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Interleukin-8 (IL-8) is a pro-angiogenic cytokine associated with aggressive prostate cancer (CaP). We detected high levels of IL-8 in sera from patients with CaP compared with healthy controls and patients with benign prostatic hypertrophy. This study examines the role of IL-8 in the pathogenesis of metastatic prostate cancer. We developed a biocompatible, cationic polylactide (CPLA) nanocarrier to complex with and efficiently deliver IL-8 small interfering RNA (siRNA) to CaP cells in vitro and in vivo. CPLA IL-8 siRNA nanocomplexes (nanoplexes) protect siRNA from rapid degradation, are non-toxic, have a prolonged lifetime in circulation, and their net positive charge facilitates penetration of cell membranes and subsequent intracellular trafficking. Administration of CPLA IL-8 siRNA nanoplexes to immunodeficient mice bearing human CaP tumours produced significant antitumour activities with no adverse effects. Systemic (intravenous) or local intra-tumour administration of IL-8 siRNA nanoplexes resulted in significant inhibition of CaP growth. Magnetic resonance imaging and ultrasonography of experimental animals demonstrated reduction of tumour perfusion in vivo following nanoplex treatment. Staining of tumour sections for CD31 confirmed significant damage to tumour neovasculature after nanoplex therapy. These studies demonstrate the efficacy of IL-8 siRNA nanotherapy for advanced, treatment-resistant human CaP.

Background/aims Prostate specific antigen (PSA) is regulated by steroid hormones, such as testosterone, the serum levels of which are altered in patients with Alzheimer's disease (AD).This pilot study compared serum levels of the free (f) PSA between AD, mild cognitive impairment (MCI), and control subjects, and evaluated the relationship between fPSA serum levels and cognitive assessment tests and neuroimaging data. In addition, in a subgroup of AD patients, we correlated fPSA serum levels with the existing data on serum levels of amyloid-beta (Aβ), and iron-related proteins, including hepcidin and ferritin. Methods Frozen serum samples from the Oregon Tissue Bank were used to measure serum levels of fPSA using enzyme-linked immunosorbent assay. Results fPSA serum levels calculated as median ± SD were higher in AD males (663.6 ± 821.0 pg/ml) compared to control males (152.0 ± 207.0 pg/ml), p = 0.003. A similar Pattern emerged when comparing MCI males (310.7 ± 367.0 pg/ml) to control males (P = 0.02). Correlation studies showed a significant association between fPSA and CDR (r = 0.56, P = 0.006) and CDR-SOB (r = 0.54, P = 0.009) in AD males. Conclusion Additional studies in a larger cohort are required for determining whether fPSA can be used as biomarker of AD disease progression and whether it has the potential to identify male subjects at risk of AD dementia.

This study is one in series determining the potential of RAGE axis (receptor for advanced glycation end products, isoforms, ligands) as a biomarker in multiple sclerosis (MS). We evaluated serum levels of RAGE ligand, the high-mobility group box (HMGB)1 in MS patients, and assessed the correlation between HMGB1 serum levels and the use of disease-modifying drugs (DMDs), and between HMGB1 serum levels and indicators of MS disease severity. HMGB1 serum levels were compared between 96 (23 males) MS patients and 34 age- and gender-matched healthy controls (HCs) using enzyme-linked immunosorbent assays. DMD-naive MS patients had significantly higher HMGB1 serum levels compared with DMD-treated (P = 0.04) and compared with HCs (P = 0.01). HMGB1 serum levels were not significantly different between total MS patients (DMD-naive plus DMD-treated) and HCs (P = 0.09). DMD-naive MS patients in clinical relapse tended to have lower HMGB1 serum levels than clinically stable RRMS patients (P = 0.07). HMGB1 serum levels showed 0.65 area under the curve (95 % CI 0.55–0.95) sensitivity/specificity for MS clinical relapse. The role of HMGB1 in MS disease pathology and DMD modulation of this protein warrant further investigations.

BACKGROUND PSA is a biomarker for diagnosis and management of prostate cancer. PSA is known to have anti-tumorigenic activities, however, the physiological role of PSA in prostate tumor progression is not well understood. METHODS Five candidate peptides identified based upon computer modeling of the PSA crystal structure and hydrophobicity were synthesized at >95% purity. The peptides in a linear form, and a constrained form forced by a di-sulfide bond joining the two ends of the peptide, were investigated for anti-angiogenic activity in HUVEC. RESULTS None of the five PSA-mimetic peptides exhibited PSA-like serine protease activity. Two of the peptides demonstrated significant anti-angiogenic activity in HUVEC based on (i) inhibition of cell migration and invasion; (ii) inhibition of tube formation in Matrigel; (iii) anti-angiogenic activity in a sprouting assay; and (iv) altered expression of pro- and anti-angiogenic growth factors. Constrained PSA-mimetic peptides had greater anti-angiogenic activity than the corresponding linearized form. Complexing of PSA with ACT eliminated PSA enzymatic activity and reduced anti-angiogenic activity. In contrast, ACT had no effect on the anti-angiogenic effects of the linear or constrained PSA-mimetic peptides. Modeling of the ACT-PSA complex demonstrated ACT sterically blocks the anti-angiogenic activity of the two bioactive peptides. CONCLUSIONS The interaction of a hydrophilic domain on the surface of the PSA molecule with a target on the cell membrane of prostate endothelial and epithelial cells was responsible for the anti-angiogenic or anti-tumorigenic activity of PSA: enzymatic activity was not associated with anti-angiogenic effects. Furthermore, since PSA and ACT are both expressed within the human prostate tissue microenvironment, the balance of their expression may represent a mechanism for endogenous regulation of tissue angiogenesis. Prostate 75:1285–1299, 2015. © 2015 Wiley Periodicals, Inc.

Background: Inflammation is known to play a role in cererovascular risk. Multiple sclerosis (MS) is a neurodegenerative disease that is initially characterized by inflammatory changes in the brain. We hypothesized that due to chronic inflammation, MS patients would present with a higher levels of cardiovascular (CV) risk factors than non-MS patients. Methods: We performed a retrospective chart review on 206 MS patients and 142 control patients suffering from meningiomas and acoustic neuromas, non inflammatory, non autoimmune diseases of the brain. The obtained data included fasting lipid profiles, plasma glucose, systolic and diastolic blood pressure (BP), serum levels of homocysteine and uric acid, data on iron status, smoking habit, and list of medications. In addition, data on indicators of MS disease severity was obtained for MS patients. Results: MS patients had significantly higher total plasma cholesterol, p = 0.01, and plasma high density lipoprotein, P

Background We investigated Fingolimod treatment effects on the RAGE (receptor for advanced glycation endproducts) axis in multiple sclerosis (MS) patients. The primary outcome of the study was whether Fingolimod treatment increases serum levels of the soluble RAGE isoforms, sRAGE and esRAGE – both being considered putative endogenous inhibitors of RAGE signaling. Additional variables were serum levels of RAGE ligands, the high mobility group box (HMGB)1 and pentosidine. Methods Serum levels of the study variables were measured by ELISA, and compared between baseline (before Fingolimod treatment) and 6 and 12 months post-drug treatment in 17 relapsing MS patients. Fingolimod treatment effects on MS disease progression were assessed by comparing pre- and post-Fingolimod values of the EDSS and rate of clinical relapse, and changes in the T1-and T2-enahncing lesions on the MRI scan.methods Results Twelve months treatment with Fingolimod increased serum levels of sRAGE and esRAGE by 32.4% (P = 0.004) and 48.5% (P = 0.007) respectively. In addition, Fingolimod treatment reduced serum levels of HMGB1 by 71.6% (P = 0.02) and pentosidine serum levels by 41.3% (P = 0.12). EDSS remained stable (baseline: 3.57 ± 1.56; post-Fingolimod: 3.54 ± 1.2, P = 0.96) and the rate of clinical relapse decreased near significantly (P = 0.094). T1-and T2-enhancing lesions remained stable, showing no significant changes pre-vs. post-Fingolimod treatment. Conclusion Fingolimod mediates modulation of the RAGE axis which apparently contributes to the Fingolimod's anti-inflammatory and neuroprotective effects. These findings may provide a rationale for the clinical efficacy of Fingolimod in pathological states other than MS, where dysregulation of the RAGE axis plays a role.

Matrix metallaprotinase-9 (MMP-9) is zinc-containing proteinase whose expression and trafficking are frequently altered in cancer. MMP-9 in the plasma membrane and the secreted forms are thought to contribute to the invasive and metastatic properties of malignant tumors. We have manipulated the expression of MMP-9 in prostate tumor cell line LNCaP and measured their capacity to invade through a basement membrane matrix. Stable expression of human MMP-9 in a poorly metastatic LNCaP prostate cancer cell line produced a 2-3-fold increase in MMP-9 activity and a comparable increase in invasiveness. Transient transfection of LNCaP stable clone expressing MMP-9 with MMP-9 antisense oligonucleotide (ASODN) produced 55-90% less MMP-9 than control cells and were proportionately less invasive. In contrast, manipulating MMP-9 levels had no effect on cell migration across an uncoated membrane. A standard MMP-9 inhibitor at a concentration ranging from 1-10 nM, caused a nearly quantitative inhibition of extracellular MMP-9 activity and had significant effect on basement membrane invasion. Collectively, these results confirm the role of MMP-9 in tissue remodeling associated with prostate tumor invasion.

BACKGROUND Human and animal studies have suggested that diet-derived flavonoids, in particular quercetin may play a beneficial role by preventing or inhibiting oncogenesis, but the underlying mechanism remains unclear. The aim of this study is to evaluate the effect(s) of quercetin on normal and malignant prostate cells and to identify the target(s) of quercetin's action. METHODOLOGY We addressed this question using cells in culture and investigated whether quercetin affects key biological processes responsible for tumor cell properties such as cell proliferation and apoptosis and also studied the effect of quercetin on the proteome of prostate cancer cells using difference gel electrophoresis (DIGE) to assess changes in the expression of relevant proteins. RESULTS Our findings demonstrate that quercetin treatment of prostate cancer cells results in decreased cell proliferation and viability. Furthermore, we demonstrate that quercetin promotes cancer cell apoptosis by down-regulating the levels of heat shock protein (Hsp) 90. Depletion of Hsp90 by quercetin results in decreased cell viability, levels of surrogate markers of Hsp90 inhibition (intracellular and secreted), induced apoptosis and activation of caspases in cancer cells but not in normal prostate epithelial cells. Knockdown of Hsp90 by short interfering RNA also resulted in induction apoptosis similar to quercetin in cancer cells as indicated by annexin V staining. CONCLUSION Our results demonstrate that quercetin down-regulates the expression of Hsp90 which, in turn, induces inhibition of growth and cell death in prostate cancer cells while exerting no quantifiable effect on normal prostate epithelial cells. Prostate © 2008 Wiley-Liss, Inc.

Prostate-specific antigen (PSA) is a serine protease secreted both by normal prostate glandular epithelial cells and prostate cancer cells. We explored "thiophilic-interaction chromatography" (TIC) to isolate tissue prostate-specific antigen (T-PSA) from fresh human prostate cancer tissue harvested by radical prostatectomy for the purpose to characterize T-PSA for its enzymatic activity and sensitivity to zinc ions. We have shown, for the first time, that T-PSA has strong affinity for the thiophilic gel (T-gel). The average recovery of T-PSA from T-gel is over 87%. The presence of PSA in the column eluate was confirmed by ELISA and SDS/PAGE. Western blot developed with monoclonal antibody to PSA revealed that T-PSA was predominantly in the "free" form having a molecular weight of 33 kDa. Furthermore, T-PSA was found to be enzymatically active. T-PSA was found to be less enzymatically active as compared to seminal plasma PSA. The inhibition of enzymatic activity of both f-PSA and T-PSA over a wide range of concentrations of Zn(2+) ions (10nM to 50 microM) was comparable. In contrast, the enzymatic activity of chymotrypsin, another serine-protease, was affected differently. At higher concentrations of Zn(2+) (10 microM and higher) the enzymatic activity of chymotrypsin was inhibited, whereas, at lower concentrations of Zn(2+) (5 microM and lower), the enzymatic activity was enhanced.

The role of prostate-specific antigen (PSA) during the onset of prostate cancer and subsequent tumor growth and metastasis is not well understood. We have developed a simple two step procedure, based on principles of hydrophobic charge-induction chromatography and molecular size chromatography to provide pure free-PSA (f-PSA) preparation that is free from all other known PSA complexes as well as human kallikrein 2 (hK2). The overall recovery of f-PSA is 72%. The isolated f-PSA consists of three known isoforms that corresponds to pI of 6.2, 6.4 and 7.2. f-PSA is enzymatically active and its enzymatic activity can be effectively neutralized by a serine protease inhibitor.

Double-stranded RNAs induce interferons and cause the development of antiviral and antiproliferative activities. Antiviral activity is related to the production of interferons and other proteins that stimulate various immunologic activities, which appear to contribute to their overall antiproliferative activity. The most active double-stranded RNA, polyI:polyC, was shown to be too toxic for therapeutic use. We conducted selective thiolation of the polyC strand at the five position of the cytosine bases, generating a partially thiolated polyC (MPC) which after annealing with a complimentary unmodified polyI, gave the thiolated double-stranded RNA, pI:MPC. We have explored antiviral and antiproliferative activities at various levels of thiolation and found that optimal responses can be obtained at 7.4% level of thiolation. This compound deserves further study of antiviral and antiproliferative responses in vivo, and eventually clinical exploration. Earlier studies have shown that this and related compounds are active against HIV-1, in human cells, and against DNA polymerases of DNA and RNA tumor viruses.

BACKGROUND Many viral and neoplastic diseases are resistant to interferon-α/β (IFN-α/β) therapy or develop resistance during the course of IFN treatment. In patients with viral diseases, the authors identified four IFN inhibitors, of which the most important, most likely is a free IFN receptor of type 1 appearing in the circulation that captures and neutralizes IFN-α/β. METHODS Ninety-one cancer patients and 25 healthy individuals were studied. Free circulating IFN receptor-α/β type 1 was studied. The patients were ages 35–75 years. The diagnoses were 24 cases of colon carcinoma, 7 cases of prostate carcinoma, 16 cases of breast carcinoma, 8 cases of ovarian carcinoma, 9 cases of uterine carcinoma, 5 cases of lung carcinoma, 3 cases of astrocytoma, 4 cases of transitional cell carcinoma of the bladder, 1 case of osteosarcoma, 3 cases of multiple myeloma, 4 cases of Hodgkin disease, 2 cases of non-Hodgkin lymphoma, 3 cases of myelodysplastic syndrome, and 2 disseminated tumors of unknown origin. RESULTS All patients were found to have increased free IFN receptor-α/β type 1 in the circulation, with the highest levels reported in patients with adenocarcinoma. CONCLUSIONS High IFN inhibitory activity in patients with cancer may be a significant factor in their increased susceptibility to progressive disease, infectious complications, and resistance to IFN therapy. Ongoing studies are being performed with the objective of overcoming this inhibitory activity. Cancer 2003;98:2730–3. © 2003 American Cancer Society.

Flavonoids are plant metabolites that are dietary antioxidants and exert significant anti-tumor, anti-allergic, anti-inflammatory and anti-viral effects. It is generally accepted that Th-1 derived cytokines such as IL-2, IFNgamma and IL-12 promote cellular immunity while Th-2 derived cytokines such as IL-4, IL-5, IL-6 exert negative immunoregulatory effects on cellular immunity while upregulating humoral immunity. The molecular mechanisms underlying the biological activities of flavonoids have not been elucidated. We hypothesize that the flavonoid, quercetin, exert significant anti-viral and anti-tumor effects possibly by modulating the production of Th-1 and Th-2 derived cytokines. Peripheral blood mononuclear cells (PBMC, 1 x 10(6) cells/ml) from normal subjects were cultured with different concentrations of quercetin (0.5-50 microM) for 24-72 h and supernates were quantitated for IFN-gamma and IL-4 by ELISA and antiviral activity of IFNgamma by bioassay. FACS analysis was done to determine the number of IFN-gamma and IL-4 positive cells and RT-PCR was done to quantitate gene expression. Quercetin significantly induces the gene expression as well as the production of Th-1 derived IFNgamma and the downregulates Th-2 derived IL-4 by normal PBMC. Further, quercetin treatment increased the phenotypic expression of IFNgamma cells and decreased IL-4 positive cells by FACS analysis, which corroborate with protein secretion and gene expression studies. These results suggest that the beneficial immuno-stimulatory effects of quercetin may be mediated through the induction of Th-1 derived cytokine, IFNgamma, and inhibition of Th-2 derived cytokine, IL-4.

BACKGROUND It is known that total PSA values are often greater than the sum total of free-PSA (f-PSA) and PSA–ACT complex. It is primarily because other PSA complexes are present in the patient serum and currently cannot be quantified. Our studies were aimed in developing methodology that will permit recovery of all molecular forms of PSA from various biological fluids. METHODS Thiophilic interaction (3S,T-gel) chromatography procedure was used to isolate various PSA molecular forms present in biological fluids. f-PSA and PSA complexes were shown to have strong affinity for T-gel. PSA forms eluted from the column were analyzed by SDS–PAGE/Western blot and were identified by immunostaining with antibody-specific to PSA and to various protease inhibitors. The identity of PSA complexes was also confirmed by Enzyme-linked Immunosorbent-assay (ELISA). RESULTS One step affinity chromatography procedure (3S,T-gel) was used to isolate different molecular forms of PSA in seminal plasma, patient sera, and in prostate cancer cell culture medium. Seminal plasma was shown to contain f-PSA, PSA–ACT, PSA–A2M, and PSA–PCI. Sera from prostate cancer patients at different stages of the disease contained f-PSA, PSA–ACT, and PSA–A2M. Besides these PSA forms, some patients at late stage of the disease (T3 and T4) also contained two additional PSA-complexes that have not been identified as of yet. Conditioned medium from LNCaP cells was shown to contain f-PSA, PSA–ACT, and PSA–A2M complexes. CONCLUSIONS Thiophilic gel has strong affinity for all known molecular forms of PSA present in any biological material. Visualization of PSA molecular forms in Western-blot analysis was feasible only after patient serum was processed through T-gel. Our procedure can be used to screen hybridoma and antibodies specific to the individual PSA complex. The PSA molecular forms isolated from patient serum after T-gel procedure may provide useful internal calibrators, and thus may significantly enhance the reliability of PSA measurements. Prostate 50: 145–153, 2002. © 2002 Wiley-Liss, Inc.

PSA is a biomarker for diagnosis and management of prostate cancer. PSA is known to have anti-tumorigenic activities, however, the physiological role of PSA in prostate tumor progression is not well understood.Five candidate peptides identified based upon computer modeling of the PSA crystal structure and hydrophobicity were synthesized at95% purity. The peptides in a linear form, and a constrained form forced by a di-sulfide bond joining the two ends of the peptide, were investigated for anti-angiogenic activity in HUVEC.None of the five PSA-mimetic peptides exhibited PSA-like serine protease activity. Two of the peptides demonstrated significant anti-angiogenic activity in HUVEC based on (i) inhibition of cell migration and invasion; (ii) inhibition of tube formation in Matrigel; (iii) anti-angiogenic activity in a sprouting assay; and (iv) altered expression of pro- and anti-angiogenic growth factors. Constrained PSA-mimetic peptides had greater anti-angiogenic activity than the corresponding linearized form. Complexing of PSA with ACT eliminated PSA enzymatic activity and reduced anti-angiogenic activity. In contrast, ACT had no effect on the anti-angiogenic effects of the linear or constrained PSA-mimetic peptides. Modeling of the ACT-PSA complex demonstrated ACT sterically blocks the anti-angiogenic activity of the two bioactive peptides.The interaction of a hydrophilic domain on the surface of the PSA molecule with a target on the cell membrane of prostate endothelial and epithelial cells was responsible for the anti-angiogenic or anti-tumorigenic activity of PSA: enzymatic activity was not associated with anti-angiogenic effects. Furthermore, since PSA and ACT are both expressed within the human prostate tissue microenvironment, the balance of their expression may represent a mechanism for endogenous regulation of tissue angiogenesis.

Earlier studies have supported a significant role for cocaine in the susceptibility to and the progression of human immunodeficiency virus type 1 (HIV-1) infection. Recently, several unique HIV-1 entry coreceptors (e.g., CCR5 and CCR3) and a trio of HIV-1-specific suppressor chemokines, namely, RANTES (regulated-upon-activation T expressed and secreted), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β, were identified. Although cocaine has been linked to the immunopathogenesis of HIV-1 infection, the corresponding cellular and molecular mechanism(s) have not been well defined. We hypothesize that cocaine mediates these pathologic effects through the downregulation of HIV-1-suppressing chemokines and/or upregulating HIV-1 entry coreceptors in HIV-1-infected subjects, resulting in disease progression to AIDS. Our results show that cocaine selectively downregulates endogenous MIP-1β secretion by normal peripheral blood mononuclear cells (PBMC), while cocaine did not affect the MIP-1β production by PBMC from AIDS patients. Cocaine also selectively suppresses lipopolysaccharide-induced MIP-1β production by PBMC from HIV-infected patients. Further, cocaine significantly downregulates endogenous MIP-1β gene expression, while it upregulates HIV-1 entry coreceptor CCR5 by normal PBMC. These studies suggests a role for cocaine as a cofactor in the pathogenesis of HIV infection and support the premise that cocaine increases susceptibility to and progression of HIV-1 infection by inhibiting the synthesis of HIV-1 protective chemokines and/or upregulating the HIV-1 entry coreceptor, CCR5.

It is now well established that parenteral drug abuse is a significant risk factor for contracting human immunodeficiency virus type 1 (HIV-1) infection and subsequently developing AIDS. Earlier studies have shown that morphine can modulate various immune responses and therefore support the premise that morphine is a cofactor in susceptibility to and progression of HIV infection. Dysregulation of interferon (IFN) production, nonspecific apoptosis of T cells, and the immune response to soluble HIV gene products have been associated with potential mechanisms of pathogenesis in HIV disease. The present study was undertaken to examine the immunomodulatory role of morphine on HIV protein-induced lymphocyte proliferative responses, Sendai and Newcastle disease virus-induced alpha IFN (IFN-alpha) and IFN-beta production by lymphocytes and fibroblast cells, respectively, and induction of apoptosis of normal lymphocytes in vitro. Our results demonstrate that HIV protein-induced human lymphocyte proliferative responses were significantly inhibited by morphine in a dose-dependent manner. Furthermore, morphine significantly inhibited both IFN-alpha and IFN-beta production by normal lymphocytes and fibroblasts but induced apoptosis of normal lymphocytes. Inhibition of IFN-alpha production by morphine could be reversed by the opiate receptor antagonist naloxone. This suggests that the immunomodulatory effects of morphine are mediated through the opioid receptor. These studies support a role of morphine as a cofactor in the pathogenesis of HIV infection and describe some of the possible pathologic mechanisms which underlie the immunoregulatory effects of morphine.

While considerable progress in examining the course of human immunodeficiency virus (HIV) infection in adults has been made, a better understanding of the natural history of perinatal HIV infection remains to be obtained. Dysregulation of the production and functions of various cytokines, especially the interferons (IFNs), during HIV infections has been reported. Using an in vitro model system, we examined the effects of the HIV type 1 envelope protein, gp120 (10, 50, and 100 ng/ml), on gamma IFN (IFN-gamma) and IFN-alpha production by lymphocytes from neonates and adults and also examined the potential regulatory effects of gp120 on phorbol 12-myristate acetate (PMA)- and Sendai virus-induced IFN-gamma and IFN-alpha production by lymphocytes. PMA at a concentration of 50 ng/ml plus 50 ng of calcium ionophore A23187 per ml was used to induce IFN-gamma, while 150 hemagglutinating units of Sendai virus was used to induce IFN-alpha production. The antiviral activity of both IFN-alpha and IFN-gamma in leukocyte culture supernatants was assayed on BG-9 cells by a dye uptake technique using vesicular stomatitis virus as a challenge virus. Placental cord blood leukocyte (CBL) samples from healthy, term infants and adult peripheral blood leukocytes (APBL) produced no IFN in response to gp120. However, CBL produced significantly decreased levels of IFN-gamma compared with APBL in response to PMA plus ionophore. gp120 significantly suppressed both Sendai virus-induced IFN-alpha and PMA-induced IFN-gamma production by both CBL and APBL in a dose-dependent manner. However, gp120-induced suppression of IFN-alpha and IFN-gamma was significantly greater with CBL than with APBL. Treatment of CBL and APBL with gp120 did not induce any phenotypic alteration of the CD45 RO+ subset. Increased suppression of IFN-alpha and IFN-gamma production by gp120 in neonates may partially explain their apparent increased susceptibility to the clinical progression of HIV infections compared with that of adults.

BACKGROUND Prostate specific antigen (PSA) is a well known biomarker for early diagnosis and management of prostate cancer. Furthermore, PSA has been documented to have anti-angiogenic and anti-tumorigenic activities in both in vitro and in vivo studies. However, little is known about the molecular mechanism(s) involved in regulation of these processes, in particular the role of the serine-protease enzymatic activity of PSA. METHODS Enzymatic activity of PSA isolated directly from seminal plasma was inhibited specifically (>95%) by incubation with zinc2+. Human umbilical vein endothelial cells (HUVEC) were utilized to compare/contrast the physiological effects of enzymatically active versus inactive PSA. RESULTS Equimolar concentrations of enzymatically active PSA and PSA enzymatically inactivated by incubation with Zn2+ had similar physiological effects on HUVEC, including inhibiting the gene expression of pro-angiogenic growth factors, like VEGF and bFGF, and up-regulation of expression of the anti-angiogenic growth factor IFN-γ; suppression of mRNA expression for markers of blood vessel development, like FAK, FLT, KDR, TWIST-1; P-38; inhibition of endothelial tube formation in the in vitro Matrigel Tube Formation Assay; and inhibition of endothelial cell invasion and migration properties. DISCUSSION Our data provides compelling evidence that the transcriptional regulatory and the anti-angiogenic activities of human PSA are independent of the innate enzymatic activity. Prostate 71:1680–1690, 2011. © 2011 Wiley-Liss, Inc.

Background: The chronic inflammation associated with multiple sclerosis (MS) may lead to the upregulation of pentosidine. Objectives: This cross-sectional study compares plasma pentosidine levels among healthy controls (HCs) and patients with MS at different disease stages. The study also determines pentosidine's usefulness as a biomarker of MS disease activity and/or severity via its correlation with a number of indicators of MS disease. Methods: Pentosidine levels were analyzed in 98 MS patients and 43 HCs using reverse-phase high-pressure liquid chromatography with fluorescence detection. Results: Plasma pentosidine levels were significantly higher in MS patients when compared with HCs ( p = 0.02). Patients on disease-modifying therapies (DMTs) had lower plasma pentosidine levels when compared with DMT-naïve patients ( p = 0.01). Pentosidine plasma levels correlated with indicators of MS disease severity, including Extended Disability Status Scale ( p = 0.03), MS Severity Scale ( p = 0.01), and MS Functional Composite ( p = 0.03). No correlation between pentosidine levels and age, rate of clinical relapse, and disease duration was observed. Conclusions: Our results suggest that pentosidine could be a novel, inflammatory biomarker in MS clinical practice. Longitudinal studies are warranted to determine any causal relationship between changes in plasma pentosidine levels and MS disease pathology. These studies may pave the way for use of advanced glycation end product (AGE) inhibitors and AGE-breaking agents as new therapeutic modalities in MS.

Extract: Interferons alpha, beta and gamma are a group of structurally and functionally related proteins, produced in response to viruses or double-stranded RNA and defined by their ability to establish an antiviral state in cells. They were originally discovered by Isaacs and Lindenmann (1957). Since their original discovery, several interferon genes and proteins have been identified. Their mechanisms of action were studied and in addition to the antiviral activity, other biological activities were also uncovered including inhibition of cell proliferation and immunomodulation. Human interferon-alpha is a family of at least 23 polypeptides coded by several related genes. The various interferons exhibit a high level of species specificity with some exceptions. For example, there is a high level of activity of human interferon-alpha subtypes on bovine cells. Interferons have been used for the treatment of certain viral infections, multiple sclerosis, certain cancers including hematologic malignancies and Acquired Immune Deficiency Syndrome (AIDS)-related cancerous complications.

The study is aimed to determine the role of luteolin (3',4',5,7-tetrahydroxyflavone), alone and in combination with human interferon-beta (IFN-beta), in modulating the immune response(s) of peripheral blood mononuclear cells (PBMCs) isolated from multiple sclerosis (MS) patients. PBMC proliferation in the presence or absence of these drugs was determined and the production of pro-inflammatory cytokines (IL-1beta, TNF-alpha), and the ratio of cell migration mediator MMP-9, and its inhibitor, TIMP-1 was assessed in the culture supernatants. Luteolin reduced, in a dose-dependent manner, the proliferation of PBMCs, and modulated the levels of IL-1beta and TNF-alpha released by PBMCs in the culture supernatants. Luteolin reduced the MMP-9/TIMP-1 ratio via lowering MMP-9 production. In the majority of cases, luteolin, when combined with IFN-beta, had additive effects in modulating cell proliferation, IL-1beta, TNF-alpha, MMP-9 and TIMP-1.

BACKGROUND Serum prostate specific antigen (PSA) levels in prostate cancer patients serve as a useful biomarker for diagnosing and monitoring prostate cancer. Recently, secreted PSA has been characterized as an autocrine survival factor through activation of Akt and induction of AR. In the normal prostate, PSA is secreted in the lumen of prostatic ducts to lyse proteins in the seminal coagulum. METHODS However, the mechanism for constitutive PSA secretion from benign prostate and its transport across the prostate-blood barrier into serum are unknown. Regulation of peptide secretion by iPLA2-β has been reported in non-prostatic tissue and in prostate tissue iPLA2-β is reported to be under androgen regulation. We investigated whether iPLA2 plays a role for in PSA secretion by comparing iPLA2 activity and expression in normal prostate epithelial RWPE-1 cells and in LNCaP prostate cancer cells. Expression of the two active iPLA2-β mRNA splice variants, LH-iPLA2 and SH-iPLA2, were increased and the inhibitory ankyrin-iPLA2 isoform was markedly reduced in LNCaP cells as compared to normal prostate epithelial RWPE-1 cells. RESULTS These changes are consistent with a higher enzymatic activity in LNCaP cells. The iPLA2-β-specific inhibitor BEL inhibited PSA secretion and induced apoptosis in LNCaP cells. iPLA2 knockdown using SiRNA inhibited PSA secretion, downregulated AR and induced apoptosis. Exogenous PSA suppressed BEL-induced apoptosis and neutralizing anti-PSA antibody blocked the survival effect of PSA. CONCLUSIONS These data indicate that iPLA2-β participates in regulating PSA secretion and supports the concept that secreted PSA provides an autocrine survival function in LNCaP cells. Prostate 69:1270–1280, 2009. © 2009 Wiley-Liss, Inc.

We have described a nonantibody type inhibitor of interferons (IFN) in the blood of patients with AIDS, advanced neoplastic disorders, and lupus erythematosus in earlier reports (1,2). In the present study we show that the semipurified inhibitor blocks the antiproliferative signal of IFN-alpha in Daudi cells and the membrane potential shifting ability of IFN-alpha is modulated by the interferon inhibitor preparation (IFI).

BACKGROUND Prostate-specific antigen (PSA) is a well-known biomarker for diagnosis and management of prostate cancer. PSA has been shown to have anti-angiogenic activity. We used the emerging proteomic research technology to identify proteins in prostate cancer cells whose expression is regulated by enzymatically active PSA. METHODS Differentially expressed proteins in PC-3M cells treated with PSA were analyzed by 2D-DIGE analysis and identified by HPLC–MS/MS and SEQUEST data mining. Biological network analysis was carried out using MetaCore integrated software designed for functional analysis of experimental data. Gene expression data for several regulated proteins were confirmed by real-time, quantitative PCR. RESULTS A total of 41 proteins were significantly (P

Objectives To compare serum levels of the receptor for advanced glycation end products (sRAGE) between multiple sclerosis (MS) patients and healthy control subjects, and to investigate whether serum sRAGE levels correlate with MS disease severity as indicated by the Kurtzke Expanded Disability Status Scale (EDSS). Method 37 patients with clinical diagnosis of MS and 22 healthy control subjects were investigated in a cross-sectional study using enzyme-linked immunosorbent assays (ELISA). Results Serum levels of sRAGE were found to be significantly lower in MS patients compared to levels in healthy controls ( p = 0.005). A trend toward lower levels of serum sRAGE was observed in female MS patients compared to their male counterparts ( p = 0.05). A relationship between sRAGE and EDSS, and sRAGE and rate of clinical relapse was observed ( p = 0.012). Conclusion The significant reduction of sRAGE in MS patients relative to healthy controls supports the potential role for RAGE axis in MS clinical pathology. Lower levels of sRAGE may be associated with enhanced inflammatory responses. Based on these observations, further investigations into the role of sRAGE in MS clinical pathology is warranted.

The study is aimed to determine the role of quercetin (3,3'4',5,7-pentahydroxy flavone), alone and in combination with human interferon-beta (IFN-beta), in modulating the immune response(s) of peripheral blood mononuclear cells (PBMC) isolated from multiple sclerosis (MS) patients and from normal healthy subjects. PBMC proliferation in the presence or absence of these drugs was determined and the production of proinflammatory cytokines (IL-1beta, TNF-alpha), and the ratio of cell migration mediator MMP-9, and its inhibitor, TIMP-1 were assessed in the culture supernatants. Quercetin reduced, in a dose-dependent manner, the proliferation of PBMC and modulated the level of IL-1beta and TNF-alpha released by PBMC in the culture supernatants. Quercetin reduced the MMP-9/TIMP-1 ratio via lowering MMP-9 production. Quercetin, when combined with IFN-beta, had additive effects in modulating TNF-alpha and MMP-9. These immunomodulatory responses to quercetin were similar between MS patients and healthy control (HC) subjects.

Synthesis of interferon (IFN) in response to Sendai virus and the development of the resulting antiviral state were studied in human (BG-9) and murine (L-929) fibroblast cell cultures in the presence of the calmodulin antagonists, trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Compared to control cultures, 16-fold and 8-fold more IFN was formed in human and murine cells, respectively, when 10 microM TFP was present in the medium for 24 h prior to IFN induction with Sendai virus. W-7 did not affect IFN production in human cells, but enhanced it in L-929 cells by 4- to 8-fold. TFP inhibited the antiviral state induced by homologous IFN in the two cell systems, and at 20 microM, there was a 3,000-fold increase in vesicular stomatitis virus (VSV) yield. It also reduced the maintenance of the antiviral state in human cells. In contrast, W-7 had no effect on the development of the antiviral state in either of the two cell systems. Thus, calcium-calmodulin dependent cellular processes are involved in both induction of IFN and its action. The several patterns response to TFP and W-7 may reflect different ligand-binding sites on calmodulin.

This is a brief review on studies of attacking HIV through a new angle. In previous studies, we have found that many patients with AIDS are resistant to interferon (IFN) therapy, and some develop resistance during therapy. Four factors were found to be responsible for the resistance of untreated patients: (a). release of free-circulating IFN-alpha/beta type 1 receptors, (b). a newly detected IFN inhibitory protein, (c). high prostaglandin E2, and (d). high levels of cAMP phosphodiesterases, particularly in AIDS-related neoplasms. This may interfere with intrinsic disease resistance and with the efficacy of IFN therapy. In an attempt to overcome this resistance, new compounds were synthesized which increase endogenous production of alpha, beta and gamma IFNs, have anti-template activity against DNA and RNA polymerases, inhibit reverse transcriptases and activates IFN-induced double-stranded RNA (dsRNA)-dependent protein kinase. It is expected that planned nonhuman primate and clinical studies will support preliminary findings. Preliminary in vitro and animal studies suggest that these new compounds may be effective against HIV, including multi-drug resistant strains.

>> Many attempts have been made to inhibit viral and neoplastic diseases by targeting the RNA system. The pathophysiologic significance of the microRNA system and the therapeutic potential of its manipulation are discussed. Studies of doublestranded RNA derivatives are reviewed. The therapeutic potential of one of these compounds, polyI:MPC, is emphasized. Studies of other related antiviral and antineoplastic agents are discussed, including 29-deoxyoligocytidilates and telomerase inhibitors. Exp Biol Med 231:1283–1286, 2006

Although the CD4 molecule is the primary receptor for HIV-1, several studies have shown that in addition to CD4, other co-receptors such as CCR3, CCR5 may be required for efficient viral entry. Recent studies show that HIV suppressing such as, RANTES, and can bind to HIV-1 entry co-receptors, and, therefore, are important host factors that can influence susceptibility to M-tropic HIV-1 (Broder and Cocchi). Cocaine is one of the most widely abused drugs in the U.S. The last decade has witnessed a great, entangled epidemic of cocaine abuse and HIV-1 infections. Several studies have described the association of cocaine use with susceptibility to and progression of HIV-1 infections (Donahoe, Webber and Bagasra). We hypothesize that cocaine can mediate these pathologic effects through modulation of HIV suppressing chemokine and their receptors. The present study examines the effect of cocaine on synthesis by lymphocytes from normal and HIV infected subjects and and CCR 5 gene expression by normal PBMC.

We hypothesize that expression of proangiogenic genes correlates with the metastatic potential of prostate cancer cells. LNCaP, DU-145, and PC-3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as we demonstrated by their capacity to invade an extracellular matrix, an established tumor invasion assay. The constitutive gene expression of the proangiogenic factors, vascular endothelial growth factor, intercellular adhesion molecule-1, interleukin-8, and transforming growth factor-β2, was significantly greater in the more metastatic DU-145 and PC-3 cells as compared with LNCaP cells. Matrix metalloproteinase (MMP)-9 is thought to contribute to the invasive phenotype of tumor cells. PC-3 cells showed increased expression of MMP-9 and membrane type 4-MMP as compared with LNCaP and DU-145. Tissue inhibitors of metalloproteinase 1 and 4 gene expression were elevated in DU-145 and PC-3 cells, but paradoxically, LNCaP cells had undetectable levels of these genes. We transfected and overexpressed MMP-9 in poorly metastatic LNCaP cells and measured their invasive activity. Transient expression of human MMP-9 in LNCaP cells produced a 3–5-fold increase in MMP-9 activity with a comparable increase in invasiveness. Antisense ablation of the expression of MMP-9 in DU-145 and PC-3 cells produced concomitant inhibition of the gene expression of the proangiogenic factors, vascular endothelial growth factor, and intercellular adhesion molecule-1 (ICAM-1). Treatment of DU-145 and PC-3 cells with a selective chemical inhibitor of MMP-9 proteinase activity also inhibited their invasive activity. These results support our hypothesis that metastatic potential of prostate cancer cells correlates with expression of proangiogenic factors.

Interferon (IFN) resistance is an important factor in the pathophysiology of neoplastic disorders, certain viral infections (e.g., AIDS), and autoimmune diseases (e.g., lupus erythematosus and Wegner's granulomatosis). In addition, in some of these disorders, there is also decreased ability to produce IFNs. The capacity of viruses and neoplastic processes to interfere with the IFN system are thought to represent a “virus-against-host” or “cancer-against-host” defense mechanism. Four resistance factors have been identified: 1) release of free IFN-α/β type 1 receptors into the circulation that, at appropriate concentrations, capture and inactivate IFNs; 2) a new IFN inhibitory protein has been isolated and its chemical structure is under study; 3) prostaglandin E2, which is produced by certain tumor cells, inhibits IFN production; and 4) high levels of cAMP phosphodiesterases present, for example in certain tumor cells, reduces cAMP, an important second messenger in IFN synthesis. Studies are under way to reverse these inhibitory effects and to increase endogenous interferon production.

Many viral and neoplastic diseases are resistant to interferon-alpha/beta (IFN-alpha/beta) therapy or develop resistance during the course of IFN treatment. In patients with viral diseases, the authors identified four IFN inhibitors, of which the most important, most likely is a free IFN receptor of type 1 appearing in the circulation that captures and neutralizes IFN-alpha/beta.Ninety-one cancer patients and 25 healthy individuals were studied. Free circulating IFN receptor-alpha/beta type 1 was studied. The patients were ages 35-75 years. The diagnoses were 24 cases of colon carcinoma, 7 cases of prostate carcinoma, 16 cases of breast carcinoma, 8 cases of ovarian carcinoma, 9 cases of uterine carcinoma, 5 cases of lung carcinoma, 3 cases of astrocytoma, 4 cases of transitional cell carcinoma of the bladder, 1 case of osteosarcoma, 3 cases of multiple myeloma, 4 cases of Hodgkin disease, 2 cases of non-Hodgkin lymphoma, 3 cases of myelodysplastic syndrome, and 2 disseminated tumors of unknown origin.All patients were found to have increased free IFN receptor-alpha/beta type 1 in the circulation, with the highest levels reported in patients with adenocarcinoma.High IFN inhibitory activity in patients with cancer may be a significant factor in their increased susceptibility to progressive disease, infectious complications, and resistance to IFN therapy. Ongoing studies are being performed with the objective of overcoming this inhibitory activity.

An inhibitor of interferon antiviral activity, which is absent in healthy HIV-seronegative persons, was detected in the sera of all 29 HIV-seropositive study participants. The relationship of the level of interferon inhibitor to CD4 count and HIV-RNA copy number was statistically significant in distinct models. Levels of interferon inhibitor declined by an average of 41-60% in patients who underwent a change in anti-retroviral therapy. Interferon inhibitor levels appear to decline as CD4 cell count rises and HIV-RNA levels fall. This suggests that interferon inhibitor may have a significant role in the host immune response to HIV infection.

Prostate-specific antigen (PSA) is currently used as a biomarker for the early diagnosis and management of prostate cancer (CaP). However, PSA generally lacks the sensitivity and specificity desired of an effective diagnostic biomarker. The goal of this study was to identify an additional biomarker or a panel of biomarkers that is more sensitive and specific than PSA in differentiating benign vs. malignant prostate disease and/or localized CaP vs. metastatic CaP. Concurrent measurements of circulating IL-8, TNF-alpha (TNF-α) and sTNFR1 were obtained from four groups of men: (1) controls (2) men with elevated PSA with a negative prostate biopsy (eIPSA_negBx) (3) men with clinically localized CaP and (4) men with castration resistant CaP (CRPC). TNF-α (AUC=0.93) and sTNFR1 (AUC=0.97) were strong predictors of elPSA_negBx vs CaP). The best predictor of elPSA_negBx vs prostate cancer was sTNFR1 and IL8 combined (AUC=0.997). The strongest single predictors of localized versus metastatic CaP were TNF-α (AUC=0.992) and PSA (AUC=0.963) levels. Conclusions: The specificity and sensitivity of a PSA-based CaP diagnosis can be significantly enhanced by concurrent serum measurements of IL-8, TNF-α and sTNFR1. In view of the concerns about the ability of PSA to distinguish clinically relevant CaP from indolent disease, assessment of these biomarkers in larger cohort consisting of Caucasians and African Americans is warranted. Citation Format: Kailash C. Chadha, Austin Miller, Bindukumar B. Nair, Stanley A. Schwartz, Donald L. Trump, Willie Underwood. New serum biomarkers for prostate cancer diagnosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 879. doi:10.1158/1538-7445.AM2014-879

Prostate-specific antigen (PSA) protein and complexes of PSA with α 1 -antichymotrypsin (PSA-ACT) or α 2 -macroglobulin (PSA-A 2 M) prepared in vitro, have strong affinity for different thiophilic gels (T-gel). Free PSA, and these PSA complexes can be isolated due to their affinity for T-gels. The average recovery of PSA from several of the T-gels, based upon ELISA measurements, was 84 to 94%. The data suggest that T-gel affinity can be explored for the purification of free and complexed PSA from various biologic fluids.

Background: Prostate-specific antigen (PSA) is currently used as a biomarker for diagnosis and management of prostate cancer (CaP). However, PSA typically lacks the sensitivity and specificity desired of a diagnostic marker. Objective: The goal of this study was to identify an additional biomarker or a panel of biomarkers that is more sensitive and specific than PSA in differentiating benign versus malignant prostate disease and/or localized CaP versus metastatic CaP. Methods: Concurrent measurements of circulating interleukin-8 (IL-8), Tumor necrosis factor-α (TNF-α) and soluble tumor necrosis factor-α receptors 1 (sTNFR1) were obtained from four groups of men: (1) Controls (2) with elevated prostate-specific antigen with a negative prostate biopsy (elPSA_negBx) (3) with clinically localized CaP and (4) with castration resistant prostate cancer. Results: TNF-α Area under the receiver operating characteristic curve (AUC = 0.93) and sTNFR1 (AUC = 0.97) were strong predictors of elPSA_negBx (vs. CaP). The best predictor of elPSA_negBx vs CaP was sTNFR1 and IL-8 combined (AUC = 0.997). The strongest single predictors of localized versus metastatic CaP were TNF-α (AUC = 0.992) and PSA (AUC = 0.963) levels. Conclusions: The specificity and sensitivity of a PSA-based CaP diagnosis can be significantly enhanced by concurrent serum measurements of IL-8, TNF-α and sTNFR1. In view of the concerns about the ability of PSA to distinguish clinically relevant CaP from indolent disease, assessment of these biomarkers in the larger cohort is warranted.

We attempted to isolate and characterize PSA from prostate tissue. The aim was to isolate tissue-specific PSA and its initial characterization. This included: (i) determination of PSA molecular forms (free and complexed PSA), (ii) enzymatic activity, and its inhibition by metal ions, (iii) and their relative anti-angiogenic activity. Prostate, tumor and non-tumor, tissue was acquired under IRB approved protocol. PSA was isolated from prostate non-tumor (NT-PSA) and tumor tissue (T-PSA) and characterized using various chromatographic techniques including ligands such as Thiophilic gel, Concanavalin-A agarose, Protein-A agarose, and other lectins. The average NT-PSA concentration was 25±6.3ng PSA/μg of DNA (p=0.0001) and the average T-PSA concentration was 13±5.7ng PSA/μg of DNA (p=0.0001). On an average, tumor PSA (EA=29.64±11.34 p=0.0001)) had twice as much enzymatic activity as compared to non-tumor PSA (EA=13.29±3.300 p=0.001). The inhibition of enzymatic activity of PSA by metal ion (Zn2+)showed that seminal plasma-PSA (SP-PSA) and NT-PSA were inhibited to the same degree [85.3% and 87.0% respectively (p Citation Format: Srikant S. Chakravarthi, Kailash C. Chadha, Gary J. Smith. Tissue-specific prostate specific antigen (PSA). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5306. doi:10.1158/1538-7445.AM2013-5306

1. 1. The immunosuppressive effects of drugs such as alcohol or hormones such as cortisol may be age-related. To test this hypothesis, the authors investigated the in vitro effects of ethanol (EtOH) and cortisol on Natural Killer (NK) cell activity of lymphocytes from normal cord blood in comparison with that of lymphocytes from normal adult peripheral blood. 2. 2. K562, an erythroleukemia cell line, was used as a target in a 4 hr 51 Cr release assay. 3. 3. Ethanol at 0.3% (V/V) and cortisol at 0.05, 0.1 and 0.2 μg/ml concentrations, added directly to a mixture of effector and target cells significantly suppressed the NK activity of cord blood lymphocytes in a dose dependent fashion, whereas similar concentrations of either EtOH or cortisol did not manifest significant immunoregulatory effects on NK cell activity of normal adult lymphocytes. 4. 4. Pre-treatment of the target with either EtOH or cortisol for 4 hours did not affect cytotoxicity. Inhibition of cytotoxicity was also not due to direct toxicity of effector cells because lymphocytes treated with either EtOH or cortisol showed normal 51 Cr release and their viability was comparable to that of untreated control cells. 5. 5. This suggests a selective inhibitory effect of EtOH and cortisol on NK activity of neonatal lymphocytes that may be of clinical significance.

A three-group design of alcoholics, heavy smokers and controls was used to investigate the status of the interferon system, including natural killer cell activity. Group differences were indicated via discriminant function analysis which correctly classified 86% of subjects. Test-retest relations were investigated for 14 subjects following a 30-day alcoholic inpatient program. Whereas significant immune suppression was indicated at the time of detoxification, recovery was evident at the 30-day follow-up. Results are discussed in terms of the significance of alcohol abuse on immune system functioning with consequences for susceptibility to viruses, bacteria and medical illnesses.

Spleen cells from C57BL/6 mice maintained on alcohol containing liquid diet for two weeks were evaluated for different immune functions. On an average, 22% fewer spleen cells were recovered from alcohol-fed mice when compared to cells from control animals. In alcohol-fed mice, the relative frequency of B cells increased, whereas total T cells including CD4 + cells decreased significantly. Alcoholic mice, when challenged with poly(rI) poly(rC), produced significantly less interferon than control mice. In vitro production of interferon alpha and gamma by the spleen cells of alcoholic mice was reduced by 67–90%. No significant differences were seen in the level of natural killer cell activity in spleen cells of control and alcoholic mice. These results suggest that chronic alcohol intake can result in not only changes in the number of immune cells, but more importantly affect their biological functions such as their ability to produce interferons.

Human leukocyte interferon (HL-IF)binds to AGPOLY(A)TM, AGPOLY(U)TM and AGPOLY(I)TM. The bound interferon could be displaced from all three polyribonucleotides by including sodium chloride in the eluant. The nature of interaction of HL-IF with polyribonucleotides is electrostatic and not hydrophobic since its binding was not prevented in the presence of 50% ethylene glycol. The binding of HL-IF on AGPOLY(I)TM is stronger at lower pH since an increase in ionic strength is required to displace it.

Tomato plants 21 days old were inoculated with Agrobacterium tumefaciens, the causative organism of crown gall, and 72 h later [3-14C]cinnamic or [carboxyl-14C]benzoic acid was administered by wick-feeding to these and to uninfected control plants. After a further 48 h both groups were examined for incorporation of label into phenolic acids. The pattern of incorporation into bound phenolic acids of the controls was in accord with biosynthetic pathways previously demonstrated in normal plants, but qualitative and quantitative divergence from this pattern was observed in the bound phenolic acids of the infected plants. The data suggest interference by the pathogen with β-oxidation of at least two phenylpropanoid acids of the lignification pathway. In infected plants, lowered incorporation of 14C from cinnamic acid into phenylpropanoid acids of the lignification pathway and their β-oxidation products was accompanied by the appearance of labelled o-coumaric acid, consistent with some diversion of cinnamic acid metabolism toward ortho-hydroxylation. Incorporation of 14C from benzoic acid into gentisic and especially salicylic acid of infected plants was much lower than in the controls.

Human leukocyte interferon (HuIFN-alpha ) was adsorbed on controlled pore glass (CPG) beads in 0.01 M potassium phosphate, pH 7.2. Partial recovery fo HuIFN-alpha from CPG, ranging from 50% to 75%, was obtained with ethylene glycol (6.8 M), ammonium chloride (1.0 M) and Tris.HCl (1.0 M). A near complete recovery (90%) was obtained with potassium phosphate (1 M), but there was no selectivity in the elution vis á vis other proteins. An efficient elution of HuIFN-alpha was accomplished with tetramethyl-, and tetraethylammonium chlorides: the recovery of HuIFN-alpha was complete at low concentrations (0.25 M to 0.50 M) of these salts at pH 8.0 and the elution of interferon was more selective than with other eluants.

Two human cell lines (HT1080 and MRC5) were found to be resistant to the antiproliferative effects of human interferon α. These cells showed also a reduced antiviral response, when virus yield was measured by a plaque assay. The binding of 125 I-labeled α interferon to HT1080 and MRC5 cells was greatly reduced, relative to another human cell line (A549) which responded well to the antiproliferative and antiviral activities of interferon. These results suggested that the poor response of HT1080 and MRC5 cells was due to the presence of fewer interferon receptors than in A459 cells. Some functional receptors, however, appeared to be present in HT1080 and MRC5 cells, since treatment with interferon resulted in a slight reduction in virus yield and in a modest increase in 2′,5′-oligo(A) polymerase activity.

The antiviral activity of exogenously applied HuIFN-alpha was significantly reduced in cells pretreated with non-toxic concentrations of adriamycin. With low concentrations of adriamycin (up to 0.125 micrograms/ml) the effect was reversible in all cells tested. In human foreskin (BG-9) cells, that are more resistant to adriamycin toxicity, higher nontoxic concentrations of adriamycin (0.625 micrograms/ml) caused an irreversible decrease in sensitivity to HuIFN-alpha. Under these conditions, adriamycin pretreatment irreversibly inhibited the rate of cellular protein synthesis in BG-9 cells by about 30%. In contrast, cellular RNA synthesis was inhibited by 80% in 4 h after exposure to the drug, but was restored to almost normal levels (97% of control) at day 4 after removal of the drug. These results suggest that the loss of sensitivity of adriamycin-treated BG-9 cells to HuIFN-alpha may be related to the inhibition of cellular protein synthesis.