The purpose of this article is to assess recent data supporting the safety and efficacy of black cohosh products for the mitigation of menopause-related symptoms. Searches of the published literature in Napralert, Cochrane Library and PubMed databases were performed from 2003 to 2006. Information from drug regulatory agencies from five different countries was obtained to evaluate safety. While there are a few contradictory studies, the majority of the clinical trials indicate that extracts of black cohosh (Actaea racemosa L.) improve menopause-related symptoms. However, to date, at least 50 cases of possible hepatotoxicity have been reported. Although previous safety reviews suggest that black cohosh is well tolerated, the increasing numbers of these case reports indicates that further preclinical toxicological evaluations of black cohosh are urgently needed. At this time, it appears prudent to advise menopausal women with underlying liver disease, autoimmune diseases or those taking medications that may impact liver function not to use products containing black cohosh.

738 Isothiocyanates (ITCs) are a class of phytochemicals that are especially abundant in cruciferous vegetables. Many ITCs have been repeatedly shown to inhibit chemical carcinogenesis in a variety of rodent organs. However, their effects on bladder carcinogenesis have been inconsistent. Dietary supplementation of 1-naphthyl-ITC or phenylhexyl-ITC (both are synthetic) has been shown to inhibit carcinogen-induced bladder tumorigenesis in rats, whereas chronically feeding rats allyl-ITC, benzyl-ITC, or phenethyl-ITC (PEITC) either enhanced or induced bladder carcinogenesis. A literature survey has led us to conclude that the bladder epithelium, where the majority of bladder tumor originates, is by far the most exposed tissue to dietary ITCs. Ingested ITCs are metabolized in vivo to N-acetylcysteine conjugates (NAC-ITCs), which are disposed and concentrated up to most likely several mM in urine, depending on the ITC dosage. Several NAC-ITCs also are known to inhibit carcinogenesis in non-bladder organs, primarily due to their facile dissociation to ITCs. We have therefore speculated that the observed carcinogenic effect of ITCs on the bladder in rodents might be related to the prolonged exposure of bladder epithelial cells to very high and toxic concentrations of NAC-ITCs/ITCs in urine stored in the bladder. In other words, overdosing of ITCs may be largely responsible for the adverse effects observed in the bladder. To support this concept, we report that AITC, BITC, and PEITC, as well as several other tested ITCs, all potently induce apoptosis, based on TUNEL assay, and arrest cell cycle progression, based on flow cytometry assay, in cultured human bladder carcinoma UM-UC-3 cells and T24 cells at 5-30 μM concentrations. Multiple caspases, including caspase-9, -8, and -3, as well as poly(ADP-ribose) polymerase, were cleaved upon ITC exposure. The ITCs blocked cell cycle progression at S and/or G2-M phases in these cells. A limited structure-activity analysis has been carried out, identifying PEITC as the most potent agent. Therefore, only very low concentrations of ITCs may be required for inhibition of bladder carcinogenesis. Interestingly, although many ITCs also are known to induce Phase 2 enzymes in a variety of cultured cells and animal tissues, only the activity of quinone reductase and the level of glutathione were somewhat elevated by the ITCs. No induction of glutathione transferase and UDP-glucuronosyltransferase were detected in the ITC-exposed cells, suggesting that ITCs may have limited effect on carcinogen metabolism in bladder cells. In summary, dietary ITCs display potent antiproliferative activity in cultured human bladder cancer cells and may inhibit bladder tumorigenesis in vivo if overdosing is avoided. Supported by NCI grant CA80962.

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Insulin-like growth factor-I (IGF-I) is a polypeptide mitogen which is regulated by growth hormone (GH). IGF-I mediates many of the biological functions of GH, including the maintenance of lymphoid mass and functions. Since GH secretion declines with age, we asked whether changes in the availability of IGF-I might contribute to age-associated alterations in immune functions. As a first step, we examined relationships between plasma levels of IGF-I andin vitrocorrelates of immunity in young and elderly subjects. Heparinized plasma and lymphocytes were collected from the peripheral blood of 34 healthy young (aged 27 ± 0.9 years, mean ± SEM) and 41 elderly (79 ± 1.3 years) volunteers (31 males and 44 females in total). Plasma levels of IGF-I, measured by radioimmunoassay after the removal of IGF-I-binding proteins, were reduced among elders compared to young controls (138 ± 8.7 ng/mL vs 80.2 ± 4.7 ng/mL,P< 0.001). The number of circulating lymphocytes did not change with age. The proliferative response ([3H]thymidine uptake into DNA) of T-cells to concanavalin A and B-cells to pokeweed mitogen were reduced among elders (P< 0.05). An increased spontaneous antitumor natural killer (NK) activity (P< 0.001) was accompanied by a higher percentage of CD16+NK cells among lymphocytes in older subjects (P< 0.001). The NK cell number was positively related to IGF-I levels in young volunteers but not among elders. Correlation analysis demonstrated a highly significant relationship between plasma IGF-I levels and T-cell (but not B-cell) proliferative response during aging (r= 0.492,P< 0.001). Our results imply that reduced immunocompetence may be one of the consequences of reduced IGF-I levels in human aging. Among the three types of immune cells tested, the T-cells were most sensitive to fluctuations in IGF-I levels. Reduced IGF-I availability may be one of the determinants of the decline in T-cell-mediated immune function in the elderly. To our knowledge, this is the first report presenting correlative data on concurrent changes in IGF-I levels and immune parameters in human aging.

It is not clear if physiological aging is accompanied by a down- or up-regulation of Th1 or Th2 cytokine levels and the Th1 or Th2 responses. Aging modulates the natural killer (NK) cell immunocompetence through alterations in their number and functions. Mt cell activational defects during aging process may be global or expressed in a segregated manner. We have tested whether or not the cytokine-inducible Th1 type of response of aged human NK cells could be boosted to the level elicited in the young. An impairment in IL-2 or IL-12-induced interferon gamma (IFN-gamma, a Th1 cytokine) secretion and a lower steady-state level of IFN-gamma mRNA by purified, 18-hr activated Mt cells from the elderly were noted under several experimental conditions. The early phase NK cell activation defect could favor the establishment of tumors or infection in the aged. The data may have implications for cytokine therapies in elderly patients. (USPHS grant supported).

We have been investigating senescence-related changes in human peripheral blood natural killer (NK2) cells. Data accumulated so far consistently and clearly show that both basal and cytokine (IL-2 and interferon α and γ) induced antitumor MHC-unrestricted cytotoxic activity of NK cells are well-preserved in the healthy elderly. To investigate if the non-cytotoxic functions of NK cells are also spared from the influence of senescence, recombinant IL-2-inducible secretion of IFN-γ, which serves as a first line of defense, was examined. The amount of IFN-γ secreted by purified, 18 h activated NK cells from the elderly was only 25 percent of that released by the cells from the young. Thus, the type 1 cytokine-inducible cytotoxic and cytokine secretory functions appear to be dissociable properties of NK cells, at least in the elderly. However, this aging-related early phase secretory deficit could be overcome by chronic stimulation with IL-2 (7 day culture). Since different subsets could perform different functions, we analyzed the NK subsets by flow cytometry. A minor CD56bright subset and a major CD56dim subset could be distinguished based on the density of expression of the cell surface CD56 molecule (N-CAM). We inquired if immunosenescence is likely to impact the steady-state level of circulating NK subsets. A significant decrease (P<0.01) in percent CD56bright among CD56+ cells was observed in the elderly with a relative sparing of the CD56dim subset. The CD56bright/CD56dim ratio, perhaps representing NK cell maturity status, declined with age. This maturation-related subset redistribution and partial loss during aging of high affinity IL-2αβγ receptor bearing `immature' CD56bright NK cells has not been reported before. It could be a consequence of the decline in the level of IL-2 during aging. It is concluded that post-adolescent age-associated modulations in human NK cells are not expressed uniformly; they are pronounced in some, subtle in others but negligible in yet other biological parameters.

A majority of natural killer (NK) cells constitutively express intermediate affinity IL2 receptors made of beta gamma chains and respond by way of enhanced cytokine secretion. However, alterations in cytokine sensitivity of purified NK cells with respect to cytokine secretion during immunosenescence have not been examined before. In view of the major immunomodulatory role of IL-2 and the anti-tumor effects of interferon gamma (IFN-gamma), we have investigated the recombinant human IL2-induced NK cell secretion of IFN-gamma in vitro. After an 18-h activation period; the secretion of IFN-gamma by the peripheral blood NK cells from the elderly was severely impaired at 80 U/ml of IL2 and above. At an optimal dose of IL2 (960 U/ml), NK cells from young and elderly showed a mean 11- and 3-fold increase in secretion, respectively. Under similar conditions, purified T cells did not respond to IL2. However, the sensitivity of NK cells to the same inductive ligand, IL2, towards a different function i.e., cytotoxic activity, was not significantly impaired in the aged. It is possible that the cytokine secretory deficiency of senescent NK cells might be an additional mechanism which could favor the establishment of tumors and viral infections in the elderly. However, as discussed here, our findings do offer alternate explanations and a potential target for experimental immunotherapy.

We investigated whether immune deficiency was associated with a fatal case of invasive cerebral aspergillosis due to Aspergillus fumigatus infection. The lymphocyte proliferative capacity to T- and B-cell mitogens was comparable to that of healthy controls. However, the natural killer (NK) cell activity of the patient was 2-6-fold lower than the activity expressed by a paired control and age-sex matched healthy controls (n = 20). No specific abnormalities were evident in the capacity to transduce either proliferative signals or cytokine (interleukin 2) secretory capacity of in vitro activated lymphocytes from the patient. The specific reduction in NK cell activity might either indicate that she was a low NK responder, or that the low NK cell activity was primarily or secondarily associated with the fungal infection caused by Aspergillus fumigatus in humans.

Previous results from this laboratory have shown the preservation of non-MHC-restricted, constitutive oncolytic activity of human peripheral blood NK cells in the elderly as assessed by the chromium release assay which quantitates the lytic endpoint at the cell population level. We have now addressed this senescence-related change at single-cell level using 101 blood samples. Both the efficiency of the initial tumor target binding step i.e., recognition of K562, the NK-sensitive erythroleukemia cell line, as well as the ability of NK cells to deliver lethal hit are highly conserved during healthy aging. In fact, the elderly exhibit a statistically significant, moderately higher frequency of active killers among circulating lymphocytes. Analyzed in another way, a majority of “high NK responders” were found to be older donors, while none in the “low NK responders” group were > 70 years old. Gamma interferon, a gene product as well as an autocrine activator of NK cells, is effective in converting non-lytic “pre-NK” cells to active killers at single-cell level. This in vitro cytokine sensitivity of NK cells is unaltered during immune senescence. The intactness of the NK cell's capacity to be modulated may be vital in both tumor resistance and host viral defenses of aged humans.

The major histocompatibility complex-unrestricted, cell-mediated, constitutive anti-tumor cytotoxic function of natural killer cells is highly preserved in healthy elderly. A study of the dynamics of expression of natural killer cell-associated phenotypes during immunosenescence shows that selective, bidirectional, and disproportionate changes in certain natural killer cell subset number and ratio take place during aging. The mean natural killer cell subset ratio (%CD16+CD57+ over %CD56+CD57-) gradually increases from a young adult level of 0.7 to 4.6 with advancing age predominantly due to a tripling of %CD16+57+ cells as opposed to a moderate decrease (-54%) in %CD56+57- phenotype. The parallel increase in natural killer phenotype ratio and cytotoxic activity might represent a shift in the maturity status of these cells. Based on these findings, a model of natural killer cell immunosenescence is proposed. It is concluded that not all immunosenescent changes need be detrimental; some may even improve the potential for survival and represent an adaptational immunosenescent change.

THA (Tacrine), a drug used in the experimental therapy of dementia of Alzheimer's disease type, and whose biochemical site of action is believed to be the neural cholinesterase, is shown, for the first time, to be an immunosuppressant in vitro on normal human peripheral blood lymphocytes in microgram quantities. THA down-regulates non-MHC restricted natural killer (NK) cell activity without affecting the general viability of cells. This down-regulation can be demonstrated at all effector and target (K562) concentrations, in purified resting NK cells as well as in lymphokine (interleukin 2) activated killer cells in 3- or 16-h NK assays and in all the blood samples tested. Kinetic analysis shows that the Vmax (maximal cytotoxic potential) and Km of NK cell-mediated cytolysis are also attenuated. Single cell assays using agarose matrix reveal that THA moderately interferes with tumor target binding/recognition events and strongly abrogates the delivery of lethal hit, thus lowering the frequency of active killer cells among THA-treated lymphocytes. THA down-regulates NK cells upon direct interaction and does not require the help of non-NK cells. The THA sensitive site(s) on NK cells does not appear to be perturbed significantly either by their proliferative status or by membrane modulations that may be normally induced by interleukin 2. The in vitro immunomodulatory pharmacological properties of THA reveal that the biological site of action of THA extends to non-neural cells also. Such non-neural models may be helpful in exploring the pathophysiological neuroimmunomodulatory properties of THA at cellular and molecular levels.

Is it possible to demonstrate experimentally, an age-associated, but not necessarily an age-dependent change (see Chapter 11 this volume), in human peripheral blood NK activity by a multitechnique approach? The resulting pattern of NK cell dynamics should be qualitatively consistent with different traditional as well as unconventional techniques and experimental conditions used to quantitate the NK activity.

Rabbit liver phosphofructo-1-kinase, designated isozyme B, and rabbit brain phosphofructokinase, which contains all three isozymes as heteropolymers, have been modified by [14C]fluorosulfonylbenzoyladenosine (FSBAdo). Several lines of evidence supported modification at the binding site for AMP. The modification proceeded to the extent of 2 to 4 mol of reagent incorporated per mol of tetramer, and AMP protected against the reaction. The kinetic properties of modified isozymes A and B and of modified brain phosphofructokinase were examined and compared to their unmodified forms. It was observed that modification greatly diminished ATP inhibition of all of the isozymes. Furthermore, equilibrium binding studies of modified phosphofructokinase B showed a greatly diminished capacity and affinity for cyclic AMP. Cyclic AMP had little or no influence on the properties of modified A isozyme or brain phosphofructokinase, but was capable of further deinhibiting modified B isozyme, apparently at sites remaining unmodified by FSBAdo. Phosphofructokinase B, modified by radiolabeled FSBAdo, was digested by trypsin, and the digest separated by high-pressure liquid chromatography. The labeled peptide was isolated and sequenced to provide the sequence: Asn-Tyr-Gly-Thr-Lys-Leu-Gly-Val-Lys, with the lysine in the fifth position being the site of modification. To isolate isozyme C, a monoclonal antibody to this isozyme was produced by injecting purified rabbit brain phosphofructokinase into mice, and subsequently selecting for those clones that recognized brain phosphofructokinase but not purified phosphofructokinases A and B. The selected monoclonal was specific for native rabbit isozyme C and would not recognize mouse or rat brain phosphofructokinases. Linking the antibody to an inert phase provided an efficient means of purifying rabbit isozyme C from rabbit brain. The enzyme so recovered retained little of its original activity, but the method provided a simple technique for the preparation of enzyme for protein chemistry studies. The modified C isozyme was isolated on the immuno-affinity column and digested with trypsin. A tryptic peptide bearing the label was isolated and sequenced to provide the structure: Asn-Phe-Gly-Thr-Lys-Ile-Ser-Ala-Arg, with position 5 being the site of modification. The sequences of isozymes B and C are homologous to the site of modification of the A isozyme by FSBAdo.

Galactosyltransferase activity (UDP-galactose:N-acetyl-D-glucosamine-D-galactosyltransferase) could be measured in thymus and sera from different strains of mice. Total thymic homogenates or thymocyte preparations obtained from thymoma carrying AKR/J mice exhibited higher enzyme activity compared to nonleukemic control mice. A similar difference was also noted in Swiss mouse thymus which develop thymic leukemia upon a single injection of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboximide. Galactosidase, which was 25 times less active than galactosyltransferase, was not responsible for this difference. These observations were extended to an evaluation of the serum level of the enzyme as a potential tumor biomarker. A 3- to 4-fold increase in the activity of galactosyltransferase was detected in serum samples obtained from both leukemic mice models (AKR/J and Swiss) compared to the controls, whereas the sera from P388 tumor-bearing DBA/2 mice showed a statistically nonsignificant increase of only 20%. The data indicate that serum galactosyltransferase (that accepts the low-molecular-weight acceptor, N-acetyl-D-glucosamine) levels are elevated in the presence of thymic leukemia, and suggest the possibility of shedding of this enzyme from the tumor cells to the systemic circulation of the host. The implications, including the potential diagnostic significance of the results, are discussed.

The activity of calcium-stimulated and magnesium-dependent adenosinetriphosphatase which possesses a high affinity for free calcium (high-affinity (Ca2+ + Mg2+)-ATPase, EC 3.6.1.3) has been detected in rat ascites hepatoma AH109A cell plasma membranes. The high-affinity (Ca2+ + Mg2+)-ATPase had an apparent half saturation constant of 77 +/- 31 nM for free calcium, a maximum reaction velocity of 9.9 +/- 3.5 nmol ATP hydrolyzed/mg protein per min, and a Hill number of 0.8. Maximum activity was obtained at 0.2 microM free calcium. The high-affinity (Ca2+ + Mg2+)-ATPase was absolutely dependent on 3-10 mM magnesium and the pH optimum was within physiological range (pH 7.2-7.5). Among the nucleoside trisphosphates tested, ATP was the best substrate, with an apparent Km of 30 microM. The distribution pattern of this enzyme in the subcellular fractions of the ascites hepatoma cell homogenate (as shown by the linear sucrose density gradient ultracentrifugation method) was similar to that of the known plasma membrane marker enzyme alkaline phosphatase (EC 3.1.3.1), indicating that the ATPase was located in the plasma membrane. Various agents, such as K+, Na+, ouabain, KCN, dicyclohexylcarbodiimide and NaN3, had no significant effect on the activity of high-affinity (Ca2+ + Mg2+)-ATPase. Orthovanadate inhibited this enzyme activity with an apparent half-maximal inhibition constant of 40 microM. The high-affinity (Ca2+ + Mg2+)-ATPase was neither inhibited by trifluoperazine, a calmodulin-antagonist, nor stimulated by bovine brain calmodulin, whether the plasma membranes were prepared with or without ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetra-acetic acid. Since the kinetic properties of the high-affinity (Ca2+ + Mg2+)-ATPase showed a close resemblance to those of erythrocyte plasma membrane (Ca2+ + Mg2+)-ATPase, the high-affinity (Ca2+ + Mg2+)-ATPase of rat ascites hepatoma cell plasma membrane is proposed to be a calcium-pumping ATPase of these cells.