Epitope mapping of human herpesvirus-7 gp65 using monoclonal antibodies.

Human herpesvirus (HHV)-7 encodes a unique 65-kDa heparin-binding glycoprotein, designated gp65. This molecule is thought to play a role in virus attachment and entry. To obtain reagents to map the structure and function of HHV-7 gp65, we produced monoclonal antibodies to this molecule. Ten monoclonal antibodies reacting with gp65 on ELISA were subdivided in four groups on the basis of their isotype and differential reactivity with (i) native versus denatured forms of gp65, and (ii) mature (virion-associated) versus immature (cell-associated) forms of the molecule. We were able to map the binding epitopes for eight of these ten antibodies, and these were found to cluster to one site on gp65 (amino acids 239-278); within this region, the antibodies reacted with at least three distinct domains (244-251, 255-262, 263-278). The reasons for the apparent immunodominance of this region are uncertain. Taken together, this panel of antibodies constitutes an extensive and well-characterized set of HHV-7 specific antibodies that may have utility for future analyses of the structure/function of gp65, and for studies on the virus life cycle.

Dendritic cells transduced with HSV-1 amplicons expressing prostate-specific antigen generate antitumor immunity in mice.

There is currently much interest in generating cytotoxic T lymphocyte (CTL) responses against tumor antigens as a therapy for cancer. This work describes a novel gene transfer technique utilizing dendritic cells (DCs), an extremely potent form of antigen-presenting cell (APC), and herpes simplex virus-1 (HSV-1) amplicons. HSV-1 amplicons are plasmid-based viral vectors that are packaged into HSV-1 capsids, but lack viral coding sequences. Amplicon vectors have been constructed that encode the model tumor antigen ovalbumin (HSV-OVA) and human prostate-specific antigen (HSV-PSA), a protein that is expressed specifically in prostate epithelium and prostate carcinoma cells. These amplicons were packaged using a helper virus-free system that produces vector stocks that are devoid of contaminating cytotoxic helper virus. Transduction of DCs with HSV-OVA or HSV-PSA and co-culture with CTL hybridomas results in specific activation, indicating that transduced DCs express these transgenes and process the tumor antigens for class I MHC presentation to CTL. Mice immunized with HSV-PSA-transduced DCs generate a specific CTL response that can be detected in vitro by a (51)Cr-release assay and are protected from challenge with tumors that express PSA. These results indicate that DCs transduced with HSV-1 amplicon vectors may provide a tool for investigation of the biology of CTL activation by DCs and a new modality for immunotherapy of cancer.

T-cell antigen discovery (T-CAD) assay: a novel technique for identifying T cell epitopes.

The identification of T cell epitopes is a critical step in evaluating and monitoring T cell mediated immune responses. Here, we describe a novel technique for simultaneously identifying class I and class II MHC restricted epitopes using a one-step protein purification system. This method uses Ni/chelate coated magnetic beads and magnetic separation to isolate poly-histidine tagged recombinant antigen from bacterial lysates. These beads, once coated with antigen, are also used to deliver antigen to APC where it is processed and presented to T cells. A colorimetric assay and ovalbumin specific, lacZ inducible, T cell hybridomas were used to validate the system. Further, using PSA specific hybrids, generated from T cells isolated from PSA secreting tumors, both class I and class II MHC restricted epitopes of PSA were identified. Additional characterization has shown that these peptides contribute significantly to the overall PSA specific response in vivo, and may represent the dominant epitopes of PSA.

The effect of pravastatin and atorvastatin on coenzyme Q10.

BACKGROUND: Coenzyme Q10 (CoQ10) is an antioxidant and plays an important role in the synthesis of adenosine triphosphate. Studies suggest that 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors reduce CoQ10 levels; however, no studies have directly compared HMG-CoA reductase inhibitors in a randomized crossover fashion. METHODS: Twelve healthy volunteers received either 20 mg pravastatin (P) or 10 mg atorvastatin (A) for 4 weeks in a randomized crossover fashion. There was a 4- to 8-week washout period between the 2 phases. CoQ10 levels and a lipid profile were obtained. RESULTS: There was no difference in CoQ10 levels from baseline to post-drug therapy for either P or A (0.61 +/- 0.14 vs 0.62 +/- 0.2 microg/mL and 0.65 +/- 0.22 vs 0.6 +/- 0.12 microg/mL, respectively; P >.05). There was a significant difference in low-density lipoprotein (LDL) levels from baseline to post-drug therapy for both P and A (97 +/- 21 vs 66 +/- 19 mg/dL and 102 +/- 21 vs 52 +/- 14 mg/dL, respectively; P <.01). There was no significant correlation between LDL and CoQ10. CONCLUSIONS: P and A did not decrease CoQ10 despite a significant decrease in LDL levels. These findings suggest that HMG-CoA reductase inhibitors do not significantly decrease the synthesis of circulating CoQ10 in healthy subjects. Routine supplementation of CoQ10 may not be necessary when HMG-CoA reductase inhibitor therapy is administered.

Regulation of human prostate-specific antigen gene expression in transgenic mice: evidence for an enhancer between the PSA and human glandular kallikrein-1 genes.

The human prostate-specific antigen (PSA) and glandular kallikrein-1 (hGK-1, also known as hK2) genes are tandemly located on chromosome 19, separated by a 12-kb intergenic region. The coordinate regulation of these two genes suggests the presence of common regulatory elements responsible for tissue specificity and/or levels of expression within this region. To identify such regulatory elements, we generated two sets of transgenic mice, which had incorporated either the PSA gene alone or together with the intergenic region. Both sets of transgenics exhibit remarkably prostate-specific expression of the transgene. However, the presence of the intergenic region abrogates the dependence on high PSA gene copy-number for high levels of PSA expression. This suggests the existence of a positive regulatory element in the intergenic region. By using a previously identified distal enhancer element of PSA (termed DEE 1) as a probe, we identified a cross-hybridizing fragment, which we termed DEE 2, in the intergenic region. Sequence analysis shows that DEE 2 is 76% identical to DEE 1, and it includes a putative androgen-responsive element. Here, we propose a model to illustrate how the two enhancers may work to regulate the transcription of PSA and hK2.

A transgenic strategy for analyzing the regulatory regions of the human prostate-specific antigen gene: potential applications for the treatment of prostate cancer (Review).

Prostate-specific antigen (PSA) has been used clinically as a marker for the diagnosis and staging of prostate cancer due to its specific expression in prostate epithelial cells. In addition to its medical importance, its complex hormonal and tissue-specific regulation makes it an attractive model to study gene regulation. Two approaches have been applied to the identification of regulatory regions which confer this specific expression pattern. In vitro analysis of the regulatory regions of the human PSA gene using promoter reporter constructs and tumor cell lines has revealed a number of the DNA sequences involved in the hormone-dependent expression of PSA. We have pursued an alternative in vivo approach using transgenic animal technology, which is the focus of this review. Using this second approach, a transgenic mouse was generated using a 14 kilobase (kb) region of the human PSA gene encompassing the coding region and intervening sequences as well as 6 kb of upstream sequence and 2 kb of downstream sequence. This genomic DNA clone confers a PSA expression pattern in mice which appears to be very similar if not identical to that of humans, allowing us to investigate tissue-specificity and developmental regulation of PSA expression. In addition, these mice, for which PSA is a self-antigen, provide a model to test the feasibility and efficacy of PSA-directed immunotherapy for prostate cancer. The further identification of the PSA regulatory regions important for tissue-specificity may ultimately allow the design of new therapeutics for the treatment of prostate cancer.

Tissue-specific expression of the human prostate-specific antigen gene in transgenic mice: implications for tolerance and immunotherapy.

Human prostate-specific antigen (PSA) has been widely used as a serum marker for cancer of the prostate. The cell type-specific expression of PSA also makes it a potential tumor antigen for prostate cancer immunotherapy. Study of the immunological aspects of PSA within either normal or malignant prostate tissue has been hampered by the lack of a mouse model, because no PSA counterpart has been identified in mice. Using a 14-kb genomic DNA region that encompasses the entire human PSA gene and adjacent flanking sequences, we generated a series of human PSA transgenic mice. In the six independent lines of transgenic mice generated, the expression of the human PSA transgene, driven by its own cis-acting regulatory elements, is specifically targeted to the prostate. Tissue distribution analysis demonstrated that PSA transgene expression closely follows the human expression pattern. Immunohistochemical analysis of the prostate tissue also showed that the expression of the PSA transgene is confined to the ductal epithelial cells. Despite expressing PSA as a self-antigen in the prostate, these transgenic mice were able to mount a cytotoxic immune response against PSA expressed by tumor cells, indicating that expression of the transgene has not resulted in complete nonresponsiveness. This transgenic mouse model will provide a well defined system to gain an insight into the mechanisms of nonresponsiveness to PSA, ultimately leading to strategies for immunotherapy of human prostate cancer using PSA as the target antigen.

The effect of ethanol and/or food restriction on coenzyme Q in liver in rats.

The individual and combined effects of ethanol and lovastatin on rats and their prevention by supplemental coenzyme Q10 (CoQ10) was studied. The ethanol and lovastatin findings are reported elsewhere. This paper focuses on the food restriction which occurred in rats fed 35% of energy as ethanol and those control rats pair-fed to the 35% of energy as ethanol group. Six groups of rats received 35% of energy as ethanol (with or without lovastatin and/or CoQ10 treatment). One group served as a 0% ethanol ad libitum control and one 0% ethanol control group was pair-fed to the 35% ethanol group. Rats receiving 35% of energy as ethanol and their pair-fed controls consumed 83% of the energy/day consumed by the ad libitum controls. This was consistent regardless of lovastatin or CoQ10 treatment. Weight gains were 84% of control. The energy reduction was consistently associated with a substantial (48%+) increase in liver CoQ9 concentrations. Reports by others of associations between food restriction and increased longevity in rodents has focused on a decrease in oxidant damage in tissues of food restricted animals. The increase in CoQ levels in the food restricted animals would result in an increase in antioxidant protection and might explain the observed increases in longevity.

Expression of human prostate-specific antigen (PSA) in a mouse tumor cell line reduces tumorigenicity and elicits PSA-specific cytotoxic T lymphocytes.

Human prostate-specific antigen (PSA) has a highly restricted tissue distribution. Its expression is essentially limited to the epithelial cells of the prostate gland. Moreover, it continues to be synthesized by prostate carcinoma cells. This makes PSA an attractive candidate for use as a target antigen in the immunotherapy of prostate cancer. As a first step in characterizing the specific immune response to PSA and its potential use as a tumor-rejection antigen, we have incorporated PSA into a well-established mouse tumor model. Line 1, a mouse lung carcinoma, and P815, a mouse mastocytoma, have been transfected with the cDNA for human PSA. Immunization with a PSA-expressing tumor cell line demonstrated a memory response to PSA which protected against subsequent challenge with PSA-expressing, but not wild-type, tumors. Tumor-infiltrating lymphocytes could be isolated from PSA-expressing tumors grown in naive hosts and were specifically cytotoxic against a syngeneic cell line that expressed PSA. Immunization with tumor cells resulted in the generation of primary and memory cytotoxic T lymphocytes (CTL) specific for PSA. The isolation of PSA-specific CTL clones from immunized animals further demonstrated that PSA can serve as a target antigen for antitumor CTL. The immunogenicity studies carried out in this mouse tumor model provide a rationale for the design of methods to elicit PSA-specific cell-mediated immunity in humans.

The effect of dietary fermented milk products and lactic acid bacteria on the initiation and promotion stages of mammary carcinogenesis.

The effects of spray-dried yogurt powder product (YPP), bifidobacteria, and Lactobacillus acidophilus were studied during the initiation and promotion phases of carcinogenesis using the 7,12-dimethylbenz[a]anthracene (DMBA)-induced mouse mammary carcinogenesis model. In two separate studies, Sencar mice were fed a diet consisting of 86%, 43%, or 0% YPP or 0% YPP, but with added cultures of bifidobacteria or L. acidophilus. When the animals were 55-63 days old, DMBA was administered by intragastric gavage at 1 mg/mouse and continued once a week for six weeks. During the initiation study, the test diets were fed for four weeks before and during DMBA administration. One week after the final DMBA treatment, all animals were switched to a basal diet based on the AIN-76 formulation. For the promotion study, the diets were introduced one week after the final dose of DMBA and fed for the remainder of the study. Palpable tumor development was monitored weekly throughout the studies. For the initiation study, mice fed 86%, 43%, or 0% YPP or 0% YPP supplemented with bifidobacteria or L. acidophilus had a histologically verified mammary tumor incidence of 15%, 35%, 19%, 30%, and 20%, respectively. The histologically verified tumor incidence for the promotion study was 48%, 58%, 36%, 59%, and 43% in the mice fed diets consisting of 86%, 43%, or 0% YPP or 0% YPP supplemented with bifidobacteria or L. acidophilus, respectively. The data indicate that neither the initiation nor the promotion phase of carcinogenesis is significantly affected by diets composed of 86% YPP, 43% YPP, 0% YPP, or 0% YPP supplemented with bifidobacteria or L. acidophilus.

Evidence of fibroblast heterogeneity and the role of fibroblast subpopulations in fibrosis.

This review article highlights the evidence supporting the concept that, like lymphocytes, fibroblasts also consist of subpopulations with unique phenotypes and functions. A new view of the fibroblast is that they are dynamic and consist of subsets which produce cytokines and interact with the immune system. For example, murine lung fibroblasts separated by fluorescence-activated cell sorting on the basis of the thymocyte-1 antigen are heterogeneous in their morphology, expression of surface markers, antigen presentation to T lymphocytes, ability to synthesize collagen, and cytokine production. Human lung fibroblasts have also been found to be heterogeneous in surface marker expression, proliferation, and collagen production. Investigation of pulmonary fibroblast heterogeneity is important since the lung is particularly susceptible to fibrosis induced by chemotherapy and radiation, inhaled particles, systemic autoimmune disease, etc. The inflammatory responses which typically precede fibrotic induction may be controlled by a subset of resident fibroblasts. Another subset may be important for the fibroblast hyperplasia and extensive extracellular matrix production which are hallmarks of fibrosis. In another model system, periodontal fibroblasts, namely those from periodontal ligament (PDL) and gingiva, also reveal heterogeneity. For example, PDL fibroblasts are composed of subpopulations based on collagen production, morphology, and glycogen pools. Subsets of gingival fibroblasts have also been obtained based on receptors for cyclosporin A and C1q. Specific fibroblast subsets may be involved in gingival repair and hyperplasia. Studies comparing fibroblasts from normal skin vs skin involved with scleroderma have found that scleroderma fibroblasts are activated and able to participate in an inflammatory response. How these fibroblasts become activated is unclear, but it is believed that a subset of fibroblasts is selectively recruited by cytokines at the inflammation site. Finally, investigation and identification of fibroblast subsets from various tissues and their interaction with the immune system could lead to strategies to prevent or reverse debilitating and potentially fatal fibrotic development.

Induction of nitric oxide synthase in subsets of murine pulmonary fibroblasts: effect on fibroblast interleukin-6 production.

The purpose of this investigation was to determine whether mouse lung fibroblast subsets have the ability to produce nitric oxide (NO), and if so, to characterize the induction and effects of its synthesis. Previously, we isolated Thy1+ and Thy1- subpopulations of mouse lung fibroblasts, which differ in terms of cytokine production, morphology, response to cytokines and radiation, and ability to present antigen to T lymphocytes. When treated with the proinflammatory cytokines IFN-gamma, TNF-alpha, and IL-1 alpha, these fibroblast lines produce micromolar quantities of NO2- and NO3-, two stable end products of the NO pathway. A combination of all three cytokines provided the greatest induction, and there was no measurable production of NO in unstimulated cells. Thy1+ fibroblasts have fewer requirements for induction of NO production than the Thy1- line, in that NO production could be induced by only two of the above cytokines, where the Thy1- fibroblasts required all three. Inducible NO synthase (iNOS) mRNA was shown to be present by the reverse transcriptase-polymerase chain reaction as early as 2 hr after cytokine treatment in both cell lines. Addition of the NO synthase inhibitors NG-monomethyl-L-arginine and aminoguanidine inhibited production of NO2- and NO3-, but not iNOS mRNA. This inhibition was partially reversed by the addition of an excess of L-arginine. Interestingly, inhibition of NO synthesis was shown to decrease IL-6 production by more than 50% in cytokine-treated Thy1+ fibroblasts. These results indicate for the first time that Thy1+ and Thy1- mouse lung fibroblast subsets have the capability to produce NO to differing extents in response to cytokines and may therefore play an important role in the inflammatory response in the lung as well as in the progression of lung disease.

Effects of ethanol, lovastatin and coenzyme Q10 treatment on antioxidants and TBA reactive material in liver of rats.

Alcohol metabolism may result in oxidant stress and free radical injury through a variety of mechanisms. Lovastatin may also produce oxidant stress by reducing levels of an endogenous antioxidant, coenzyme Q (CoQ). The separate and combined effects of ethanol, 20 EN% in a total liquid diet, and lovastatin, 67 mg/kg diet, on alpha-tocopherol, retinol palmitate, CoQ9 and thiobarbituric acid reactive (TBAR) material in liver from rats were determined. The effect of exogenous CoQ10 on these treatment groups was also determined. Food consumption, weight gain, liver lipid and TBAR material were similar between treatment groups. Compared to control animals, ethanol reduced retinol palmitate significantly, from 143 to 90 micrograms/g wet weight. Lovastatin had no effect on retinal palmitate nor did it act additively with ethanol. Ethanol decreased liver alpha-tocopherol from 28 to 12 micrograms/g wet weight and lovastatin diminished it to 12 micrograms; no additive effect was evident. Ethanol had no effect, but lovastatin decreased CoQ9 from 83 to 55 micrograms/g wet weight. Supplementation with CoQ10 did not modulate the effect of ethanol on retinal palmitate, but it did reverse the effect of lovastatin on CoQ9. Supplementary CoQ10 did not alter control levels of alpha-tocopherol, but it appeared to reverse most of the decrease in alpha-tocopherol attributable to ethanol or lovastatin separately. It only partially reversed the effect of ethanol and lovastatin combined on alpha-tocopherol, however. As expected, lovastatin had no effect on CoQ10 levels in supplemented animals. Ethanol, either separately or in combination with lovastatin, diminished liver stores of CoQ10 by almost 40%. We conclude that 20 EN% ethanol given in a liquid diet for 5 weeks is sufficient to lower retinol palmitate and that lovastatin reduces CoQ9. Both diminish alpha-tocopherol, an effect largely overcome by CoQ10 supplementation with either drug alone, but not with the combination. Since many individuals chronically consume the levels of ethanol represented by this experiment, and since a certain number of those also take lovastatin, further research into the possible clinical significance of these observations is warranted.

Expression of alpha 6 beta 1 integrin, the laminin receptor, on subsets of normal murine lung fibroblasts and its upregulation by the inflammatory cytokines IFN-gamma and TNF-alpha.

The purpose of this investigation was to ascertain whether the alpha 6 integrin subunit was present on normal murine lung cells and fibroblasts, and if so, to determine the identity of the beta subunit coordinately expressed with alpha 6 and whether or not these integrin subunits could be regulated by cytokines. Previously, our laboratory isolated populations of Thy 1+ and Thy 1- fibroblasts from normal murine lung tissue. These cells differed in surface marker expression and in response to, and production of, pro-inflammatory cytokines. Research defining the properties of these two populations has led to the hypothesis that unique groups of fibroblasts exist within the murine lung. Though alpha 6 beta 1 is known to be expressed by platelets, lymphocytes, and epithelial cells, its presence and regulation on lung fibroblast subsets has not been explored. We now report the following findings: 1) the laminin receptor, alpha 6 beta 1, is present on 20-30% of freshly isolated normal murine lung cells in all three murine strains tested; 2) established Thy 1+ and Thy 1- murine lung fibroblast subsets and clones constitutively express alpha 6 beta 1 at varied levels; and 3) alpha 6 beta 1 expression on fibroblast lines and clones can be upregulated by treatment with IFN-gamma or TNF-alpha. Since these T cell and macrophage derived cytokines are known to be present during an inflammatory response, upregulation of alpha 6 beta 1 expression may facilitate recruitment and retention of lung fibroblasts in regions undergoing repair.(ABSTRACT TRUNCATED AT 250 WORDS)

Lovastatin decreases coenzyme Q levels in rats.

Lovastatin is used for the treatment of hypercholesterolemia. It functions by inhibiting the enzyme, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34), that is required for the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A to mevalonic acid. Since biosynthesis of both cholesterol and coenzyme Q (CoQ) requires mevalonic acid as a precursor, it was considered that lovastatin therapy would also result in a lowering of cellular CoQ levels. This study was conducted to determine whether lovastatin treatment does decrease CoQ levels and whether such decreases can be prevented by CoQ supplementation. Forty-five adult male Holtzman rats were randomly assigned to one of three treatment groups. Controls were fed ground laboratory rat chow ad libitum. The other two groups were fed ground laboratory rat chow containing 400 mg of lovastatin per kg of diet ad libitum. One of the lovastatin-fed groups received CoQ10 (15 mg per kg of body weight) daily via stomach intubation. After 4 weeks, samples of heart, liver, and blood were analyzed for CoQ concentrations. Results indicated that CoQ concentrations in all tissues analyzed were decreased in lovastatin-treated rats. Lovastatin-treated animals that were supplemented with CoQ10 had blood, heart, and liver CoQ10 concentrations that approximated or exceeded those of control animals. It is concluded that lovastatin does indeed lower tissue concentrations of CoQ and that a return to normal can be achieved by supplementation with CoQ.

Faecal incontinence--Willis Home Bowel Washout Programme.

A simple procedure for managing bowel incontinence among children with spina bifida and other disabilities is described. The results of treatment offered to 100 children shows that the majority achieved reliable continence.

Manganese balance and clinical observations in young men fed a manganese-deficient diet.

A balance study was conducted to determine the minimum requirement for manganese (Mn) and to examine the effects of Mn depletion. Seven male subjects, age 19-22, were fed a Mn-adequate diet of conventional foods (2.59 mg Mn/d, 135 mg cholesterol, and P:S ratio of 0.86) for 3 wk to establish base-line data. Then a purified diet containing 0.11 mg Mn/d was fed for 39 d (depletion), followed by two 5-d periods of 1.53 and 2.55 mg Mn/d (repletion). Diets, feces, urine, and integument were analyzed for Mn, and blood was analyzed for Mn, cholesterol, and other constituents. Plasma levels of cholesterol decreased from 170 to 152 mg/dL during the base-line period, and then to 142 mg/dL at the end of depletion, but did not respond to 10 days of repletion. A fleeting dermatitis, Miliaria crystallina, developed in five of the seven subjects at the end of depletion, but disappeared as repletion began. The minimum requirement for Mn on this purified diet, calculated by the factorial method using Mn balance at three levels of intake was 0.74 mg/d. This requirement would be increased to 2.11 mg/d if the obligatory loss was combined with the lowest individual percentage of retention.

Effects of soybean polysaccharide on plasma lipids.

Free-living volunteers with mild to moderate hypercholesteremia added 25 gm soybean polysaccharide or starch placebo in crouton or cookie form to their normal, daily diets. A total of 31 persons completed the blind, crossover design, 8-week, experimental protocol. Subjects ingesting soybean polysaccharide prior to placebo showed an 11% decrease (from 252 to 224 mg/dl) in total plasma cholesterol; those who followed placebo with fiber showed a 5% decrease (from 241 to 230 mg/dl). Starch placebo was associated with a 2% decrease in total cholesterol when consumed first and a 4% increase when consumed following the fiber consumption period. High-density-lipoprotein (HDL) cholesterol decreased 8% and 6% from initial values during the first period for the fiber and starch groups, respectively. HDL cholesterol increased 2% but decreased 1% during the second period for starch and fiber, respectively. No significant changes in triglyceride levels occurred. The data indicate that soybean polysaccharide fiber promotes a significant decrease in plasma cholesterol in persons with mild to moderate hypercholesteremia. The addition of fiber may represent an important adjunct to traditional fat- and cholesterol-controlled diets for such persons.