target genes IL6 (IL-6), Cxcl10 (IP- 10), and Tnf (TNFα) in RAW cells, as indicated by qPCR. Furthermore, overexpression of wild type STAT3 in Stat3-deficient murine embryonic fibroblasts significantlyreduced LPS-stimulated expression of IL6, Cxcl10, and IL12p35. In addition, in aprimary murine osteoclast differentiation assay, a STAT3-specific SH2 domain inhibitor led to significantly increased levels of osteoclast-specific gene expression. These results suggest that STAT3 serves as a negative regulator of NFκB-mediated gene expression, and furthermore imply that STAT3 mutations associated with HIES contribute to the osteopenia and inflammation observed in HIES patients.

Experimental models for p53, hepatitis B and aflatoxin hepatocarcinogenesis

The major risk factors for liver cancer in Southeast Asia: HBV infection, aflatoxin exposure and p53 expression/mutation, were examined in experimental models. Four groups were examined for development of hepatocellular carcinoma (HCC) with and without neonatal exposure to aflatoxin (AFB$\sb1)$: (Group I.) Transgenic HBsAg mice with one p53 allele. (Group II) Transgenic HBsAg mice with two p53 alleles. (Group III) Non-transgenic litter mates with one p53 allele. (Group IV) Non-transgenic litter mates with two p53 alleles. HCC developed in Group I animals exposed to aflatoxin at an earlier time and were of a higher grade than those seen later in other groups. These results provide an explanation for as to why p53 is a target for deletion and/or mutation in human HCC especially when found in high risk areas where HBV infection and Aflatoxin B1 food contamination is high, and nicely illustrates a synergistic interaction among these three factors. None of the tumors analyzed had loss or mutation in the p53 gene.^ To determine the significance of the specific p53ser249 mutation found in HBV/aflatoxin associated human hepatomas in an in-vivo experimental model using transgenic mice, a two-nucleotide change in the mouse p53 gene at amino acid position 246, which is equivalent to that of 249 in human p53, was introduced. Transgenic mice with mutant p53 controlled by the albumin promoter were generated and shown to express the p53ser246 mutant RNA and protein specifically in liver. Three groups were examined for development of HCC with and without neonatal exposure to aflatoxin: (Group V) Transgenic p53ser246 mice with two p53 alleles. (Group VI) Transgenic p53ser246 mice with one p53 allele. (Group VII) Double transgenic for p53ser246 and HBsAg with two p53 alleles. One hundred percent of male mice with the three risk factors injected with aflatoxin developed high grade liver tumors, compared to 66.6% from group VI and only 14.2% of group V suggesting synergistic interaction between HBsAg and this particular ser246 p53 mutation.^ In order to examine the growth properties of hepatocytes and correlation with p53 loss and/or mutation, cell proliferation and ploidy analysis of liver from normal heterozyous, homozygous null mice and from transgenic mutant p53ser246, mice were studied. Loss of wild-type p53 increased G1/G0 ratios of cells as well as proliferation and decreased cell ploidy. The mutant p53ser246 did not show a significant effect on cell ploidy or proliferation. However a striking 5-10X increase in G1/G0 ratio suggests that this specific mutation specifically induces G0 to G1 transition, which in turn further predisposes hepatocytes to the damaging effect of Aflatoxin. (Abstract shortened by UMI.) ^

The role of plasmacytoid dendritic cells in the regulation of adaptive immunity

Plasmacytoid dendritic cells (pDCs) selectively express TLR7 which allows them to respond to RNA viruses and TLR9 which allows them to respond to DNA viruses and CpG oligonucleotides. Upon exposure to virus pDCs produce vast amounts of type I interferon (IFN) directly inhibiting viral replication and contributing to the activation of other immune cells. The ability of pDCs to promote B and T cell differentiation through type I IFN has been well documented although the role of additional factors including tumor necrosis factor (TNF) family members has not been thoroughly addressed. Here the expression of selected TNF family members in pDCs was examined and the role of TNF receptor-ligand interactions in the regulation of B and T lymphocyte growth and differentiation by pDCs was investigated. Upon stimulation with CpG-B, pDCs exhibit strong and stable expression of CD70, a TNF family ligand that binds to its receptor CD27 on memory B cells and promotes plasma cell differentiation and Ig secretion. Using an in vitro pDC/B cell co-culture system, it was determined that CpG-B-stimulated pDCs induce the proliferation of CD40L-activated human peripheral B cells and Ig secretion. This occurs independently of IFN and residual CpG, and requires physical contact between pDCs and B cells. CpG-stimulated pDCs induce the proliferation of both naive and memory B cells although Ig secretion is restricted to the memory subset. Blocking the interaction of CD70 with CD27 using an antagonist anti-CD70 antibody reduces the induction of B cell proliferation and IgG secretion by CpG-B-stimulated pDCs. Published studies have also indicated an important role for CD70 in promoting the expansion of CD4+ and CD8+ T cells and the development of effector function. CpG-B-stimulated pDCs induce naïve CD4+ T cell proliferation and production of multiple cytokines including IFN-γ, TNF-α, IL-10, IL-4, IL-5 and IL-13. Blocking the function of CD70 with an antagonist anti-CD70 antibody significantly reduced the induction of naïve CD4+ T cell proliferation by CpG-B-stimulated pDCs and the production of IL-4 and IL-13. Collectively these data indicate an important role for CD70 in the regulation of B and T lymphocyte growth and differentiation by pDCs. ^

BLyS/BAFF-R signaling pathway in human aggressive non Hodgkin's lymphoma B cell and normal peripheral blood B lymphocytes

B-lymphocyte stimulator (BLyS also called BAFF), is a potent cell survival factor expressed in many hematopoietic cells. BLyS levels are elevated in the serum of non-Hodgkin lymphoma (NHL) patients, and have been reported to be associated with disease progression, and prognosis. To understand the mechanisms involved in BLyS gene expression and regulation, we examined expression, function, and regulation of the BLyS gene in B cell non-Hodgkin's lymphoma (NHL-B) cells. BLyS is constitutively expressed in aggressive NHL-B cells including large B cell lymphoma (LBCL) and mantle cell lymphoma (MCL) contributing to survival and proliferation of malignant B cells. Two important transcription factors, NF-κB and NFAT, were found to be involved in regulating BLyS expression through at least one NF-κB and two NFAT binding sites in the BLyS promoter. Further study indicates that the constitutive activation of NF-κB and BLyS in NHL-B cells forms a positive feedback loop contributing to cell survival and proliferation. In order to further investigate BLyS signaling pathway, we studied the function of BAFF-R, a major BLyS receptor, on B cells survival and proliferation. Initial study revealed that BAFF-R was also found in the nucleus, in addition to its presence on plasma membrane of B cells. Nuclear presentation of BAFF-R can be increased by anti-IgM and soluble BLyS treatment in normal peripheral B lymphocytes. Inhibition of BLyS expression decreases nuclear BAFF-R level in LBCL cells. Furthermore, we showed that BAFF-R translocated to nucleus through the classic karyopherin pathway. A candidate nuclear localization sequence (NLS) was identified in the BAFF-R protein sequence and mutation of this putative NLS can block BAFF-R entering nucleus and LBCL cell proliferation. Further study showed that BAFF-R co-localized with NF-κB family member, c-rel in the nucleus. We also found BAFF-R mediated transcriptional activity, which could be increased by c-rel. We also found that nuclear BAFF-R could bind to the NF-κB binding site on the promoters of NF-κB target genes such as BLyS, CD154, Bcl-xL, Bfl-1/A1 and IL-8. These findings indicate that BAFF-R may also promote survival and proliferation of normal B cells and NHL-B cells by directly functioning as a transcriptional co-factor with NF-κB family member. ^

STUDIES ON THE CLASS I GLYCOPROTEINS OF THE MURINE MAJOR HISTOCOMPATIBILITY COMPLEX (TRANSPLANTATION ANTIGEN, LYMPHOCYTE, CELL SURFACE MOLECULE)

A series of studies were undertaken to analyze and compare various aspects of murine class I glycoproteins. An initial area of investigation characterized the Qa-1 alloantigens using two-dimensional gel electrophoresis. Analysis of the products of the Qa-1('b), Qa-1('c) and Qa-1('d) alleles indicated that these were distinct molecules as determined by their lack of comigration upon comparative two-dimensional gel analysis. The importance of asparagine-linked glycosylation in the cell surface expression of class I molecules was also examined. These studies employed tunicamycin, an inhibitor of N-linked glycosylation. Tunicamycin treatment of activated T lymphocytes diminished the surface expression of Qa-1 to undetectable levels; the levels of other class I molecules exhibited little or no decrease. These results indicated that N-linked glycosylation has a differential importance in the cell surface expression of various class I molecules. The molecular weight diversity of class I molecules was also investigated. Molecular weight determination of both the fully glycosylated and unglycosylated forms of H-2 and Qa/Tla region encoded molecules established that there is a significant variation in the sizes of these forms of various class I molecules. The most significant difference ((TURN)9,000 daltons) exists between the unglycosylated forms of H-2K('b) and Qa-2, suggesting that the structural organization of these two molecules may be very different. A comparative two-dimensional gel analysis of various class I glycoproteins isolated from resting and activated T and B lymphocytes indicated that class I molecules expressed on activated T cells exhibited an isoelectrophoretic pattern that was distinct from the isoelectrophoretic pattern of class I molecules expessed on the other cell populations. This difference was attributed to a lower sialic acid content of the molecules expressed on activated T cells. Analysis of cell homogenates determined that activated T cells contained a higher level of endogenous neuraminidase activity than was detected in the other populations, suggesting that this may be the basis of the lower sialic acid content. The relationship of the Qa-4 and Qa-2 alloantigens was also examined. It was established that upon mitogen activation, the expression of Qa-4 was greatly decreased, whereas Qa-2 expression was not decreased. However, an anti-Qa-2 monoclonal antibody blocked the binding of an anti-Qa-4 monoclonal antibody to resting cells. These studies established that Qa-4 is a determinant restricted to resting cells, which is closely associated on the surface with the Qa-2 molecule. ^

2005 ACIP recommendations effect on newborn hepatitis B vaccination rates and the factors affecting the successful implementation of the recommendations in the US

Background. Hepatitis B virus infection is one of major causes of acute and chronic hepatitis, cirrhosis of the liver, and primary hepatocellular carcinoma. Hepatitis B and its long term consequences are major health problems in the United States. Hepatitis B virus can be vertically transmitted from mother to infant during birth. Hepatitis B vaccination at birth is the most effective measure to prevent the newborn from HBV infection and its consequences, and is part of any robust perinatal hepatitis B prevention program following ACIP recommendations. Universal vaccination of the new born will prevent HBV infection during early childhood and, assuming that children receive the three dosages of the vaccine, it will also prevent adolescent and adult infections. Hepatitis B vaccination is now recommended as part of a comprehensive strategy to eliminate HBV transmission in the United States. ^ Objective. (1)To assess if the hepatitis B vaccination rates of newborn babies have improved after the 2005 ACIP recommendations. (2) To identify factors that affects the implementation of ACIP recommendation for hepatitis B vaccination in newborn babies. These factors will encourage ongoing improvement by identifying successful efforts and pinpointing areas that fall short and need attention. Additional focus areas may be identified to accelerate progress in eliminating perinatal HBV transmission.^ Methods. This review includes information from all pertinent articles, reviews, National immunization survey (NIS) surveys, reports, peer reviewed literature and web sources that were published after 1991.The key words to be used for selecting the articles are: "Perinatal Hepatitis B Prevention program", "Universal Hepatitis B vaccination of newborn babies", "ACIP Recommendations." The data gathered will be supplemented with an analysis of vaccination rates using the National Immunization Survey (NIS) birth dose coverage data.^ Results. The data collected in the NIS of 2009 reveals that the national coverage for birth dose of HepB increased to 60.8% from 50.1% in 2006. The largest increase observed for the birth dose in the past 5 years is from 2008 which increased from 55.3 % to 60.8% in 2009. By state, coverage ranged from 22.8% in Vermont to 80.7% in Michigan. %. Overall, in 2009 the estimated vaccination rates are in higher ranges for most states compared to the estimated vaccination rates in 2006. States vary widely in hepatitis B vaccination rates and in their compliance with the 2005 ACIP recommendation. There are many factors at various stages that might affect the successful implementation of the new ACIP recommendation as revealed in literature review. ^ Conclusions. HBV perinatal transmission can be eliminated, but it requires identifying the gaps and measures taken to increase the current vaccination coverage, ensuring timely administration of post exposure immunoprophylaxis and continued evaluations of the impact of immunization recommendations.^