WO2005007082A2

WO2005007082A2 - Treatment of chronic bacterial infection

Info

Publication number: WO2005007082A2
Application number: PCT/US2004/016780
Authority: WO
Inventors: Anthony A. Azenabor
Original assignee: Wisys Technology Foundation, Inc.
Priority date: 2003-06-03
Filing date: 2004-05-27
Publication date: 2005-01-27
Also published as: WO2005007082A3; WO2005007082A8

Abstract

The co-administration of antibiotics and calcium channel blockers in appropriate dose can produce a synergistic antimicrobial effect due to the ability of the calcium channel blocker to convert cryptic forms of chronic bacterial infections to a non-cryptic form. In particular, the effectiveness of L-type calcium channel blockers has been demonstrated with respect to converting chronic bacterial infections to non-cryptic forms. However, decrease or increase of intracellular calcium below or above certain levels results in a decrease in NO production. Since NO can produce desirable effects including, for example, desirable vascular changes and antimicrobial effects, it can be advantageous to control administration of calcium channel blockers to transform cryptic bacterial infections to non-cryptic forms while providing for desirable levels of NO production.

Description

TREATMENT OF CHRONIC BACTERIAL INFECTION AND RELATED PATHOLOGIES

CROSS REFERNECE TO RELATED APPLICATION This application claims priority to copending U.S. Patent Application serial number 60/475,562 to Azenabor filed on June 3, 2003, entitled "Treatment of Chronic Bacterial Infection," incorporated herein by reference.

FIELD OF THE INVENTION The invention relates to the treatment of chronic bacterial infections and conditions associated with chronic bacterial infections, such as atherosclerosis. In particular, the invention relates to improving the effectiveness of antibiotics through the administration of calcium channel inhibitors. BACKGROUND OF THE INVENTION Chlamydial diseases are significant medical conditions and usually have a chronic nature in that the bacteria enter macrophages and form an obscure inclusion within the cells. Chlamydia pneumoniae, a respiratory pathogen (Hahn et al., 2002), also has been implicated in atherosclerosis, the cause of coronary artery disease (CAD) and a significant cause of mortality in western world (McMillan, 1995; Meijer et al., 2000). C. pneumoniae exhibits the capability of infecting prominent cells at atheroma sites, including macrophages (Gaydos et al., 1996), smooth muscle cells (Shor et al., 1998), and endothelial cells (Kaukoranta et al., 1994). Improvements in the treatment of chronic Chlamydia infections can result in reduced pathogenic symptoms associated with the chronic infections including, for example, the cure or reduction of the pathogenicity of respiratory disease, coronary artery disease and other associated diseases. Generally, Ca²⁺ influx signals have significant effects on cell functions (Berridge et al., 1998) including the proliferation and function of the cells of the immune system. For instance, the importance of Ca²⁺ signaling in immune cell function is highlighted by the fact that immunosuppressant action of cyclosporin is achieved by inhibiting the Ca²⁺ - dependent activation of NF-ATc (de la Pompa et al., 1998). A molecular marker of bacterial viability can be mRNA gene expression of selected genes, which has been used to detect the presence of metabolically active microorganism in atherosclerotic tissue (Sheridan et al., 1998; Khan et al., 1996; Morre et al., 1998). For instance, an increase in the expression of Chlamydial stress protein HSP-60 is recognized as an index of chronic course of Chlamydia while a relative increase in the expression of Chlamydial major outer membrane protein (MOMP) is associated with non- chronic, more usual growth pattern (Kol et al., 1999; Valassina et al., 2001). The initial events in atherogenic processes include fatty streak formation expressed by lipid-laden macrophages, referred to as foam cells, and a disturbance in extracellular lipid distribution. Foam cell lipid vesicles consist of modified cholesterol, phospholipids and cholesteryl esters (modified low density lipoprotein) accumulated in macrophages, endothelial cells and smooth muscle cells (Ross 1990). Classically, atherogenic low density lipoprotein is rich in saturated cholesteryl esters with a transition temperature frequently above body temperature. This lipid-crystalline cholesteryl ester core of low density lipoprotein modulates the conformation of apo-B on the surface, affecting the interaction of low density lipoprotein with the cellular receptor (Therond et al., 2000) and Wang et al., 2000), a process that contributes considerably to atherogenesis. Nitric oxide (NO) is an important signaling agent in humans. In particular, NO is thought to have a vasoactive activity that can improve immune functions through enhanced blood flow. In addition, NO can react with superoxide O₂ ^" to form peroxynitrite, a toxic radical that, together with its decay products, are potent antimicrobials. Also, nitric oxide can counter some of the effects of atherosclerosis. Thus, nitric oxide production can be beneficial from a therapeutic standpoint.

SUMMARY OF THE INVENTION In a first aspect, the invention pertains to a composition comprising a calcium channel blocker and an antibiotic. The calcium channel blocker is present in a therapeutically effective amount to enhance the efficacy of the antibiotic against a chronic bacterial infection. In an additional aspect, the invention pertains to a dose of medicinal composition comprising a selected quantity of a calcium channel blocker formulated with a time release agent. The quantity of calcium channel blockers can be selected to administer an appropriate amount of calcium channel blockers to enhance intracellular NO production for a period of time for a patient within a prescribed weight range following introduction into the patient. In another aspect, the invention pertains to a method for treating a chronic bacterial infection, the method comprising administering a therapeutically effective amount of a calcium channel blocker and an antibiotic agent. Moreover, the invention pertains to a method for formulating a composition comprising a calcium channel blocker with a time-release agent. The method comprises selecting a formulation of the calcium channel blocker with the time-release agent to maintain the concentration of calcium channel blocker within a patient's blood vessels within a selected range for a period of time wherein the patient has a weight within a prescribed range. In a further aspect, the invention pertains to a method for the treatment of atherosclerosis, the method comprising administering a therapeutically effective amount of a calcium channel blocker and an antibiotic agent to a patient exhibiting symptoms of atherosclerosis. In addition, the invention pertains to a kit comprising a calcium channel blocker or an antibiotic along with instructions for the administration of a calcium channel blocker and an antibiotic for the treatment of a chronic bacterial infection and associated conditions.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a plot of fluorescence as a function of BAPTA-AM doses for cHSP-60 and MOMP gene expression. Fig. 2A is a histogram of fluorescence of cHSP-60 gene expression in macrophages under five conditions to show the effects of calcium channel blockers. Fig. 2B is a histogram of fluorescence of MOMP gene expression in macrophages under the same five conditions in Fig. 2A. Fig. 3 is a plot of mRNA gene expression as a function of doses of L-type calcium channel blockers for the cHSP-60 gene and the MOMP gene for two different calcium channel blockers. Fig. 4 is a plot of IFU as a function of antibiotic concentration for three different antibiotics and two different calcium channel blockers. Fig. 5 A is a plot of cHSP-60 gene expression as a function of dose of antibiotic for cells either treated with a calcium channel blocker or not. Fig. 5B is a plot of MOMP gene expression as a function of dose of antibiotic for the same four sets of conditions as in the plot of Fig. 5 A. Fig, 6A is a plot of fluorescence as a function of time for macrophages under three sets of conditions. Fig. 6B is a histogram showing fluorescence from macrophages infected with different levels of C. pneumoniae. Fig. 7 is a histogram showing LpL activity as a function of the degree of infection with C. pneumoniae. Fig. 8 is a plot of fluorescence as function of time for macrophages under three sets of conditions to explore the effects of lipopolysaccharides. Fig. 9A is a plot of fluorescence as a function of BAPTA-AM concentration for infected or non-infected cells. Fig. 9B is a plot of fluorescence as a function of time for infected or non-infected macrophages with intracellular calcium ion concentrations shown for reference. Fig. 10 is a plot of fluorescence as a function of time of macrophages under three sets of conditions to show the effects of calcium channel blockers. Fig. 11 is a plot of fluorescence as a function of LDL concentration under two sets of conditions for infected and un-infected cells. Fig. 12A is a plot of fluorescence as a function of BAPTA-AM concentration for macrophages under four different conditions to show the effects of calcium chelation after treatment with estradiol. Fig. 12B is a plot of intracellular calcium ion concentrations as a function of

BAPTA-AM concentration for macrophages under the same four sets of conditions as in Fig. 12A. Fig. 12C is a plot of fluorescence as a function of BAPTA-AM concentrations for macrophages under two sets of conditions from Fig. 12A with intracellular calcium ion concentrations further indicated. Fig. 13 is a plot of fluorescence as a function of time for macrophages under six sets of conditions. Fig. 14 is a plot of fluorescence as a function of time for macrophages under six sets of conditions to show the effects of different concentrations of estradiol. Fig. 15A is a plot of fluorescence as a function of time for macrophages treated with a calcium channel inhibitor under three sets of conditions to show the effects of different estradiol concentration after treatment with a calcium channel blocker. Fig. 15B is a plot of fluorescence as a function of time for macrophages treated with a calcium channel inhibitor and a calcium store inhibitor under three sets of conditions with both a calcium channel blocker and a calcium store inhibitor. Fig. 16 is a western blot showing nitric oxide synthase gene expression.

DETAILED DESCRIPTION OF THE INVENTION It has been discovered that calcium channel blockers can have a synergistic effect for the treatment of chronic bacterial infections, especially Chlamydia infections. In particular, calcium channel blockers are found to block response pathways in macrophages that both reduce the efficacy of immunological responses against bacteria in a macrophage and make the bacteria cryptic with respect to antibiotics. By blocking these response pathways, antibiotics in combination with non-cryptic immunological responses of the macrophages can be more effective to kill bacteria chronically infecting macrophages. The calcium channel blockers restrict the influx of calcium into the macrophages such that Chlamydia forms in macrophages are non-chronic and less refractory to antibiotics, thus enhancing the effectiveness of co-administered antibiotics. A synergistic response results from the co-administration of L-type calcium channel blockers and antibiotics. Due to bell shaped response curve of NO production as a function of intracellular calcium ion concentration, a controlled dose of calcium channel blockers may yield improved results in comparison with higher or lower values. Thus, a controlled dose of calcium channel blockers along with an antibiotic can result in synergistic improvement through the improved effectiveness of the antibiotic along with improved immunological responses associated with enhanced NO production. These approaches can be also effective for the treatment of atherosclerosis. The synergistic effects induced by calcium channel blockers with respect to antibiotics can be understood in relation to the physiological changes associated with intracellular calcium ion concentrations. The Chlamydia infection induces an enhanced flow of Ca⁺² within an infected macrophage, which modifies the physiology of the macrophage. The administration of a calcium channel blocker counters an enhanced flow of calcium ions (Ca⁺²) into a macrophage. The flow of calcium into the macrophage inhibits immunological response against the bacteria and has been associated with a decrease production of nitric oxide. This altered state of the macrophage exhibiting enhanced flow is also associated with the formation of a form of Chlamydia inclusion that resists treatment. With respect to atherosclerosis, the involvement of low density lipoprotein (LDL) accumulation in foam cells associated with atherogenic processes points to LDL metabolism for the manipulation of the etiology of atherosclerosis. Alongside classical risk factors involved in atherosclerosis, infectious agents like cytomegalovirus or C. pneumoniae have been implicated in the etiology of the disease (Thorn et al., 1992; Grayston et al., 1993; Melnick et al., 1994; Sorlie et al., 1994) and of the two infectious agents, C. pneumoniae; a respiratory pathogen is particularly implicated. Seroepidemiological studies have shown a relationship between C. pneumoniae antibody titer and atherosclerotic processes or even future cardiovascular events (Saikku et al., 2002; Muhlestein et al., 1996). Chlamydia pneumoniae has been isolated from atheroma sites (Shor et al., 1992; Campbell et al., 1995 and Kuo et al. 1995). Also, experimental animal models for C. pneumoniae-induced atherogenesis are available (Ramirez, 1996; Moazed et al., 1997; Kuo and Campbell. 1998; Fong et al., 1999 and Blessing et al., 2000). Furthermore, antibiotic intervention has proven beneficial (Fong, 2000; Meier, 2000; and Rothstein et al., 2001). Thus, the improvement of the effectiveness of antibiotic agents against chromic infections, as described herein, can have a significantly improved outcome with respect to the treatment of atherosclerosis induced by infectious bacterial agents. Specialized L-type Ca²⁺ channel in macrophages regulates Ca²⁺ influx signals in a manner favoring C. pneumoniae chronic forms that are refractory to antibiotic therapy. The evidence indicates that application of L-channel inhibitors may upset fonnation of refractory forms of infection. Examples presented below involve the examination of infected macrophages for the role of [Ca²⁺]i availability and L-type Ca²⁺ influx signals in C. pneumoniae MOMP and HSP-60 mRNA gene expression. The bacterial susceptibility to selected antibiotics assessed by inclusion counts or MOMP and HSP-60 mRNA gene expression. As shown in the Examples, intracellular calcium ([Ca²⁺]i) chelation using varying doses of 1,2-bis (o-aminophenoxy) ethane-N,N,N'N' -tetraacetic acid (acetoxymethyl) ester (BAPTA-AM) induced an increase in MOMP and a decrease in HSP-60 mRNA gene expression. L-type Ca²⁺ channel antagonists produced a similar but enhanced effect. These findings associate specialized Ca²⁺ channel function to Chlamydia chronicity. In additional examples, it was further determined that manipulations of Ca²⁺ channel functions had an effect on the usual antibiotic refractory form of C. pneumoniae in macrophages. Inhibition of macrophage L-type Ca²⁺ channel operation improved C. pneumoniae antibiotic susceptibility assessed by decreased inclusion count or down regulated MOMP and HSP-60 mRNA gene expression. These findings provide molecular insights into how specialized Ca²⁺ channel influences Chlamydia chronic course in macrophages and demonstrates a role for L-type Ca²⁺ channel inhibitors in enhanced C. pneumoniae susceptibility to antibiotic therapy. The molecular evidence for Chlamydia chronicity (upregulation of stress protein HSP-60 and down-regulation of major outer membrane protein genes) has been used in this study in elucidating the role of macrophage L-type Ca²⁺ channel in the outcome of Chlamydia pneumoniae infection of macrophages. Calcium channel antagonists prompted the growth of forms of C. pneumoniae that were more susceptible to antibiotics. The present series of data consistently uphold the hypothesis that manipulation of Ca²⁺ influx signals in macrophages could improve the efficacy of antibiotic intervention. Immobilization of intracellular Ca²⁺ by chelation using BAPTA-AM, thus preventing its fluxes, produced a mRNA gene expression pattern consistent with reversion from persistence to more usual form. Unusual morphological appearance of C. pneumoniae can be prompted to more normal inclusions with BAPTA-AM treatment (Azenabor & Chaudhry, 2003a). A dose-dependent, decreased shedding of cHSP-60 with BAPTA-AM treatment dose dependently (a feature consistent with normal growth pattern (Kleindienst et al., 1993)) indicates that control of Ca²⁺ influx mechanism induced by Ca²⁺ channel inhibitors may render C. pneumoniae non-cryptic and non-chronic. The discovery of an association between a specialized Ca²⁺ channel and Chlamydial lipopolysaccharide mediated Ca²⁺ influx signal in macrophages (Azenabor & Chaudhry, 2003b) has provided opportunity for further assessment of the regulatory effect of such channel on the outcome of Chlamydia infection of macrophages. The observation that L-type Ca²⁺ channel antagonists such as nifedipine or nimodipine alter cHSP-60 and MOMP production to a more usual form found in infected HEp-2 cells validates the speculation that the manner of Ca²⁺ influx signals operating in C. pneumoniae infected macrophages favor Chlamydia chronicity. This involvement of calcium channel influx also provides prospects for exploring possible therapeutic benefits when the specialized Ca²⁺ channel is appropriately manipulated. An interesting feature of C. pneumoniae infection of macrophages and their precursor is the establishment of forms that are not susceptible to anti-chlamydial treatment. For this reason, a method of reversion of Chlamydia to forms that are non- chronic is an attractive approach in the treatment of Chlamydia diseases. The upsetting of Chlamydia chronic course by macrophage L-type Ca²⁺ channel antagonist provides a prospect for their use along with antibiotics in the treatment of Chlamydia diseases. This combination treatment can result in an in vivo benefit of Ca²⁺ channel blockers with respect to reversing Chlamydia chronicity and enhancing antibiotic susceptibility. Due to the association of Chlamydia with atherosclerosis, this synergistic relationship from co- administaration of calcium channel blockers with an antibiotic provides greater prospect for efficacious use of antimicrobial agents to which C. pneumoniae is susceptible in the treatment and management of coronary artery disease patients. Understandably, the eradication of Chlamydia from endothelial cells, smooth muscle cells, or adventitial cells (Gieffers et al., 1999) may be easily achieved, but in the face of macrophage induced persistence, only temporary relief of atherogenic processes is provided. Therefore, the ameliorating effects of L-type Ca²⁺ channel blockers can be used in combination with antibiotic therapy in the treatment of C. pneumoniae induced atherosclerosis. Through the elucidation of the synergistic effect of calcium channel blockers and antibiotics, improved treatment of Chlamydia infections can be provided for chronic infections that otherwise could not be effectively treated. Also, the chronic forms assumed by Chlamydia in macrophages are usually refractory to antibiotic therapy, making it difficult to propose proper anti-microbial therapy to patients suffering from coronary heart disease, respiratory infection or other conditions related to Chlamydia infection. Thus, some infections may be cured that otherwise can persist for extended periods of time even after administration of extremely high doses of antibiotics. Similarly, the reliance on antibiotics can be reduced such that reduced dosages of antibiotics can be equally effective or more effective than antibiotic doses administered without a calcium channel blocker. Large doses of antibiotics, especially when administered over significant periods of time, can have severe side effects that can significantly reduce the quality of life for the patient. Also, due to the synergistic relationship of the calcium channel blocker and an antibiotic, certain antibiotics can be used for the treatment of Chlamydia and similar bacteria that may not be effective without the calcium channel blockers. By increasing the number of effective antibiotic compositions available for patients, side-effects can be reduced by selecting a good choice for a particular patient. Also, for patients who cannot tolerate conventional antibiotics due to allergies or sensitivities, additional treatment options are available. While a link has been established between C. pneumoniae and atherosclerosis, the evidence below provides direct evidence that interruption of the infection affects foam cell activity and in particular low density lipoprotein (LDL) influx into the macrophages. The oxidation of LDL associated with foam cell formation cannot be explained by extracellular generation of superoxide (Azenabor and Chaudhry, 2003 a). Specifically, superoxide production is too low given that extracellular fluids have reserves of antioxidants. The evidence presented in the examples below indicates that C. pneumoniae infection results in an upregulation of lipoprotein lipase (LpL), a lipid-metabolizing enzyme. Calcium ion influx signals evidently relate to the upregulation of the LpL gene. The upregulation of the LpL gene evidently results in the favoring of atherogenic properties of the cell. Thus, the effective treatment of the infection using the synergistic combination of calcium channel blockers and antibiotics can block the atherogenic properties of the macrophages to reduce atherosclerosis. With most therapeutic agents, excessive dosages can lead to undesirable side effect such that there is an appropriate range of doses. However, the data below exhibiting a bimodal response with respect to NO production as a function of calcium ion concentration suggests that improved antimicrobial performance is obtainable by using an antibiotic with a controlled dose of calcium channel blocker that remains within a defined range within a treatment course. Regardless of the presence of infection, using calcium channel blockers to increase NO production can provide desirable vascular events. Approaches know in the art, such as use of controlled release agents, can be used to obtain a more effective treatment with calcium channel blockers by maintaining the dose of calcium channel blockers within a selected range over the course of treatment or a suitable portion thereof.

Changes Associated With Chronic Bacterial Infections Of Macrophages Chlamydia infection of macrophages can produce an unusual inclusion, which can be obscure or cryptic, i.e., persistent, thus unsusceptible to immune responses of the macrophages. Similarly, Chlamydia with these unusual inclusions can remain dormant for extended periods of time, which leads to observed chronic infection. As disclosed herein, the inclusions within macrophages become more susceptible to immune responses as well as antibiotics upon exposure to a calcium channel blocker. The extent of impairment of Ca²⁺ influx signal can correlate with alteration of C. pneumoniae course in macrophages from persistent, cryptic form, to the more usual form, which is susceptible to immune response. Chlamydia lipopolysaccharaides induces Ca ⁺ influx signal in a unique manner (Azenabor & Chaudhry, 2003b), making C. pneumoniae infection of macrophages to only elicit minimal nitric oxide generation, an event potentially rendering C. pneumoniae cryptic. A link between C. pneumoniae infection of macrophages and its probable chronic course can arise from free calcium signaling events (Azenabor & Chaudhry, 2003a). Further evidence linking Ca²⁺ signaling to macrophage function have revealed that macrophage Ca²⁺ influx signal is dependent on a specialized L-type Ca ⁺ channel (Azenabor & Chaudhry, 2003b), which has modulatory effect on C. pneumoniae inclusion formation in macrophages. Chlamydia lipopolysaccharides induces Ca²⁺ influx signal in an unusual manner, such that C. pneumoniae infection of macrophages reduces nitric oxide generation. The reduction of nitric acid production could render C. pneumoniae cryptic in the sense that the bacteria become hidden from the immunological response function. Since Chlamydia lipopolysaccharide induced Ca²⁺ influx signal is through an L-type Ca²⁺ channel, blockage of the effects of this influx signal have been demonstrated to improve the antibiotic susceptibility of C. pneumoniae in macrophages. The discovery of the correlation of calcium regulation and a decrease in nitric oxide and reactive-oxygen-species production for immunological response is described further in Azenabor et al., "Chlamydia pneumoniae Survival in Macrophages is Regulated by Free Ca⁺² Dependent Reactive Nitrogen and Oxygen Species," Journal of Infection 46:120-128 (2003), incorporated herein by reference. This observed result is consistent with a link between C. pneumoniae infection of macrophages and the probable chronic course of the bacteria. The modulation of a specialized L-type Ca²⁺ channel has a corresponding modulatory effect on C. pneumoniae inclusion formation in macrophages, which provides further evidence linking Ca²⁺ signaling to macrophage function. Also, the chronic forms assumed by Chlamydia in macrophages are usually refractory to antibiotic therapy, making it difficult to propose proper anti-microbial therapy to patients suffering from coronary heart disease or other forms of chronic Chlamydia infection. Therefore, the modulation of the specialized Ca²⁺ channel mediated regulation of Chlamydia from a chronic course to antibiotic susceptible form can greatly improve therapeutic effectiveness against the chronic disease. Measurements of Chlamydia MOMP and HSP-60 mRNA gene expression as molecular markers provides evidence for macrophage L-type Ca²⁺ channel regulatory effects on C. pneumoniae forms. The results presented in the Examples below confirm that a specialized L-type Ca²⁺ channel in macrophages regulates Ca²⁺ influx signals in a manner favoring C. pneumoniae chronic forms that are refractory to antibiotic therapy. The application of L-channel inhibitors is observed to reverse the chronic form of infection such that the bacteria are again susceptible to immune response. This result was verified by examining infected macrophages for the role of [Ca²⁺]i availability and L-type Ca²⁺ influx signals in C. pneumoniae MOMP and HSP-60 mRNA gene expression, and the bacterial susceptibility to selected antibiotics assessed by inclusion counts or MOMP and HSP-60 mRNA gene expression. The data reported here provide insights into the relationship between chronic forms and antibiotic susceptibility. In particular, there is a feedback mechanism in which Chlamydia lipopolysaccharides induce the L-type calcium channel to pump relatively high concentrations of Ca⁺² into the macrophages, and the large amounts of calcium ions are correlated with a physiological change in the bacterial membrane that is correlated with the acute form of bacterial infection. This feedback mechanism is broken by the treatment with calcium channel blockers.

Lipase-Based Connection Between Infection and LDL Uptake As shown in the Examples, lipoprotein lipase upregulation is significantly different in C. pneumoniae-in&cted cells compared with uninfected cells. The relative effectiveness of C. pneumoniae in inducing LpL mRNA gene expression compared with the capability of homocycteine, a known activator of LpL, to cause the up regulation demonstrates that C. pneumoniae is a more efficient inducer of LpL gene expression. There is a well accepted role for LpL-mediated lipoprotein metabolism and induction of LDL uptake by macrophages (Rumsey et al., 1992; Obunike et al., 1994). It is shown in the Examples below that the LpL gene is up-regulated in C. pneumoniae- infected macrophages and that this up-regulation is accompanied by elevated gene product generation. The mechanism of the induction appears to come into play early in the infection process, spanning a time interval that corresponds to the internalization period and strongly implicate in part functions involving Chlamydial lipopolysaccharides. This Chlamydial lipopolysaccharides up-regulatory effect on macrophage LpL is significant because it is consistent with a role for C. pneumoniae in the etiology of atherosclerosis, and is unexpected since previous findings with Escherichia coli lipopolysaccharides demonstrated a down-regulatory effect. (White et al., 1988). The present findings point to the heterogeneity of lipopolysaccharides regulation across bacterial genera and suggest a relationship between structure, activity and disease association. Chlamydial lipopolysaccharides are less active than typical endotoxins, which can be attributed to the higher hydrophobicity of its lipid A moiety with long chain fatty acids and to the presence of non-hydrated fatty acids in ester linkages with the sugar backbone. Chlamydial lipopolysaccharides are thought to present a genus-specific epitope composed of 3-deoxy-D-manno-oct-2-ulopyranosonic acid trisaccharide, which is surface exposed and known to be immunogenic (Brade et al., 1986; Ingalis et al., 1995; and Brade, 1999). While not wanting to be limited by theory, this unique epitope of the Chlamydial lipopolysaccharides may account for the different physiological effects on macrophages of C. pneumoniae compared to E. coli with respect to LpL gene expression. At atheroma sites, macrophages contribute to lesion progression, cell necrosis and apoptosis (Rosenfeld et al., 2000). Monocytes, macrophage precursors within the blood stream, can serve as vehicles for C. pneumoniae transportation from respiratory sites to remote vascular sites where they can become monocyte-derived macrophages. Through the mechanism for disturbing lipoprotein metabolism through early up-regulation of LpL, as well as through migration within the body, the increase in foam cell formation can be linked to C. pneumoniae infection of macrophages which contributes to the atherogenic properties of C. pneumoniae. The up-regulation of the LpL gene provides a biochemical link between C. pneumoniae and the disease process of atherosclerosis. Ca ⁺ influx is correlated with the up-regulation of the LpL gene, such that LpL gene expression was abrogated by L-type Ca²⁺ channel antagonists. The highest levels of LpL expression are reached at calcium ion influx levels roughly half of the Ca²⁺ capability of infected macrophages of 732.57 nM. C. pneumoniae-indacβd LpL upregulation triggers LDL modification and generates unregulated LDL uptake. Unregulated LDL uptake is significant in cases of existing risk factors such as hypocholesteromia and high LDL levels. The regulatory role of Ca²⁺ influx signaling in macrophage LpL expression during C. pneumoniae infection indicates an approach of using Ca²⁺ channel antagonists in ameliorating C. pneumoniae-^' duced atherogenic processes. Thus, the administration of L-type Ca²⁺ channel antagonists offer a direct treatment of the atherogenic processes through blockage of LPL up-regulation as well as making the infection susceptible to antibiotic treatment for the termination of chronic infection. In other words, the beneficial effects of calcium channel blockers can be increased through the synergistic effects of combining calcium channel blockers with antimicrobial agents, i.e. antibiotics, for the treatment of atherosclerosis. As described below, control of the [Ca²⁺]i levels within selected levels can provide beneficial effects through increased NO production with or without co-administration of antibiotics.

Calcium Channel Activity and Biological Response As described above, Ca²⁺ influx at high levels is associated with chronic intracellular bacterial infection of macrophages. Blockage of the calcium ion influx can be used to revert a chronic bacterial infection to a non-chronic form that is not cryptic, i.e., is susceptible to antibiotic treatment. Furthermore, calcium ion influx is associated with up-regulation of LpL gene expression that induces foam cell formation and atherogenic processes associated with blood vessel disease. However, other results suggest a correlation between the calcium channel activity and NO release resulting from activity of nitric oxide synthase. NO has a vasoactive function that may impair some adverse immune functions through enhanced blood flow. Also, NO interaction with superoxide ^'O₂ can generate peroxynitrite, a toxic radial, which together with its decay products are potent killers of microbes (Halliwell et al. 1992; Hibbs Jr. 2000). Since NO release is associated with toxicity against microorganisms and beneficial vascular effects, these results suggest an improved control of calcium channel activity to reduce high influx levels induced by chronic intracellular bacterial infection without decreasing calcium ion concentrations below levels in which NO is produced through nitric oxide synthase activity. The female steroid 17β-estradiol has been identified with a range of beneficial biological responses in animal physiology. Specifically, estradiol can have positive effects on neuronal cells as well as having a significant effect on immune response. Specifically, with respect to effects on immune response, 17β-estradiol has been observed to inhibit monocyte adhesion to endothelial cells (Suzuki et al. 1997), reduce cardiac leukocyte accumulation in myocardial ischaemia reperfusion injury (Yamada et al. 1996), and impair leukocyte phagocytosis (Suzuki et al. 1997; al-Afaleq and Homeida 1998). The mechanism by which estrogen influences immune response may be related to its relationship with NO production due to the effects on coronary arteries (Barrett-Connor and Bush 1991; Williams et al. 1994; Collin et al. 1994 and Guetta et al., 1997). Testosterone is an estradiol analog with some overlapping functions. Thus, testosterone would be expected to have an analogous signaling role with respect to calcium channel function and NO production as estradiol, although quantitative differences in these roles have not yet been evaluated. Nevertheless, achieving comparable calcium ion concentrations should produce desired ranges of NO release in males and females at physiological hormone levels. Estradiol signaling is correlated with Ca²⁺ signaling in unknown ways. However, observations indicate a biphasic pattern of NO release in relation with Ca²⁺ influx signal with respect to L-type calcium channels in macrophages (Azenabor and Chaudhry 2003b; Azenabor and Chaudhry 2003c). This suggests a desirable range of Ca²⁺ influx for nitric oxide synthase up-regulation and associated desirable NO generation. This is confirmed, as described in the Examples below, through the correlation of release of NO and induction of nitric oxide synthase gene expression in macrophages treated with physiological doses of 17β-estradiol. This data supports the proposition that Ca²⁺ channel inhibitors can play a significant role in modulating macrophage NO release. In particular, it is desirable to mediate Ca²⁺ to reduce high influxes into monocytes but not to reduce intracellular Ca²⁺ concentrations below levels in which nitric oxide synthase is up- regulated. Thus, the administration of calcium channel blockers can be selected to yield macrophage intracellular calcium ion concentrations between about 190 and 350 nanomolar (nM), and in some embodiments from about 190 to about 300 nM and in further embodiments from about 190 and 280 nM. The intracellular calcium ion concentrations can be evaluated from harvested cells as described in the Examples below or by estimates from in vitro cell culture studies based on vascular concentrations of calcium channel blockers and hormone levels. In addition, appropriate dosages can be evaluated empirically by administration of estimated doses with adjustments made by measurements of vascular NO concentrations. The measurement of nitric oxide levels is described, for example, in U.S. patent 5,885,842 to Lai, entitled "Methods for the Detection of Nitric Oxide in Fluid Media," incorporated herein by reference.

Therapeutic Agents The synergistic effects of a combination of calcium channel inhibitors and antibiotics may result from the effectiveness of the calcium channel inhibitors to convert inclusions of Chlamydia within a macrophage from an unsusceptible, cryptic form to a form that is more susceptible to antibiotic treatment as well as improving a macrophage mediated immune response. Thus, an appropriate dosage of the calcium channel inhibitor can be selected to yield the desired physiological conversion of the Chlamydia inclusions within a macrophage. Suitable dosages of antibiotics include, for example, conventional dosages, although in some embodiments reduced dosages can be appropriately effective. Also, a broader range of antibiotic compounds can be used effectively to treat and/or cure a patient from a chronic Chlamydia infection and related disease, such as atherosclerosis. In particular, with respect to calcium channel inhibitors, therapeutically effective amounts of generally reduce HSP-60 mRNA amounts to within 20%, in additional embodiments within about 15% and in other embodiments within 10% of the amounts of HSP-60 mRNA in normal Chlamydia, as determined by the methodology described in the Examples. In summary, HPS-60 levels are returned closer to values in non-cryptic forms of the bacteria. A person of ordinary skill in the art will recognize that additional ranges of mRNA amounts relative to normal amounts within the explicit ranges above are contemplated and are within the present disclosure. Normal Chlamydia refers to bacteria propagated in non-macrophage cell types, such as HEp-2 cells available from (ATCC). Suitable doses can be evaluated from in vitro studies and scaled to in vivo amounts using, for example, conventional scaling approaches. Specifically, approaches are described in published PCT application serial number WO 04/025393A to Arakelyan et al., entitled "An Interactive Technique for Optimizing Drug Development from the Pre-Clinical Phases through Phase IV," incorporated herein by reference. In general, the approaches described herein can provide statistically improved effectiveness of an antibiotic in comparison with the same antibiotic administered without a calcium channel blocker. Specifically, when combined with a suitable dose of an L-type calcium channel blocker, a dose antibiotic can be at least as effective as double the dose applied without the calcium channel blocker. Also, the efficacy of the new regimen can be assessed by failure to demonstrate Chlamydia in monocytes or monocyte derived macrophages, after withdrawal of calcium channel blockers and antibiotics. Suitable calcium channel blockers generally include, for example, L-type calcium channel blockers. L-type calcium channels are characterized by a high depolarization threshold for activation, a large per channel ion conductance, a greater permeability to Ba⁺² than to Ca⁺², and blockage of the channel by dihydropidine class of calcium channel agonists. Several classes of L-type channel blockers are available including, for example, dihydropyridines, such as nifedipine (sold under the trade name Procardia™ and in time released form Procardia XL™), felodipine (sold under the trade name Plendil™), isradipine (sold under the trade name DynaCirc™) and amlodipine (sold under the trade name (Norvasc™), benzothiazepines, such as ditiazem, and phenylalkylamines, such as verapamil. Suitable does for these are described further below. Also, retinoids have been found to be L-type calcium channel inhibitors, as described further in U.S. Patent 6,437,003 to Roullet et al., entitled "Use of Retinoids to Treat High Blood Pressure and Other Cardiovascular Disease," incorporated herein by reference. Other broadly directed calcium channel blockers are described, for example, in U.S. Patent 5,312,928 to Goldin et al., entitled "Calcium Channel Antagonists And Methodology For The Identification," U.S. Patent 6,117,841 to Hu et al, entitled "Substituted Peptidylamine Calcium Channel Blockers," and U.S. Patent 6,310,059 to Snutch, entitled "Fused Ring Calcium Channel Blockers," all three of which are incorporated herein by reference. Suitable calcium channel blockers include, for example, combinations of two or more calcium channel blockers. Certain antibiotics have been identified for their relative effectiveness against

Chlamydia infections. In addition, when co-administered with an appropriate calcium channel blocker, a broader range of antibiotics can have appropriate effectiveness against Chlamydia and similar chronic infections. Broadly, appropriate classes of antibiotics, i.e., antimicrobial agents, include, for example, tetracyclines, macrolides, quinolones, chloramphenicol, rifamycins, sulfonamides, co-trimoxazole, oxazolidinones (such as linezolid and oxazolidinone) and oxazolidinones. Suitable tetracycline antibiotics include, for example, tetracycline, oxytetracycline, doxycycline, demeclocycline, chlortetracycline, methacycline and minocycline. Suitable macrolides antibiotics include, for example, erythromycin, spiramycin, oleandomycin, triacetyloleandomycin, josamycin, kitsamycin, midecamycin, miocamycin, rokitamycin, rosarimycin, flurithromycin, dithromycin, azalide, ketolide, azithromycin, clarithomycin and roxithromycin. Suitable quinolone antibiotics include, for example, nalidixic acid, oxolinic acid, norfloxacin, pefloxacin, amifloxacin, ofloxacin, ciporofloxacin, enoxacin, lomefloxacin, fleroxacin, temafloxacin, sparfloxacin, tosulfoxacin, clinafloxacin, cinoxacin, trovafloxacin, levofloxacin, nadifloxacin and refloxacin. Suitable rifamycin antibiotics include, for example, rifampicin, rifabutin and rifapentin. Suitable sulfonamides include, for example, sulfϊsoxazole, sulfamethoxazole, sulfadiazine, sulfadoxine, sulfasalazine, sulfaphenazole, dapsone and sulfacytidine. Suitable antibiotics include combinations of two or more antibiotic compositions, such as those listed above. Suitable doses are described below. Additional antibiotics are continuously under development. The therapeutic approaches described herein are applicable for the treatment of infections of Chlamydia bacteria and may be similarly applicable against other intracellular bacterial infections. Chlamydiae are considered obligate intracellular bacteria that are known to multiply within an infected eukaryotic cell. Chlamydiae are in the order Chlamydiales having one family Chlamydiaceae with one genus Chlamydia. Four Chlamydia species are presently known, C. trachomatis, C. pneumoniae, C. psittaci and C. pecorum. Other intracellular bacteria include, for example, Mycobacteria, Rickettsia, Mycoplasma, Neisseria and Legionella. Chlamydiae are known to cause chronic infections. Similarly, obligate intracellular bacterial Coxiellia burnetii also are known to have species with chronic forms of infection that are often fatal. The present approaches can be effective against infections of intracellular bacteria in chronic stages that modulate calcium channel function to reduce efficacy of immunological response. For the treatment of atherosclerosis, calcium channel blockers as well as antibiotics can be combined with other suitable drugs, such as cholesterol lowering drugs such as Lupron™. Due to the effectiveness of calcium channel blockers and antibiotics at countering physiological changes associated with foam cell formation, a synergistic effect with respect to vascular health has been established from the co-administration of L-type calcium channel blockers and antibiotics. As described above, the appropriately selected controlled release of a calcium channel blocker to obtain significant periods of time with calcium channel blockers at levels within a suitable range to stimulate NO production and up-regulation of nitrogen oxide synthase.

Administration and Pharmacological Formulations In some embodiments, pharmacological formulations combine calcium channel blockers and antibiotics for improved treatment of Chlamydia and other infections that modulate the immune response by modifying calcium channel function. The combination of calcium channel blockers and antibiotics can have a synergistic effect since the calcium channel blockers make chronic infections susceptible to anti-microbial treatment. Similarly, for the treatment of atherosclerosis, calcium channel blockers along with an antibiotic and/or other medications for artherosclerosis, such as cholesterol lowering drugs. The pharmacological formulations can be delivered, for example, by way of injection, inhalation, oral ingestion, rectal placement, intravaginal placement and/or the like. Suitable injection approaches generally include, for example, intravenous, intramuscular, and/or subcutaneous injection, generally with a pharmaceutically acceptable solution and sterile vehicles, such as physiological buffers (e.g., saline solution or glucose serum). Further, delivery through a catheter or other surgical tubing is possible. Inhalation, for example, nasal or oral, generally can be performed with an aerosol formulation optionally along with a suitable vehicle suitable for this mode of administration. Improved inhaler systems for medicaments do not use a propellant gas. See, for example, U.S. Patent 6,681,768 to Haaije de Baer et al, entitled "Powder Formulation Disintegrating System and Method for Dry Powder Inhalers," incorporated herein by reference. Alternative routes include, for example, nebulizers for liquid formulations, and inhalers for lyophilized or aerosolized pharmaceutical formulations. For administration, for example, by oral ingestion (tablets, capsules, pills, liquids or the like), rectal suppositories or intravaginal pastry, the pharmacologically active agents can be combined with pharmaceutically acceptable solid and/or liquid excipients. In some embodiments, a carrier or excipient is desirable for administration of the pharmacological agents e.g., to solubilize an insoluble compound for liquid delivery; to buffer the pH of the substance to preserve its activity; or to dilute the substance for improved handling. Furthermore, the formulations of the therapeutic agents can further include processing aids, inert diluents, colorants, and other additives known in the art for the particular type of formulation, e.g., pills, liquids, aerosols and the like. In other cases, however, the carrier is for convenience, e.g., a liquid for more convenient administration. Pharmacological formulations are described further in U.S. Patent 6,475,518 to Baumgart et al., entitled "Methods and Compositions for Treatment of Disorders Associated with Chlamydia and Similar Bacterial Infections," incorporated herein by reference. In general, the particular daily dosage generally can be administered in a single dose or a plurality of dosages spread through the day. The nature of the compounding of the composition, for example, the use of a time release encapsulation, may influence the appropriate timing of administration. Suitable structuring of the dosages can be evaluated by procedures known in the art. In general, the administration of the calcium channel blocker and antibiotic can be continued until the chronic bacterial infection is eliminated. In general, the length of the administration can range from one day to several months, and in some embodiments from three days to two months. Various lengths of administration of the therapeutics within these explicit ranges can be used as appropriate. Methods have been developed for the sustained release of drugs for administration to a patient. Such approaches can involve, for example, coating of the drug or portions of the drug within a tablet with a coating, such as a bioresorbable agent. More complex approaches have been developed to obtain greater control over the release kinetics. For example, more sophisticated approaches that can be adapted for the present use are described, for example, in U.S. Patent 6,110,500 to Kim, entitled "Coated Tablet With Long Term Parabolic And Zero-Order Release Kinetics," and U.S. Patent 5,837,226 to Jungherr et al., entitled Ocular Microsphere Delivery System," both of which are incorporated herein by reference. Approximately zero order release kinetics can be desirable to yield roughly constant doses over a desired period of time. As used herein, approximately zero order release kinetics implied that the release varies by less than a factor of 35 percent from the average release rate over the release of 90 weight percent of the therapeutic agent. An initial small dose can be delivered for immediate release into the patient's blood stream followed by the controlled release version to maintain the therapeutic agent at the selected levels over the passage of time. In general, the dosage of the calcium channel blocker and the antibiotic may be correlated to elicit a desire therapeutic effect. The calcium channel blocker can be present in an amount at least high enough to modify at a statistically significant level back toward a natural level the increased calcium ion influx into infected macrophages. While this amount can be determined in vitro and correlated by known approaches with an m vivo therapeutic dose, the dosage can similarly be determined by the synergistic improvement of the effectiveness of an antibiotic to eliminate the chronic bacterial infection. Higher doses of calcium channel blocker beyond the threshold for inducing a synergistic improvement in the effectiveness of the antibiotic can further improve the effectiveness of co-administered antibiotics to reduce the effective antibiotic dosage up to an amount that decreases Ca²⁺ flow to undesirably low levels. Specifically, the levels of calcium channel inhibitors can be selected to obtain intracellular calcium ion concentrations to yield desired rates of NO production. In general, the dose of calcium channel blocker for a human patient can be in the range of at least about 0.01 milligrams (mg) per day, and in some embodiments, at least about 0.25 mg per day, in further embodiments from about 0.5 to about 100 mg per day and in other embodiments from about 1 mg to about 20 mg per day. The selected dose may depend on the mode of administration and the particular composition. Under present recommendations, nifedipine is administered in amounts from 30-60 milligrams per day while felodipine is administered in amounts from 2.5 to 10 milligrams per day, according to the 2003 Home Edition of the Physician's Desk Reference, incorporated herein by reference. To yield improved levels of NO production, it may be desirable to adjust the dose more finely in a time release formulation to obtain particular serum levels based on the patient's specific body weight. Additional ranges of calcium channel blocker dosages and concentrations within the particular ranges above are contemplated and are within the present disclosure. With respect to the antibiotic, the dose of antibiotic for a human patient generally is in the range of less than about 1 gram per day, in some embodiments, less than about 500 milligrams (mg) per day, in additional embodiments, from about 5 mg to about 300 mg, and in other embodiments from about 10 mg to about 250 mg per day. In general, the specific dosage may depend on the specific antibiotic and on the weight and health of the patient. A person of ordinary skill in the art will recognize that additional ranges of dosages within the particular dosages above are contemplated and are within the present disclosure. Selection of suitable dosages are described further in Martindale, The Extra Pharmacopoeia, Thirty-First Edition (The Royal Pharmaceutical Society, London, 1996), incorporated herein by reference. While it is advantageous to co-formulate a calcium channel blocker with an antibiotic to simplify the administration of the agents in the desired proportions, the calcium channel blocker and the antibiotic can be administered separately, e.g., in separate pills or aerosols, or in different forms from each other, such as one as a pill and the second as an aerosol or any other form. If the therapeutic agents are separately administered, the each agent generally is stored in a separate container, and the separate containers may or may not be packaged separately. In some embodiments, the separate containers of therapeutic agents are packaged together in a kit along with instructions for the proper administration of the therapeutic agents. In other embodiments, the separate containers of therapeutic agents are not packaged together, but nevertheless the patient is provided with instructions for the proper administration of the calcium channel blocker and the antibiotic to provide a synergistic effect.

EXAMPLES The Examples describe the desirable effects of calcium channel blockers with respect to modulating the calcium influx into Chlamydia infected macrophages and a corresponding change in the membrane structure of the bacteria that are more like the acute form of the bacteria than the chronic form of the bacteria. The synergistic effect of the calcium channel blockers and antibiotics is also demonstrated. In addition, the correlation between calcium ion signaling is correlated with the induction of lipoprotein lipase gene expression. Furthermore, results are presented showing that nitric oxide synthase gene expression is also correlated with intracellular calcium ion concentration.

Materials and Methods In the following examples, the following materials and methods are used to conduct the experiments. Unless otherwise specified, chemicals are obtained from Sigma (Milwaukee, WI). Cell Culture and Propagation of C. pneumoniae. Murine macrophage cell line - RAW-264.7 (American Type Culture Collection, Rockville, MD. USA), was grown in RPMI- 1640 medium with L-glutamate, supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, Utah), and treated with 50μg/ml vancomycin, lOμg/ml gentamicin. The cell culture was maintained in a 37°C, 5% CO₂ incubator and sub-cultured every 48hr after gentle scraping. Macrophages at density 1.2 xlO⁶ cells/well in 6 well plate, 5 xlO⁵ cells/well in 24 well plate or corresponding subfluency in 96 well plate (Corning Costar Corporation, Cambridge, MA) were used for experiments. In some cases, for nitric oxide and Ca²⁺ assays, 5x10⁴ macrophages /well in 96 well plate (Corning Costar Corporation, Cambridge, MA) were used, while for iNOS gene expression and iNOS protein expression, lxl 0⁶ macrophages/well in 6 well plate were used. In some cases, cells were tested for mycoplasma contaminants periodically by staining with 4-6-diamine-2-phenyl indole dihydrochloride (Boehringer Mannheim, Mannheim, Germany). In instances where macrophages were treated with Ca²⁺ regulating drugs, antibiotics, O₂ ^" inhibitors, or estradiol receptor antagonist, cell viability was assessed using trypan blue exclusion. Chlamydia pneumoniae (AR-39), was obtained from ATCC, and stock organisms propagated in HEp-2 (ATCC) cell monolayer by centrifugation (1864x g, Sorvall RC5C, SH-3000 rotor) driven infection for lhr, followed by rocking in a humidified incubator at 37°C and 5% CO₂ for 3hr. After medium replacement using fresh Iscove's modified Dulbecco's medium containing 10% FBS (prescreened for Chlamydia antibodies) and 2 μg/ml cycloheximide, it was returned into the incubator at 37° C and 5% CO₂ for 72 h. Chlamydia was harvested, sonicated, loaded onto a discontinuous gradient of urografin (Schering, Berlin, Germany), and elementary bodies (EBs) were pelleted at 17 211 g (Sorvall RC5C, ss-34 rotor) for 1 h at 4°C. Harvested EBs were stored at -70°C in sucrose- phosphate-gutamate buffer (0.22 M sucrose, 10 mM sodium diphosphate, 5 mM glutamic acid, pH 7.4) in small aliquots and thawed when needed. Chlamydia inclusion forming units (IFU) were determined by thawing a frozen aliquot of harvested EBs and infecting confluent (5 x 10⁵ cells/well) Hep-2 cell monolayers in a 24 well plate, with ten fold serial dilution in medium using the centrifugation -assisted procedure already described. Infected cells treated with cycloheximide were incubated at 37°C for 72 h, washed with PBS, fixed with methanol, and stained with an indirect fluorescence method using anti-lipopolysaccharide antibody (Chlamydia identification Kit, Bio-Rad, Woodinville, USA). The number of IFU was determined by counting ten microscope fields using an inverted fluorescence microscope (Olympus, Melville, USA). Chlamydia pneumoniae was aliquoted into 70 μl vials and stored at -70°C. This process is described further in (Azenabor & Chaudhry, 2003a). After washing the monolayers of macrophages with phosphate-buffered saline (PBS), macrophages were infected, for example, with 50 μl (96-well plates) or 1 ml (6- wellplates), of multiples of infection (MOI) of 3 EBs per cell. This inoculum was adequate for our studies and did not cause host cell cytotoxicity by trypan blue exclusion assays. For experiments involving inclusion count determination in infected macrophages, centrifugation driven infection was carried out. However, after 72hr incubation or after thawing, C. pneumoniae was harvested from macrophages and used to infect HEp-2 cells to allow for distinct inclusion counting. Generally, in all experiments multiples of infection (MOI) of 3 per cell was used. Chlamydia inclusions were visualized and counted as infection fonning units (IFU)/ml by counting 10 microscope fields with an inverted fluorescence microscope (Olympus, Melville, N.Y), after staining with an indirect method using fluorescene isothiocyanate (FITC) labeled anti-LPS antibody (Chlamydia identification Kit, Bio-Rad, Woodinville, Washington) (Azenabor & Chaudhry, 2003a). Assay of Macrophage Lipoprotein Lipase Gene Expression Raw-264.7 cells were seeded into 6-well plates and infected with C. pneumoniae at MOI=3/cell. In some experiments, infection ampoules were treated with L-type Ca²⁺ channel inhibitors methoxyverapamil or nifedipine (Azenabor and Chaudhry, 2003b). Lipoprotein lipase mRNA gene expression was determined by RT-PCR following an initial extraction of macrophage RNA. Macrophage RNA Extraction. Macrophages were scraped from wells after appropriate treatment and incubation time, and RNA extraction was done by standard methods using Trizol (Invitrogen, Life Technologies, Carlsbad, CA) and a modification of the method described by Chomczynski and Sacchi (1987). Briefly, Trizol was used to initiate cell disruption and dissolution, and then chloroform was added and centrifuged to produce aqueous and organic phases. RNA retained in the aqueous phase was withdrawn, precipitated with isopropanol and washed twice in 75% ethanol. RNA was quantified spectrophotometrically at 260 nm using a UN-VIS spectrophotometer (Perkin Elmer). All extracted RΝA was stored at -70° when not used directly for PCR. Chlamydia RΝA Extraction. Chlamydia pneumoniae was harvested from macrophages by standard harvest technique (Kaukoranta et al., 1994; Azenabor & Chaudhry, 2003a). Briefly, macrophages were scraped from wells at appropriate time and sonicated. The elementary bodies (EBs) were pelleted at 17,21 lx g (Sorvall RC5C, SS-34 rotor) for lhr at 4°C. Harvested EBs were purified by loading onto discontinuous gradient of urografin (Schering, Berlin, Germany) and centrifuged at 17,21 lx g for lhr at 4°C. RΝA extraction was done on purified EBs. This was by standard methods using TRIZOL (Invitrogen, Life Technologies, Carlsbad, CA) and a modification of the method described by Chomzynski and Sacchi, 1987. Briefly, TRIZOL was used to disrupt and dissolve EBs, and then chloroform was added and centrifuged to produce aqueous and organic phases. RΝA contained exclusively in the aqueous phase was precipitated with isopropanol and washed twice in 75% ethanol. RΝA was quantified spectrophotometrically at 260nm using UV-VIS spectrophotometer (Perkin Elmer). All extracted RΝA was stored at -70°C, when not used directly for RT-PCR. Quantitative RT-PCR. Reverse transcription of RΝA and cDΝA amplification were performed using Superscript One-step RT-PCR with Platinum Taq (Invitrogen, Life Technologies, Carlsbad, CA) according to manufacturer's instructions. Briefly, cDΝA synthesis was performed at 50°C for 30 minutes. After 2 minutes of initial denaturation at 94°C, the samples were subjected to 25 cycles of denaturation (94°C, 15s), annealing (50°C, 30s) and extension (72°C, 30s). The final extension was at 72°C for 7 minutes. Twenty five cycles was found to be the number of cycles to obtain desired levels of fluorescence. Targets were specific for the particular example. In one example, the targets were genes of major outer membrane protein and the 60-kDa heat shock protein. In another example, the targets were the mouse LpL gene. Primers (Integrated DΝA Technologies, Carolville, IA) for MOMP mRΝA were Cpn 201 (5¹ TGG TCT CGA GCA ACT TTT GAT G 3 ') and Cpn 202 (5¹ AGC TCC TAC AGT AAC TCC ACA 3 ') as originally described by Gaydos et al., 1992. Primers for HSP-60 mRΝA were GE-1 (5¹ AGC TCA CGT AGT TAT AGA TAA GAG 3 ') and GE-2 (5¹ AAG TAG CTG GAG AGG TAT CCA CGG 3 ') as described by Airenne et al., 1999. Primers (Integrated DNA Technologies, Carol ville, USA) for mouse LpL mRNA were sense: 5'-CCT TAG AAA ACG GAT AGA CTA CTC AAC-3' and antisense: 5'-CCG CTC GAG CAC TCT TCT CGC TTC TAG AGG CGT CTG-3' from GenBank as described by Zhang et al. (2001). Primers (Integrated DNA Technologies, Carolville, IA) for genes of iNOS were sense - 5¹ iNOS - CCC TTC CGA AGT TTC TGG CAG CAG C 3 ^■ and antisense - 5' iNOS - GGC TGT CAG AGC CTC GTG GCT TTG G 3 ' as described by Huang et al. 2002. The primers were designed for kinetic RT-PCR methodology by coupling to dual labeled probes, one a fluorescent marker, 6 carboxy fluorescein amine reactive succinimidyl ester, (6FAM), situated at the 5¹ end of primer and the other a quencher, 6- carboxy tetramethyl-rhodamine (TAMRA™) at the 3 ' end. Extension nullifies the energy dissipating effect of the quencher, and the fluorescence produced is a reflection of RNA present in the sample (Bustin, 2000; Bustin, 2002). Fluorescence readings were recorded as relative fluorescence unit (RFU) using fluorimetric plate reader (Cytofluor-4000, Applied BioScience, Woodinville. CA). In some instances (Figs 2B and 3), percentage gain in fluorescence reading on the instrument were adjusted to obtain appropriate values. As control for the absence of DNA in the RNA preparation, each sample was additionally amplified without prior reverse transcription. Assay of Lipoprotein Lipase (LpL) Activity. Lipoprotein lipase activity was determined on cell lysate by fluorimetric assay, based on the catalytic action of enzyme on fluorophore labeled triglyceride substrate. Fifty micro liters cell lysate (obtained by freeze thawing), or lipoprotein lipase standards (200 - 500 units/ml), or blank (reaction buffer) were put in black, flat bottom 96 wells plate (Corning Costar Corporation, Cambridge, MA. USA). Reaction buffer (100 μl) consisting of 100 mM glycine and 19 mM sodium deoxycholate, adjusted to pH 9.5 using 1 M sodium hydroxide was added to each well. This was followed by addition of 50 μl of 2 mM substrate reagent, fluorescent 1,2- dioleoyl-3-pyrene decanoyl-rac-glycerol (Molecular Probes, Eugene, OR. USA). The mixture was rocked for 30 min in the dark at 27°C and fluorescence read at excitation wavelength of 360 nm and emission wavelength of 470 nm. Calibration curve of enzyme standard in units/ml against fluorescence was generated, and values for the samples were read from the calibration curve. Specific enzyme activity was determined as units/mg protein. Ca²⁺ Assay. Imaging of intracellular Ca²⁺ was carried out as described (Azenabor and Hoffman-Goetz, 2001) except that the fluorogenic Ca²⁺ indicator Fluo-4 was used instead of Fluo-3, because it exhibits an increased fluorescence excitation with no accompanying spectra shift, thus expressing a higher signal level for microplate application. Briefly, macrophages (5x10⁴ cells/well) were seeded in 96 well plates and treated with C. pneumoniae at MOI=3/cell. At the appropriate time, intracellular Ca²⁺ was monitored by loading macrophages with cell permeant acetoxymethyl (AM) ester of Fluo- 4 calcium indicator (Molecular Probes, Eugene, OR. USA) at a final concentration of 4 μM/well and incubated at 27°C for 30 min. Then, the cells were washed in PBS and re- incubated for 30 min. Fluorescence readings were recorded using CytoFluor 4000 (Applied Bioscience, Woodinville, CA. USA) at an excitation wavelength of 488 nm and emission wavelength of 530 nm. Intracellular Ca²⁺ was computed from the equation [Ca²⁺]i = Kd x [F-Fmin]/[Fmax-F]. The dissociation constant Kd for Fluo-4-Ca²⁺ complex, Fmin and Fmax were determined in our laboratory using calcium calibration buffer kit (Molecular Probes, Eugene, OR. USA). Assay of LDL Uptake. The determination of low density lipoprotein uptake by macrophages under various experimental conditions was determined by monitoring fluorescence emitted (Wang et al., 1995) by macrophages exposed to LDL labeled with the fluorogenic probe BODIPY FL complex (Molecular Probes, Eugene, OR. USA). Cells (5.0 x 10⁴ cells/well) were seeded into 96 well plates and were either uninfected, or infected as well as homocysteine (5 mM/well) treated or inhibited for LpL gene expression by Calphostin-C (0.5 μg/ml) treatment. In some experiments, cells were not treated with any drugs but infected with C. pneumoniae. After infection protocol where appropriate, cells were incubated with fluorescence probe labeled LDL for 5 h, and fluorescence intensity was measured at excitation wavelength 488 nm and emission wavelength 530 nm. The probable disparity in cell number following growth was corrected by protein estimation and results were recorded as relative fluorescence unit/mg protein. Assay of NO. A nitric oxide assay made use of NO-specific fluorogenic probe 4- amino-5-methylamino-2^l,7^l,-difluorescein diacetate (DAF-FM diacetate) (Molecular Probes, Inc. Eugene, OR) to monitor intracellular NO generation (Lopez-Figueroa et al. 2000). Briefly, macrophages (5x10⁴ cells/well) were seeded into flat bottom black 96 well plate and incubated at 37°C for 24hr in CO₂ incubator. They were exposed to 117nM estradiol or 20μg/ml lipopolysaccharide or the estradiol receptor antagonist lOμM ICI 182,780 (Tocris, Balwin, MO. USA) or untreated, after an initial pre-incubation with varying doses of intracellular Ca²⁺ chelator 1,2-bis (o-aminophenoxy) ethane-NNiN'N¹- tetraacetic acid tetra (acetoxy methyl) ester (BAPTA-AM) (Whitehead et al. 2001), and the release of NO was monitored by incubating with 5μM/well DAF-FM for 30min at 37°C. Cells were washed with PBS and re-incubated for 30min. Fluorescence readings were recorded at an excitation wavelength 488nm and an emission wavelength 530nm using CytoFluor 4000 (Applied Bioscience, Woodinville, CA). In some experiments, NO release was measured after blockage of xanthine oxidase activity (using lμM allopurinol) and NADPH-oxidase activity (using lOOμM apocynin) (Beswick et al. 2001). Calcium Assay: Macrophage [Ca²⁺]i was assayed as previously described (Azenabor and Hoffman-Goetz 2001) except that instead of Fluo-3, its analog Fluo-4 was the calcium indicator of choice, since it exhibits an increased fluorescence excitation with no accompanying spectra shift, thus expressing a higher signal level for microplate applications. Briefly, macrophages (5xl0⁴/well) were preheated with varying doses of BAPTA-AM and [Ca ⁺]i was imaged by rapidly loading macrophages with cell permeant acetoxymethyl (AM) ester of Fluo-4 calcium indicator (Molecular Probes, Eugene, OR) at a final concentration of 5μM, and incubated at 27°C for 30min. Cells were washed in Ca²⁺ free PBS and incubated in the dark for 30min at 27°C. Fluorescence readings were recorded using CytoFluor-4000 (Applied Bioscience, Woodinville, CA) at an excitation wavelength 488nm and an emission wavelength 530nm. The [Ca²⁺]i was computed from the equation [Ca²⁺]i = Kd x [F-Fmin]/[Fmax-F]. The dissociation constant for Fluo-4-Ca²⁺ complex (Kd), Fmax and Fmin were determined in our laboratory using calcium calibration buffer kit (Molecular Probes, Eugene, OR). Assay of iNOS Gene Expression: Macrophage cell line RAW-264.7 was seeded into 6 well plates as already described. Cells were pretreated with lOμM BAPTA-AM for 30min, or treated with the L-type Ca²⁺ channel blocker 20μM nifedipine or treated with 20μM nifedipine and intracellular Ca²⁺ store blocker 7μM SKF-96365 followed in each of the set of procedures stated by exposure to doses of 17β-estradiol (15.3nM, 58.5nM and 117nM). Inducible nitric oxide synthase mRNA gene expression was determined by RT- PCR following an initial extraction of macrophage RNA. Assay of iNOS Protein: Western blot was run on macrophages treated with 117nM estradiol after pretreatment with lOμM BAPTA-AM reflecting optimum [Ca ⁺]i concentration for iNOS protein expression, or 15μM BAPTA-AM and 20μM BAPTA- AM reflecting dose effect of [Ca²⁺]i concentration on iNOS protein expression, or no BAPTA-AM treatment reflecting [Ca²⁺]i concentration higher than optimum concentration for iNOS protein expression. In some experiments, pretreatment with lOμM BAPTA-AM was followed by treatment with the estradiol receptor antagonist lOμM ICI 182,780, serving as control for the effect of estradiol on iNOS protein expression. Cells were scraped and lysed by freeze thawing. Protein was precipitated with 10% trichloroacetic acid and resuspended in assay buffer (BioRad Laboratories, CA, USA). Protein estimation was done on sample to determine the volume of sample that contains lOμg. Western blot was done on protein according to manufacturer's instructions (BioRad Laboratories, CA. USA). Briefly, lOμg protein was spotted per lane on 10% SDS-polyacrylamide gel. After electrophoresis, protein bands were electrophoretically transferred to 0.2μm Immun- Blot™ PVDF membrane (BioRad Laboratories, CA, USA). To avoid nonspecific binding blocking was done with 3% blocker provided with the kit, washed and incubated with primary antibodies (anti-iNOS) (Upstate Cell Signaling Solutions, Lake Placid, NY, USA), at a dilution of 1:600 for lhr, then washed and incubated with GA_X-HRP (horseradish peroxidase) (BioRad Laboratories, CA, USA), at a dilution of 1:6000 for lhr, then washed with PBST buffer. Colorimetric detection was done according to manufacturer's instructions. Protein Estimation. Protein assay was done on each sample where required by bicinchoninic acid - copper (II) sulphate reagent system as described by Smith et al. 1985. Statistics. Data were analyzed using a repeated measures analysis of variance

(ANOVA) model for all studies involving LpL mRNA gene expression and for other studies (including those on LPL ) data were analyzed by student's t-test using difference between means of two treatments, and p value of 0.05 was considered significant. Unless stated otherwise, all values represent group means ± 1 standard error (n = 4).

Example 1 - Cell Permeant Ca²⁺ Chelator (BAPTA-AM) Mediates Regulation of Chlamydia HSP-60 and MOMP mRNA Gene Expression. Chlamydia infection of macrophages produces an unusual inclusion, which is obscure or cryptic unless, for example, exposed to the protein inhibitor cycloheximide. Also, Ca"⁺ chelator applied during infection prompts Chlamydia inclusion formation even in the absence of cycloheximide (Azenabor & Chaudhry, 2003a). This example presents the results of an investigation of molecular changes initiated by Ca²⁺ chelation in relation to cHSP-60 and MOMP mRNA genes expression (proteins associated with Chlamydia viability and persistence). As shown in Fig. 1, treatment of macrophage - RAW - 264.7 cells with intracellular Ca²⁺ chelator 1, 2-bis (o-aminophenoxy) ethane - N,N,N'N' - tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM) during infection with C. pneumoniae down regulated cHSP-60 mRNA gene ( — ♦ — ) from levels that produced fluorescence readings of 145 RFU to 80 RFU dose dependently and up-regulated MOMP mRNA gene (— ■ — ) expression from levels that produced a fluorescence reading of 150

RFU to 240 RFU. These changes were significant (P < 0.016 and 0.003 respectively using student-t -test). While cHSP-60 mRNA gene expression was down regulated, dose dependently, MOMP mRNA gene was up-regulated. These results are indicative of an increase in gene products elaboration. Thus, BAPTA-AM applied at time of infection, allow reduced Ca²⁺ influx signal since it requires about 30 minutes to be effective as a chelator of intracellular Ca²⁺. Early reduced Ca²⁺ influx signal is required for Chlamydia infection, but it is insufficient for greater macrophage bactericidal activity (Azenabor & Chaudhry, 2003a). The extent of impairment of Ca²⁺ influx signal may explain the alteration of C. pneumoniae course in macrophages from persistent, cryptic form, to the more usual form.

Example 2 - Macrophage L-type Ca²⁺ Channel Inhibitors Down-regulate Chlamydia pneumoniae HSP-60 and Up-regulate MOMP mRNA Gene Expression. Ca²⁺ has a significant role on the out come of Chlamydia in macrophages. This example presents results relating to the effect of the specialized Ca²⁺ channel (L-type Ca²⁺ channel) on cHSP-60 or MOMP mRNA gene expression. Nimodipine or nifedipine inhibitory effect caused down-regulation of cHSP-60 mRNA gene expression in Chlamydia pneumoniae grown in macrophages to about the same extent obtained in normal growing Chlamydia in HEp-2 cells (positive control). The effect of specialized Ca²⁺ channel, (L-type Ca²⁺ channel) on Chlamydia viability in macrophages was assessed by use of L-type Ca²⁺ channel inhibitors, 20 μM/well nimodipine, or 20μM/well nifedipine. Macrophages not treated with Ca²⁺ channel blockers showed significantly higher levels of cHSP-60, as shown in Fig. 2A. However, MOMP mRNA gene expression was upregulated in macrophages in which nimodipine or nifedipine was used to inhibit L- type Ca²⁺ channel operation, marking a restoration of Chlamydia to viable forms from persistent forms, as shown in Fig.2B. Results obtained when Ca²⁺ channel antagonists were applied to macrophages were similar to those obtained for C. pneumoniae grown in HEp-2 cells. This finding validates the suggestion that Ca²⁺ influx signals regulate onset of C. pneumoniae persistence or chronicity in macrophages. These results were further confirmed in a nimodipine or nifedipine dose response studies shown in Fig. 3. The regulatory effect of L-type Ca²⁺ channel blockers nifedipine or nimodipine on cHSP-60 mRNA gene expression were dose dependent. Nimodipine

(— ■— ) or nifedipine ( — 0 — ) produced the same pattern. While MOMP was upregulated

by nimodipine (— O— ) or nifedipine ( — A — ). Down regulation of cHSP-60 mRNA gene or upregulation of MOMP gene was significant, when each dose was compared to macrophages not treated with L-type channel blocking drugs. However, it was noted that nifedipine has a mild toxic effect on macrophages at 40μM-50μM concentrations, causing a mild decline in MOMP mRNA gene expression.

Example 3 - Macrophage L-type Ca²⁺ Channel Inhibitor Improves C. pneumoniae

Susceptibility to Antibiotics. Since macrophage L-type Ca²⁺ channel greatly affected C. pneumoniae form, it was determined how the form of inclusions obtained with L-type Ca²⁺ channel inhibition behaved on exposure to antibiotics. Anti-chlamydial agents - erythromycin, doxycycline and rifampin were used. As shown in Fig. 4, the relevance of macrophage L-type Ca²⁺ channel to Chlamydia pneumoniae antibiotic susceptibility was assessed, by counting Chlamydia pneumoniae inclusion in HEp-2 after harvesting Chlamydia pneumoniae EBs from infected macrophages treated with L-channel inhibitors. The IFU/ml obtained from macrophages exposed to erythromycin or doxycycline or rifampin 48 hrs post infection/treatment with L-channel antagonist and left for 48 hrs again are represented.

Cells were either treated with 20μM/well nifedipine and exposed to erythromycin ( — ■ — ),

or doxycycline ( — O — ), or rifampin ( — 0 — ), or not treated with L-channel antagonist and

exposed to erythromycin ( — ♦ — ), or doxycycline ( — Δ — ), or rifampin ( — • — ). There was a significant change in susceptibility of Chlamydia pneumoniae to antibiotic dose dependently. Generally there was evidence of nifedipine mediation improved C. pneumoniae susceptibility to antichlamydial agents at lower doses of 0.05 μg/ml to l.Oμg/ml, compared to results obtained in cells not treated with nifedipine. The finding that macrophages not treated with nifedipine had C. pneumoniae forms that were refractory to antichlamydial agents at lower doses and relatively refractory at higher doses suggest an important role for macrophage L-type Ca²⁺ channel blockers in antibiotic treatment of C. pneumoniae associated diseases. The pattern of susceptibility amongst the three antibiotics (erythromycin, doxycycline and rifampin) was such that nifedipine effect minimized the differences in antibiotic effectiveness. Since macrophage L-type Ca²⁺ channel inhibitor appreciably improved C. pneumoniae susceptibility to antibiotic assessed by inclusion counting, cHSP-60 and MOMP mRNA gene expression was determined in an equivalent experimental setting. Fig. 5A shows mRNA gene expression for the stress protein cHSP-60 in the presence of nifedipine and doses of either erythromycin ( — ■ — ), or rifampin ( — • — ), or without nifedipine treatment but exposed to doses of erythromycin ( — ♦ — ), or rifampin ( — A — ). Fig. 5B shows MOMP mRNA gene expression in the presence of nifedipine and either erythromycin ( — ■ — ), or rifampin ( — • — ), or cells without nifedipine but treated with erythromycin ( — ♦ — ), or rifampin ( — Δ — ). Chlamydia HSP-60 mRNA or MOMP mRNA gene were significantly down regulated within 30 hrs in nifedipine treated cells in a manner consistent with antibiotic efficacy. The untreated cells exhibited refractory outlook of Chlamydia pneumoniae since mRNA gene expression did not show significant down regulation with varying antibiotic doses. These results indicate that nifedipine mediated decline in cHSP-60 and MOMP mRNA gene expression, during antibiotic treatment, differ significantly compared with results obtained from infected macrophages that were not exposed to nifedipine but treated with antibiotics. These findings serve as validations for Fig.4 in which decrease in IFU/ml was observed.

Example 4 - Chlamydia pneumoniae Induces Upregulation of Lipoprotein Lipase mRNA Gene Expression in Infected Macrophages. This example explores induction of lipoprotein lipase (LpL) upregulation accounting for unregulated uptake of low density lipoprotein. Lipoprotein lipase upregulation was significantly different in C. pneumoniae infected cells compared with uninfected cells. Referring to Fig. 6A, the upregulation of lipoprotein lipase mRNA gene expression in Chlamydia pneumoniae (MOI = 3/cell) infected macrophage ( — ■ — ) compared with uninfected macrophage ( — ♦ — ) or macrophage treated with a known inducer of LpL-homocysteine ( — A — ) is depicted in Fig 6. Chlamydia pneumoniae significantly increased LpL gene expression P<0.005 compared to uninfected cells. Also Chlamydia pneumoniae significantly increased LpL gene expression compared to homocysteine induction of these genes. The relative effectiveness of C. pneumoniae in inducing LpL mRNA gene expression compared with the capability of homocysteine (a known activator of LpL) (Beauchamp and Renier, 2002), to cause the effect showed that C. pneumoniae is a more efficient inducer of LpL gene expression. To validate this finding, the difference in LpL gene expression elicited in macrophages exposed to varying doses of C. pneumoniae was determined. The expression of LpL gene is Chlamydia pneumoniae dose dependent as shown in Fig 6B where MOI = 5/cell produced the greatest gene expression. Higher doses appeared toxic to macrophages. Results showed an increase in LpL gene expression with increase in C. pneumoniae multiple of infection (MOI), until a value of MOI = 5/cell. Higher MOI produced cytotoxic effect (determined by trypan blue exclusion studies). Chlamydia pneumoniae induction of LpL mRNA gene up-regulation is accompanied by enhanced LpL activity. To investigate if up-regulation of LpL gene is followed by translation into gene product, the specific activity of LpL was measured. Referring to Fig. 7, the specific activity of lipoprotein lipase in C. pneumoniae infected macrophages is depicted. Chlamydia pneumoniae doses (MOI=l-5) produced enhanced enzyme activity with higher activity recorded at MOI=5. Further higher MOI produced cytotoxicity. Results show a C. pneumoniae dose dependent enhancement of LpL activity with a significant difference from enzyme activity obtained in uninfected macrophages, thereby implying a gene upregulation accompanied by enhanced generation of gene product.

Example 5 - Chlamydia pneumoniae Lipopolysaccharide (cLPS) is the Active Moiety Responsible for LpL Gene Up-regulation. The enhanced expression of LpL gene in infected macrophages was early (within 3 h), considering the fact that C. pneumoniae life cycle last 48 to 72 h. It was therefore desirable to investigate if LPS component of C. pneumoniae was responsible. Results showed that when C. pneumoniae was heated to destroy protein content and preserve the LPS component, patterns were equivalent to those obtained with live C. pneumoniae. Referring to Fig. 8, the role of Chlamydia lipopolysaccharide (cLPS) as the moiety responsible for enhanced LpL gene expression in Chlamydia pneumoniae infected macrophage is represented. Chlamydia pneumoniae was heat killed by boiling to annul the effect of its protein without destroying lipopolysaccharide content. The cLPS alone

( — ♦ — ) induced LpL gene expression significantly compared to cLPS effect on cells treated with anti-CD 14 (blocker of LPS receptor sites on macrophages) ( — A — ), at PO.005. Live Chlamydia pneumoniae ( — ■ — ) induced LpL gene expression was higher than that induced by cLPS but not statistically significant. The pretreatment of cells with anti-CD14 abrogated the cLPS mediated LpL expression. This is significant compared to finding in Fig. 6 where cells untreated with anti-CD 14 produced higher LpL upregulation.

Example 6 - Ca²⁺ Influx Signals Activate LpL Gene Expression in C. pneumoniae Infected Macrophages. Lipoprotein lipase gene expression in C. pneumoniae infected macrophages was rapid, it was therefore reasoned that a signaling event may be operating. The effect of free intracellular Ca²⁺ on LpL mRNA gene expression was assessed using the intracellular Ca²⁺ chelating agent presented as l,2-bis(o-aminophenoxy) ethane-NNiN'N'-tetraacetic acid tetra (acetoxy methyl) ester (BAPTA-AM) which permeates plasma membrane and its acetoxymethyl (AM) group is cleaved off by cellular esterase to release the calcium chelator. In uninfected macrophages, BAPTA-AM dose dependently induced a decline in LpL mRNA gene expression. This differed significantly from results obtained in similarly treated cells but infected with C. pneumoniae. Fig. 9A depicts the regulatory role of free intracellular Ca in LpL gene expression in Chlamydia pneumoniae infected macrophages compared to uninfected cells. Application of varying doses of BAPTA-AM simultaneously with Chlamydia pneumoniae infection ampoule (— ■ — ) produced a significantly different LpL gene expression compared with similarly treated macrophage but uninfected ( — ♦ — ), P<0.005. In those cells the capacity of C. pneumoniae to induce

LpL gene expression was replicated at low BAPTA-AM dose, this was followed by a decline which was not significant despite increased BAPTA-AM doses. Thus, despite the Ca²⁺ chelating characteristics of BAPTA, C. pneumoniae was able to induce Ca²⁺ influx signals in macrophages. This finding is consistent with previous observation (Azenabor and Chaudhry, 2003b), but in this instance, Ca²⁺ influx signal resulting in enhanced LpL expression was greater than those observed in uninfected cells. To further explore the relevance of intracellular Ca²⁺ in LpL gene expression, the time course of LpL gene expression was related to time course of Ca²⁺ influx signals. Ca²⁺ influx signaling effect on LpL gene expression in Ca²⁺ rich medium is shown in Fig

9B. Chlamydia pneumoniae infected cells ( — ■ — ) produced significantly different LpL gene at an optimal Ca²⁺ influx signal of 303 nM compared to uninfected cells ( — ♦ — ) at

P<0.005. Higher Ca²⁺ influx mediated by Chlamydia pneumoniae did not produced correspondingly higher LpL gene expression. Results showed that an optimum Ca²⁺ influx signal (303 nM) favored LpL gene expression. When this Ca²⁺ signal was exceeded, a decline sets in. Thus a biphasic LpL expression in relation to Ca²⁺ influx signal is evident. The effect of L-type Ca²⁺ channel antagonists 50 μM methoxyverapamil or 20 μM nifedipine on Chlamydia pneumoniae infected cells compared to Chlamydia pneumoniae infected cells but without Ca²⁺ channel blocker treatment is depicted in Fig. 9. Referring to Fig. 10, cells infected with Chlamydia pneumoniae but untreated with Ca²⁺ channel antagonist ( — A — ) produced significantly higher LpL gene expression PO.005, compared to cells infected with Chlamydia pneumoniae but treated with verapamil

( — O — ), or nifedipine ( — D — ). These results show that a free Ca²⁺ influx signal is required for LpL gene expression in C. pneumoniae infected macrophages.

Example 7 - Chlamydia pneumoniae Induced LpL Upregulation in Macrophages Accounts for Unregulated LDL Uptake. Since the LpL gene is up-regulated in C. pneumoniae infected macrophages, it was determined if this observation could adequately account for unregulated LDL uptake in C. pneumoniae infected macrophages. A drug Calphostin-C (Beauchamp and Renier, 2002), which inhibits LpL gene expression by way of inhibition of protein kinase C signaling process, was used at a minimal dose of 0.5 μg/ml along with C. pneumoniae inoculum to prevent the upregulatory effect of C. pneumoniae on LpL. Interestingly, Calphostin-C prevented LDL uptake in such experimental set up, producing results comparable to those obtained in uninfected cells, but differing significantly from results obtained when C. pneumoniae infected macrophages were exposed to LDL or when an inducer of LpL - homocysteine was used to activate LpL in macrophages and cells were subsequently exposed to LDL. Referring to Fig. 11, the assessment of LDL uptake mediated by upregulated LpL in Chlamydia pneumoniae infected macrophage is represented. Chlamydia pneumoniae infected cells ( — ■ — ) exhibited significant uptake of BODIPY- labeled LDL, producing result comparable to those obtained for homocysteine treated cells ( — • — ), but different from results obtained for cells treated with the inhibitor of LpL activity Calphostin C but infected with Chlamydia pneumoniae ( — Δ — ) or uninfected cells ( — ♦ — ). Thus, up-regulation of the LpL gene in C. pneumoniae infected macrophages enhanced LDL uptake.

Example 8 - Regulated Ca²⁺ oscillation induced abbreviated NO generation in macrophages stimulated with 17β-estradiol. This example examines the generation of NO in macrophages treated with varying doses of intracellular Ca²⁺ chelator BAPTA-AM and stimulated with 117mM 17β- estradiol, or 20 μg/ml LPS, or lOμM ICI 182,780. Since a significant aim of this study is that an suitable Ca²⁺ signal may be correlated with iNOS gene expression, the effect of Ca²⁺ on generation of NO in macrophages stimulated with 17β-estradiol was examined by differentially chelating [Ca²⁺]i using BAPTA-AM, thus creating varying [Ca²⁺]i oscillation. With respect to the results shown in Fig. 12A, macrophages were pre- incubated with BAPTA-AM prepared in RPMI 1640 media at indicated concentration, and after 30 min. Some wells were further treated with ICI 182,780 (— ■ — ). The residual medium was withdrawn and replaced with either estradiol (-Δ-), or LPS (positive control) (-•-)_> or untreated (baseline) (-♦-). The cells were incubated for 5 min and NO determined. Values are mean ± SEM from three separate experiments, each performed in duplicate (n=6). Fig. 12A shows that 17β-estradiol induced NO release depending on the amount of free [Ca²⁺]i. There was however evidence that NO release was greatest at lOμM BAPTA- AM treatment. At greater concentration, there was a decline in NO generation. These findings are consistent with those obtained when macrophages were treated with 20μg/ml of lipopolysaccharide and significantly different from NO release in untreated macrophages. The monitoring of intracellular NO levels was terminated at 20μM BAPTA- AM dose per well since higher BAPTA-AM dose was toxic to cells (assessed by trypan blue exclusion studies). However, the application of estradiol receptor antagonist ICI 182,780 resulted in a decline in NO release, a finding consistent with the involvement of estradiol receptor in the signaling process. As shown in Fig. 12B, significant difference was obtained at

PO.05 (t-test) when estradiol or LPS treated cells are compared to untreated cells in similar treatment, [Ca²⁺]i levels were determined for untreated (-♦-), or ICI 182,780 treated (— ■ — ), or Estradiol treated (-Δ-), or LPS treated cells(-»-). Lower doses of

BAPTA-AM which produced higher [Ca²⁺]i concentration, also produced little NO generation, while higher BAPTA-AM level beyond lOμM appears to attenuate NO generation, thereby strongly suggesting that lOμM BAPTA-AM, approximately equivalent to 237nM Ca²⁺ may be the within the range of [Ca²⁺]i levels that are correlated with the largest levels of NO generation. This finding is supported by results obtained with lipopolysaccharide control. Significantly, the binding effect of BAPTA-AM on [Ca²⁺]i appears to exert some Ca ⁺ influx effect. This effect however is significantly aggravated by the additional Ca ⁺ ionophore characteristics of estradiol or lipopolysaccharide. It is such development that accounts for significant increase in [Ca ⁺]i observed at 3μM and 5μM BAPTA-AM. This effect is not sustained at higher BAPTA-AM doses (lOμM and 15μM) probably because these doses provided excess intracellular BAPTA (unbound to Ca²⁺), thus causing any freshly influxed Ca²⁺ to be chelated by excess BAPTA within the cell, resulting in the decline in free cytosolic Ca²⁺ despite the effect of estradiol. This is also replicated in untreated cells exposed to higher BAPTA-AM concentration only. The observation that estradiol/estradiol receptor interaction elicited the observed [Ca²⁺]i elevation, followed by a decline in Ca²⁺ influx when ICI 182,780 was applied, shows a link between estradiol, Ca²⁺ influx and NO release. The effectiveness of the data in Figs. 12A and 12B with regards to the relationship between [Ca²⁺]i levels and NO generation in macrophages stimulated with 17β-estradiol in Ca²⁺ rich medium is illustrated in Fig. 12C. Fig. 1C shows a superimposition plot of Ca²⁺ on BAPTA doses and iNOS release in macrophage either exposed to 117mM estradiol (-Δ-), or unexposed cells(-*-). Here, [Ca²⁺]i is superimposed on NO released from macrophages treated with varying doses of BAPTA-AM and exposed to 117nM estradiol.

Example 9 - Modulation of NO in 17β-estradiol - stimulated macrophages is not due to attenuation of NO by O2^". In order to verify if the decline in NO released in macrophages stimulated with higher estradiol concentrations resulted from the quenching effect of O₂ ^" due to peroxynitrite formation, the time course release of NO in macrophages pretreated with lOμM, or 15μM, or 20μM BAPTA-AM and stimulated with 117nM of estradiol was measured and compared with NO released in similarly treated cells but also pretreated with lμM allopurinol (xanthine oxidase inhibitor) and apocynin (NADPH-oxidase inhibitor). Macrophage cell line RAW-264.7 cells were incubated with or without lμM (allopurinol) and 100 μM (apocynin) as inhibitors of xanthine oxidase and NADPH oxidase respectively for 1 h, then residual medium aspirated and replace with medium containing 117nM estradiol. Nitric oxide levels were assayed. In Fig. 13,results are depicted of time-dependent release of NO in macrophages exposed to 117nM estradiol after pretreatment with lOμM BAPTA-AM (-A-), or 117nM estradiol after pretreatment with lOμM BAPTA-AM and blocking of xanthine oxidase and NADPH-oxidase activities

(-Δ-), or 117nM estradiol after pretreatment with 15μM BAPTA-AM (-■-), or 117nM estradiol after pretreatment with 15μM BAPTA-AM and blocking of xanthine oxidase and NADPH oxidase activities (-D-), or exposure to 117nM estradiol after pretreatment with 20μM BAPTA-AM (-♦-), or exposure to 117nM estradiol after pretreatment with 20μM BAPTA-AM and blocking of xanthine oxidase and NADPH-oxidase (-0-). Values are means ± SEM from three separate experiments, each performed in duplicate (n=6) and significant at PO.05 (student t-test). Results in Fig. 13 show a consistent but transient increase in NO released. It was unaffected by blocking of ^"O₂ ^" generation, implying that [Ca²⁺]i influx may have a pivotal role in the modulation of NO generation. The conclusion that differences in levels of [Ca²⁺]i regulates the release of NO is evident since BAPTA-AM concentration of lOμM produced NO levels which are significantly different from levels obtained at 20μM BAPTA-AM.

Example 10 - 17β-Estradiol Up-regulates iNOS Gene Expression in Macrophages at an Appropriate Ca²⁺ Level (237nM). The pattern of induction of NO reported in Figs. 12 and 13 imply that iNOS gene may be up-regulated by estradiol. To examine this suggestion, macrophage [Ca²⁺]i was regulated to concentration commensurate with 237nM or 700nM by treating cells with lOμM or 4.5μM BAPTA-AM respectively, and the time course iNOS mRNA gene expression was determined. Macrophages were pre-incubated with 4.5 μM or 10 μM BAPTA-AM for 30 min, then residual medium was withdrawn and replaced with estradiol doses (15.3nM, or 58.5nM, or 117nM). Inducible nitric oxide synthase was assayed at the time indicated. Time-dependent expression of iNOS gene in macrophages treated with low concentration of BAPTA-AM (4.5 μM) and exposed to 15.3nM estradiol (-0-), or 58.5nM estradiol (-□-), or 117nM estradiol (-Δ-), or optimum concentration (10 μM) of BAPTA-AM and exposed to 15.3nM estradiol (-♦-), or 58.5nM estradiol (-■-), or 117nM estradiol (-A-) is plotted in Fig. 14. Values are means ± SEM from three separate experiments, each performed in duplicate (n=6). Significant differences was at P<0.05 (t- test) between either cells treated with 4.5 μM BAPTA-AM or those treated with 10 μM BAPTA-AM and exposed to varying doses of estradiol. The results in Fig. 14 show that lOμM BAPTA-AM chelation of [Ca²⁺]i produced appropriate Ca ⁺ for up-regulation of iNOS while 4.5μM BAPTA-AM caused insignificant up-regulation.

Example 11 - Ca²⁺ channel inhibitors down regulate iNOS gene expression. Since [Ca²⁺]i influx had a significant influence on iNOS gene expression, the possible role of inhibition of the specialized macrophage L-type Ca²⁺ channel on iNOS gene in estradiol stimulated macrophages was studied. Inhibition of macrophage membrane L-type Ca²⁺ channel was performed using 20 μM nifedipine, residual medium was aspirated and replaced with doses of estradiol and at the indicated time, iNOS was measured. Referring to Fig. 15 A, results are plotted of the induction of iNOS gene in macrophage pretreated with Ca²⁺ channel inhibitors and stimulated with varying doses of estradiol 15.3nM (-♦-), or 58.5nM (-■-), or 117nM (-A -). Results of Fig. 15A compared with results of Fig. 14 show that 20μM nifedipine down-regulated iNOS gene despite exposure to estradiol, thus validating the observation that Ca²⁺ influx signals mediated by 17β-estradiol is the modulator of NO generation. There was however a significant difference in estradiol dose response, with 117nM estradiol producing higher up-regulation than 58.5nM. Therefore, it was examined if intracellular Ca²⁺ stores channel operations had some contributory role in iNOS gene expression. In Fig. 15B, the effect of simultaneous treatment of macrophage with L-type Ca²⁺ channel blocker 20μM nifedipine and intracellular Ca²⁺ store inhibitor 7μM SKF- 96365 on iNOS gene expression in macrophages stimulated with 15.3nM estradiol (-♦-), or 58.5nM estradiol (-■-), or 117nM estradiol (- A -) dose is reported. Values are mean ± SEM from three separate experiments, each performed in duplicate (n=6). Significant differences was reported at P<0.05 (student t-test). The combined effects of nifedipine and SKF-96365 (intracellular Ca²⁺ store channel inhibitor) showed a drastic down- regulation of iNOS, thus confirming the fact that estradiol induction of iNOS gene is not only dependent on extracellular Ca²⁺ influx signal but intracellular stores channel operations.

Example 12 - 17β-Estradiol upregulates macrophage iNOS protein expression. The induction of iNOS gene in estradiol treated macrophages showed that an appropriate [Ca²⁺]i level results in expression of the gene. To detennine if the genes expressed are translated into proteins, iNOS protein expression was demonstrated by Western blot. The expression of iNOS protein in macrophages exposed to estradiol after differential chelation of [Ca²⁺]i is depicted in Fig. 16. At lOμM BAPTA-AM, iNOS protein expression was higher than results obtained in cells not treated with BAPTA-AM, or treated with 15μM, or 20μM BAPTA-AM. Estradiol receptor antagonist ICI 182,780 abrogated the effect of 17β-etradiol. Cells treated with lOμM BAPTA-AM showed optimum iNOS protein expression compared to those treated with 15μM BAPTA-AM, or 20μM BAPTA-AM, or those untreated. This implies an adequate translation of upregulated genes to proteins. The fact that 17β-estradiol is the mediator of the signal responsible for the observed events was demonstrated by the lack of measurable iNOS protein expression in the experiments involving estradiol receptor antagonist ICI 182,780. The embodiments described above are intended to be illustrative and not limiting.

Additional embodiments are within the claims. Although the present invention has been described with reference to particular embodiments, workers skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention.

References These references are incorporated herein by reference in their entirety as well as for any specific disclosure noted herein.

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Claims

What is claimed is:

1. A composition comprising calcium channel blocker and an antibiotic wherein the calcium channel blocker is present in a therapeutically effective amount to enhance the efficacy of the antibiotic against a chronic bacterial infection.

2. The composition of claim 1 wherein the calcium channel blocker comprises a L- type calcium channel blocker.

3. The composition of claim 2 wherein the calcium channel blocker is selected from the group consisting of dihydropyridines, phenylalkylammes, benzothiazepines and combinations thereof.

4. The composition of claim 2 wherein the calcium channel blocker is selected from the group consisting of nifedipine, felodipine, isradipine, amlodipine, ditiazem, verapamil and combinations thereof.

5. The composition of claim 1 wherein the antibiotic is selected from the group consisting of tetracyclines, macrolides, quinolones, chloramphenicol, rifamycins, sulfonamides, co-trimoxazole, oxazolidinones and combinations thereof.

6. The composition of claim 1 wherein the calcium channel blocker is associated with a controlled release agent.

7. The composition of claim 6 wherein the calcium channel blocker is formulated with the controlled release agent to have approximately zero order release kinetics.

8. The composition of claim 1 further comprising a pharmacologically acceptable carrier.

9. The composition of claim 8 wherein the pharmacologically acceptable carrier comprises a solid.

10. The composition of claim 8 wherein the pharmacologically acceptable carrier comprises a liquid.

11. A medicinal tablet comprising the composition of claim 1.

12. A medicinal liquid comprising the composition of claim 1.

13. An aerosol dispenser comprising the composition of claim 1.

14. A dose of medicinal composition comprising a selected quantity of a calcium channel blocker formulated with a time release agent wherein the quantity of calcium channel blockers is selected to administer an appropriate amount of calcium channel blockers to enhance intracellular NO production for a period of time for a patient within a prescribed weight range following introduction into the patient.

15. The dose of medicinal composition of claim 14 wherein the calcium channel blocker is an L-type calcium channel blocker.

16. The dose of medicinal composition of claim 15 wherein the calcium channel blocker is selected from the group consisting of dihydropyridines, phenylalkylamines, benzothiazepines and combinations thereof.

17. The dose of medicinal composition of claim 14 wherein the composition releases the calcium channel blocker within a typical human patient with approximately zero order release kinetics over at least an hour.

18. The dose of medicinal composition of claim 14 wherein the composition releases the calcium channel blocker within a typical human patient with approximately zero order release kinetics over at least three hours.

19. The dose of medicinal composition of claim 14 wherein the medicament has from about 0.5 mg to about 100 mg of calcium channel blockers.

20. The dose of medicinal composition of claim 14 wherein the time release agent is formulated with the calcium channel blocker to maintain the calcium channel blocker at a vascular concentration within a prescribed range for a period of time within a patent having a weight within a specific range.

21. The dose of medicinal composition of claim 14 wherein the composition stimulated NO up-regulation within an average human patient.

22. The dose of medicinal composition of claim 14 further comprising an antibiotic.

23. The dose of medicinal composition of claim 21 wherein the antibiotic is selected from the group consisting of tetracyclines, macrolides, quinolones, chloramphenicol, rifamycins, sulfonamides, co-trimoxazole, oxazolidinones and combinations thereof.

24. A method for treating a chronic bacterial infection, the method comprising co- administering a therapeutically effective amount of calcium channel blocker and an antibiotic agent.

25. The method of claim 24 wherein the therapeutically effective amount of calcium channel blocker produces a synergistic improvement in the efficacy of the antibiotic agent.

26. The method of claim 24 wherein the chronic bacterial infection is caused by intracellular bacteria.

27. The method of claim 24 wherein the chronic bacterial infection is caused by Chlamydia.

28. The method of claim 24 wherein the administering of the therapeutically effective amount of calcium channel blocker and an antibiotic agent is performed by delivering a medicament comprising a calcium channel blocker and an antibiotic.

29. The method of claim 28 wherein delivering the medicament is performed through oral ingestion.

30. The method of claim 29 wherein the oral injection involved the taking of a tablet.

31. The method of claim 28 wherein delivering the medicament is performed through an injection.

32. The method of claim 28 wherein delivering the medicament is performed through inhalation of an aerosol.

33. The method of claim 24 wherein the administering of the therapeutically effective amount of calcium channel blocker and an antibiotic agent is performed by delivering two medicaments.

34. A method for formulating a composition comprising a calcium channel blocker with a time-release agent, the method comprising selecting a formulation of the calcium channel blocker with the time-release agent to maintain the concentration of calcium channel blocker within a patient's blood vessels within a selected range for a period of time wherein the patient has a weight within a prescribed range.

35. The method of claim 34 wherein the vascular concentrations of calcium channel blockers is selected to yield up-regulation of intracellular NO production.

36. The method of claim 34 wherein the vascular concentration is selected to produce an increase in intracellular NO concentration of at least about 20 percent relative to values without the calcium channel blocker.

37. The method of claim 34 wherein the calcium channel blocker comprises L-type calcium channel blocker.

38. The method of claim 37 wherein the L-type calcium channel blocker comprises dihydropyridines, phenylalkylamines, benzothiazepines and combinations thereof.

39. The method of claim 34 wherein the composition further comprises an antibiotic.

40. The method of claim 34 wherein the formulation is placed within a tablet.

41. A method for the treatment of atherosclerosis, the method comprising administering a therapeutically effective amount of calcium channel blocker and an antibiotic agent to a patient exhibiting symptoms of atherosclerosis.

42. The method of claim 41 wherein the calcium channel blocker is an L-type calcium channel blocker.

43. A kit comprising calcium channel blocker or an antibiotic along with instructions for the administration of calcium channel blocker and an antibiotic for the treatment of a chronic bacterial infection and associated conditions.

44. The kit of claim 43 comprising a calcium channel blocker and an antibiotic each stored in separate containers.

45. The kit of claim 43 further comprising a package holding the calcium channel blocker or antibiotic, and the instructions.