- Email: [email protected]
Chlamydia pneumoniae and Chronic Skin Wounds: A Focused Review
Chlamydia pneumoniae and Chronic Skin Wounds: A Focused Review Lloyd E. King Jr,* Charles W. Stratton,² and William M. Mitchell²
Departments of *Medicine (Dermatology) and ²Pathology, and *Nashville Veterans Administration Medical Centers, Nashville, Tennessee, U.S.A.
The genus, Chlamydophilia, as obligate intracellular pathogens, induce chronic scarring in humans. Chlamydia pneumoniae, a common cause of pneumonia, infects endothelial cells and circulating macrophages. Evidence that C. pneumoniae is an opportunistic pathogen in chronic skin ulcers and
other in¯ammatory skin conditions analogous to its role in atherosclerosis is reviewed. Key words: Chlamydia/chronic skin ulcers/diabetes mellitus/vasculopathy. Journal of Investigative Dermatology Symposium Proceedings 6:233±237, 2001
C
CHLAMYDIA PNEUMONIAE ± ``INNOCENT BYSTANDER'', INDICATOR OF PRIOR INFECTION, OR PERSISTENT INTRACELLULAR PATHOGEN?
hronic skin ulcers have common characteristics that include intermittent or persistent in¯ammation, infection, and the failure to heal (Lazarus et al, 1994; Mostow, 1994; Phillips, 1994; Margolis, 1996, 1999a; Eaglstein et al, 1997). A nonhealing wound may be due to genetic, environmental, or idiopathic factors, or an imbalance of bacteria (Robson, 1997). Bacterial colonization of stage 2±4 skin ulcers is assumed to be present irrespective of etiology, so infection is a potential source of in¯ammation in all ulcers such as pressure sores. How and if such in¯ammation sustains the most common types of chronic skin ulcers is controversial and needs further clari®cation. The possible role(s) of Chlamydia pneumoniae in initiating or sustaining chronic skin conditions, including skin ulcers, has not been extensively investigated. Abrams et al (1999) claimed that 12 of 27 patients with cutaneous T cell lymphoma had a C. pneumoniae-associated protein that activated Sezary T cells. Vannucci et al (2000) detected C. pneumoniae serologically in a diabetic patient with pyoderma gangrenosum-like lesions that responded dramatically to antibiotics directed against Chlamydia. Sams et al (2001) identi®ed by serologic, immunohistochemical, and culture methods C. pneumoniae in a patient with pyoderma gangrenosum that responded to prolonged antichlamydial antibiotic therapy with decreases in anti-C. pneumoniae antibody titers. Serologic evidence of C. pneumoniae was retrospectively detected by PCR and anti-C. pneumoniae IgG and IgM methods in 13 of 20 patients with a clinical diagnosis of pyoderma gangrenosum (King et al, unpublished observations). King et al (2001) also cultured C. pneumoniae from foot ulcer specimens from four of nine diabetic patients. We therefore propose that C. pneumoniae is present in chronic skin conditions both as an ``innocent bystander'' and as an opportunistic pathogen capable of maintaining in¯ammation.
Numerous studies have detected the presence of C. pneumoniae in endovascular structures as well as peripheral blood mononuclear cells (Maass et al, 1998; Boman et al, 1998; Blasi et al, 1999). The presence of viable C. pneumoniae in peripheral blood mononuclear cells suggests that C. pneumoniae may accompany these white blood cells to in¯amed tissue sites and cause a secondary infection in the in¯amed tissue; however, these studies do not in and of themselves prove a direct pathogenic role for C. pneumoniae in atherosclerosis, stroke, or other vasculopathies. For example, the prevalence rate of C. pneumoniae in cardiovascular atheroma differing human populations varies from very low (0%; Weiss et al, 1996; Lindholt et al, 1998) to very high (>90%; Jackson et al, 1997) in selected populations. Recent approaches focus on ®nding reliable prognostic tests such as immunoreactive proteins and lipopolysaccharide (LPS) or PCR detectable DNA and RNA from C. pneumoniae that would be indicative of active infection that is pathogenetically highly relevant (Blasi et al, 1999; Shor and Phillips, 1999; Russell, 1999). The data variability is due to the multiple dif®culties in culturing C. pneumoniae (Maass and Dalhoff, 1995; Maass and Harig, 1995) and technical problems with immunohistochemical methods (Taylor-Robinson and Thomas, 1998). Maximizing the detection of reaction product by antigen retrieval strategies using immunochemical methods may be necessary in some antiC. pneumoniae antibody staining procedures in formalin-®xed tissues as ®xation can denature antigenic epitopes of C. pneumoniae. MICROBIOLOGIC FEATURES OF CHLAMYDIA The genus, Chlamydophilia, Chlamydiaceae is rapidly expanding and now includes nine species, ®ve of which were recently been added in 1999 (see below). The various Chlamydia species are readily distinguished by analysis of signature sequences in the 16S and 23S ribosomal genes (Everett et al, 1999; Kalman et al, 1999). Two species of Chlamydia are pathogenic in humans (C. pneumoniae, C. trachomatis) and seven are primarily pathogenic in other vertebrates (four species of C. psittacci, C. pecorum, C. muradarum sp. nov, and C. suis sp. nov) (Everett et al, 1999). Chlamydia trachomatis induces conjunctivitis, keratitis and is the most common preventable cause of blindness worldwide (Ward, 1995). These organisms also cause
Manuscript received February 14, 2001; revised May 18, 2001; accepted for publication June 8, 2001. Reprint requests to: Dr. Lloyd E. King Jr, Division of Dermatology, 3900 The Vanderbilt Clinic, Nashville, TN 37232. Email: [email protected] Abbreviation: CP, Chlamydia pneumoniae. 1087-0024/01/$15.00
´ Copyright # 2001 by The Society for Investigative Dermatology, Inc. 233
234
KING ET AL
pelvic in¯ammatory disease, endometrosis, vaginitis, urethritis, and infertility. Chlamydia pneumoniae is a common pathogen in acute human upper and lower respiratory infections worldwide (Ward, 1995). As a genus the Chlamydia are obligate intracellular microorganisms that cause chronic and relapsing diseases in humans and other animals. Chlamydia have a unique biphasic life cycle with functionally and morphologically distinct dimorphic forms. The extracellular form, the elementary body, is infectious but metabolically inactive. After endocytosis, elementary bodies differentiate into the metabolic active reticulate body that replicates by binary ®ssion. Chlamydiae have been thought to be intracellular ATP scavengers as they contain nucleotide transport proteins and lack many enzymes of the electron transport chain necessary for de novo ATP biosynthesis (Stratton and Mitchell, 1997); however, recent data indicate that enough glucose-catabolizing enzymes are present in Chlamydia that have the functional capacity to produce some of their own ATP and reducing power (Tjaden et al, 1999; Iliffe-Lee and McClarty, 1999). The pathogenic signi®cance of this recent ®nding is unclear as Chlamydiae are known to stimulate glucose transport into the host cell to compensate for the extra energy load on the infected cell (Ojicius et al, 1998). Moreover, cells infected with C. pneumoniae have depleted energy and thus may be dysfunctional (Shemer-Arni and Lieberman, 1995). PATHOBIOLOGY OF CHLAMYDIAL INFECTIONS Chlamydia pneumoniae is a common cause of pneumonia in humans (Grayston et al, 1986) and is associated with in¯ammatory arthritis and atherosclerosis (see below). The antibody response to C. pneumoniae increases with age as measured in otherwise unselected human populations (Grayston, 1992). Since its discovery just over a decade ago, C. pneumoniae has been associated with a number of chronic diseases including some presumed to be autoimmune, such as multiple sclerosis (Sriram et al, 1998, 1999; Stratton et al, 2000) and Reiter's disease (Stratton, 1998; Gerard et al, 1999). Chlamydial infections including those caused by C. pneumoniae are remarkable because of their persistence in tissues. This feature has been best studied in humans with trachoma that has periodic exacerbations and remissions with progressive conjunctival in¯ammation and corneal scarring (Beatty et al, 1994). Chlamydia trachomatis is detected in the quiescent phase of trachoma even in the absence of histologic and immunologic features of in¯ammation. The mechanism(s) of chlamydial-induced tissue injury is unclear but likely is relatively unique compared with gram(+) and gram(±) bacteria. Its lipopolysaccharide, lipid A, has unique structural features that induce only minimal endotoxin effects (Kosma, 1999; Rund et al, 1999). Chlamydia induce in¯ammation and clotting abnormalities, but no known chlamydial toxins have been positively identi®ed likely to be responsible for their tissue injury-inducing effects. Read et al (2000) reported a potential type III secreted toxin with homology to the Shiga toxin produced by E. coli 0157:H7. Chlamydia have type III secretion mechanisms that enable it to secrete and inject pathogenicity proteins into the cytosol of eukaryotic host cells, although the identity of the proteins secreted into the cytosol are unknown (Hackstadt et al, 1997; Braavoil and Hsia, 1998; Hueck, 1998). Whether the in vivo activation of the immune system by Chlamydia in vivo is due to heat shock proteins (Kol et al, 1998; LaVerda et al, 1999), type III secreted proteins (see above, Braavoil and Hsia, 1998), or other T cell activation mechanisms (Igieseme, 1996) is unknown. CHLAMYDIA PNEUMONIAE AND INFLAMMATION Signal transduction pathways are activated in endothelial cells following infection with C. pneumoniae (Krull et al, 1999). Infection of human endothelial cells with C. pneumoniae stimulates transendothelial migration of neutrophils and monocytes (Moazed et al, 1998; Molestina et al, 1999). Chlamydia pneumoniae infection of vascular smooth muscle and endothelial cells activates NF-kB and induces tissue factor and plasminogen activator inhibitor 1 (PAI-1)
JID SYMPOSIUM PROCEEDINGS
expression (Dechend et al, 1999). Other pathogenic mechanisms that may be involved include chlamydial heat shock proteins (Kol et al, 1998; LaVerda et al, 1999) and Toll receptors that are involved in the innate immune responses (Muzio et al, 2000). Endothelial cytotoxicity immune reactions mediated by serum antibodies to heat shock proteins of Escherichia coli and C. pneumoniae and other outer membrane proteins, were proposed as a possible link between infection and atherosclerosis (Christiansen et al, 1999; Mayr et al, 1999). Chlamydial heat shock protein 60, and potentially outer membrane proteins (Christiansen et al 1999), are involved in the localization to and in human atheromas and regulates macrophage tumor necrosis factor-a and matrix metalloproteinase expression (Kol et al, 1998; Christiansen et al, 1999). A speci®c C. pneumoniae lipopolysaccharide, lipid A, induces macrophage foam cell formation (Kalayoglu and Byrne, 1998). A plausible explanation for the chronic and relapsing nature of C. pneumoniae infection despite antibiotic therapy was lacking until recently. Chlamydia pneumoniae antigens remain accessible to immunocytes for at least 4 wk and are capable of sustaining in¯ammation when no viable C. pneumoniae are present (Wyrick et al, 1999). CHLAMYDIA PNEUMONIAE, VASCULAR INFLAMMATION, AND ATHEROSCLEROSIS Atherosclerosis is now classi®ed as an in¯ammatory disease (Ross, 1999) so potentially it could be induced or modulated by an infectious agent. The association of C. pneumoniae with vascular disease was ®rst demonstrated serologically in a Finnish population (Saikku et al, 1988). Chlamydia pneumoniae was ®rst identi®ed as a respiratory pathogen by Grayston et al (1986), It was con®rmed as a common cause of respiratory disease that was commonly detected in asymptomatic healthy adults (Hyman et al, 1995). This organism was ®rst directly associated with atherosclerotic lesions in South African patients (for a review see Shor and Phillips, 1999). Recent evidence indicates that C. pneumoniae and potentially other microbes are associated with atherosclerosis in cardiovascular and cerebrovascular diseases (for reviews see Danesh et al, 1997; Cheng et al, 1998; Epstein et al, 1999; Chiu, 1999; Grayston and Campbell, 1999; Leinonen and Saikku, 1999; Bartels et al, 2000). Convincing data that other infectious agents such as Mycoplasma pneumoniae and Helicobacter pylori may be involved are not available (Blasi et al, 1996). In contrast, culture, serologic, immunocytochemical, polymerase chain reaction (PCR), in situ hybridization, and transmission electron microscopy data combined with epidemiologic data show that C. pneumoniae is detected in a statistically signi®cant number of samples from human arterial tissues including peripheral vessels (Shor and Phillips, 1999). Chlamydia pneumoniae infection is detectable in patients with cerebrovascular accidents (stroke) (Cook et al, 1998) as well as Alzheimer's disease (Balin et al, 1998) and diabetic nephropathy (Kanuchi et al, 2000). Therefore, C. pneumoniae is likely to be widely prevalent in the vasculature. PATHOGENETIC SIGNIFICANCE OF ATHEROSCLEROSIS AND C. PNEUMONIAE IN DIABETICS It has been claimed that the endovascular presence of C. pneumoniae DNA is a generalized phenomenon in atherosclerotic vascular disease (Maass et al, 1998). These data are intriguing as atherosclerosis is the underlying cause of chronic limb ischemia and often is the cause of chronic ulcers in many patients. The progression of atherosclerosis is accelerated by the coexistence of hypertension, lipoprotein abnormalities, tobacco addiction, and diabetes mellitus (Kempczinski and Bernhard, 1995). Although atherosclerosis is not qualitatively different in diabetics, it appears at an earlier age and progresses more rapidly. Peripheral vascular disease (PVD) in diabetics most commonly affects the tibial, popliteal, and profunda femoris arteries rather than the aorta and iliac arteries (Kempczinski and Bernhard, 1995). Atherosclerosis is generally assumed to be the cause of chronic ischemia of the lower extremities, including diabetic foot ulcers (Chait and Bierman, 1994). There are very few
VOL. 6, NO. 3 DECEMBER 2001
collateral vessels in the lower leg so even nonsegmental occlusion of the distal and proximal tibial arteries may result in gangrene or severe ischemia requiring arterial reconstruction (Kempczinski and Bernhard, 1995). IS C. PNEUMONIAE LIKELY TO BE THE ONLY PATHOGEN INVOLVED IN ATHEROSCLEROSIS? Multiple infections of atherosclerotic plaques may be present (Epstein et al, 1999; Chiu, 1999; Zhu et al, 2000). Serum levels of high-sensitivity C reactive protein (hs-CRP), an acute phase reactant, correlated well with the total pathogen burden or number of chronic endogenous pathogens and coronary artery risk (Zhu et al, 2000). In a much larger study, the levels of hs-CRP were as accurate a predictor of coronary artery disease in a long-term study of the coronary risk factors as cholesterol and lipid pro®le in a population of nurses (Ricker et al, 2000). DETECTING C. PNEUMONIAE IN CHRONIC LEG AND DIABETIC FOOT ULCERS The previous inability to detect these organisms may be due to the dif®culty of recovering viable C. pneumoniae organisms that require special methods for their isolation and propagation (Stephens, 1992). Studies on C. pneumoniae were aided by the discovery of a human lung cancer cell line, HL, that allowed isolation and propagation of the TWAR strain of C. pneumoniae (Kuo and Grayston, 1990). Chlamydia pneumoniae persistently infects and replicates in epithelium, endothelium, smooth muscle cells, macrophages (Sriram et al, 2000), and neural tissue in vivo and in vitro (Balin et al, 1998; Sriram et al, 1998, 1999; Stratton et al, 2000). The ability to isolate and propagate these organisms in HL cells (Kuo and Grayston, 1990; Pruckler et al, 1999; Sriram et al, 2000) requires additional centrifugation steps and a 7 d culture time, resulting in a 500±5000-fold increase in the number of detectable inclusion-forming units. As punch or excisional biopsies are relatively contraindicated in patients with evolving or resolving diabetic foot ulcers, routine curettage samples of diabetic foot ulcers are especially helpful as a standard source for culture. SOURCE OF C. PNEUMONIAE IN CHRONIC SKIN ULCERS If C. pneumoniae is to be considered as a source of infection for chronic skin ulcers, then the origin of the C. pneumoniae infection is germane. Chlamydia pneumoniae is common in the environment (``innocent bystander''), dif®cult to eradicate, and may be spread by contact with seemingly healthy humans (Hyman et al, 1995). Alternatively and, we believe, most likely, C. pneumoniae in chronic skin ulcers may be initiated by parasitized mononuclear cells (Airenne et al, 1999). In a mouse model, C. pneumoniae infectivity was demonstrated to be mediated by parasitized monocytes (Moazed et al, 1998). A possibility that has not been previously considered is that extracellular elementary bodies may be noncovalently bound or attached to circulating RBC via interaction with membrane molecules such as heparin. IS A SPECIFIC SKIN TYPE CELL THE SOURCE OF C. PNEUMONIAE IN CHRONIC SKIN ULCERS? Chlamydia pneumoniae was detected in human keratinocytes, endothelial cells, and histiocytes that contained intracellular inclusions stained with anti-MOMP (outer membrane protein) and antiLPS (Abrams et al, 1999). These authors con®rmed the presence of C. pneumoniae DNA and RNA in skin by PCR and RT-PCR and productively infected keratinocytes in vitro with C. pneumoniae (Abrams et al, 1999). Recently, we detected C. pneumoniae serologically and by PCR in chronic cutaneous ulcers in a diabetic who responded dramatically to appropriate antibiotic therapy (Vanucci et al, 2000). We cultured C. pneumoniae from chronic
CHLAMYDIA PNEUMONIAE AND CHRONIC SKIN WOUNDS
235
skin wounds in diabetic and nondiabetic patients such as those with pyoderma gangrenosum (King et al, 2001; Sams et al, 2001). DOES C. PNEUMONIAE INFECTION PREDIPOSE PATIENTS TO DEVELOP CHRONIC SKIN ULCERS? This is the fundamental question that underlies the debate over whether C. pneumoniae is an ``innocent bystander'' or an opportunistic pathogen (see reviews cited above). As demonstrated by the con¯icting data and opinions over the role of C. pneumoniae in atherosclerosis and coronary artery disease, it is important to ®rst document that an active infection by C. pneumoniae is unequivocally present at the site of tissue damage. Similarly, the ability to document the evolving pathologic responses or nonresponses to C. pneumoniae is critical to interpretation of epidemiologic data. Studies on the vasculopathy of coronary arteries, the aorta, and the abdominal aorta are severely limited by their accessibility for de®nitive biopsies and long-term follow-up. Epstein Barr virus, Cytomegalovirus, Herpes simplex type I and II, and potentially other members of the herpes virus family as well as Hepatitis A virus were proposed as opportunistic agents or copathogens for chronic vasculopathies (Zhu et al, 2000). Antibody titers, PCR titers, and culture results do not always directly correlate especially with patients receiving high doses of immunosuppresants (Sams and King, unpublished data). No published data are currently available to document the presence of chronic C. pneumoniae skin infections in diabetics or other causes of chronic leg ulcers so more study is required to evaluate this possibility. CHLAMYDIA PNEUMONIAE, SKIN INFECTIONS, OR ULCERATIONS AND DIABETES No direct data are available to con®rm that there is an increase in C. pneumoniae skin infections in the chronic leg ulcer population; however, there are known associations between DM and atherosclerosis as well as between C. pneumoniae and atherosclerosis (Muhlestein, 1998; Shor and Phillips, 1999). Indirect evidence suggests that increased blood glucose due to DM may help sustain persistent C. pneumoniae infections to compensate for the extra energy load on infected cells (Ojicius et al, 1998). Chlamydial infections increase glucose consumption, lactate production, glutamate synthesis, glycogen accumulation, and an associated increase expression of the glucose transporter, GLUT-1, in vitro (Ojcius et al, 1998). Conversely, diabetics are assumed to be very susceptible to infections so that it is not unreasonable to assume that C. pneumoniae are among the unusual and/or opportunistic pathogens that might be detected in some diabetics with chronic ulcers. There may be an association, moreover, between chronic C. pneumoniae infections and diabetic nephropathy based upon antiC. pneumoniae serum IgG antibody titers in an ELISA (Kanuchi et al, 2000). CHLAMYDIA PNEUMONIAE, SERUM LIPIDS, DM, AND ATHEROSCLEROSIS Abnormal cholesterol and lipoproteins are major risk factors associated with atherosclerosis in diabetic as well as and nondiabetic patients. Atherosclerotic plaques contain foam cells and evidence of altered lipid composition (Ross, 1999). Animal models (Moazed et al, 1997; Saikku et al, 1998; Muhlestein et al, 1998; Fong et al, 1999) show that C. pneumoniae can induce atheromas and mimic in¯ammatory processes present in human atherosclerosis. Elevated C. pneumoniae antibody titers were associated with altered serum lipid pro®le in humans (Laurila et al, 1998). In vitro cellular oxidation of low-density lipoproteins (LDL) is induced by C. pneumoniae (Kalayoglu et al, 1999). The atherogenic effects of Chlamydia are claimed to be dependent on serum cholesterol and speci®c to C. pneumoniae (Hu et al, 1999).
236
KING ET AL
JID SYMPOSIUM PROCEEDINGS
THERAPY Published data to con®rm the ef®cacy of chronic antibiotic therapy in the treatment of coronary artery disease to eliminate chronic infection are inconclusive. Chlamydia pneumoniae use monocytes as a transport for systemic dissemination and enter a persistent state not covered by an otherwise effective antichlamydial treatment, so prevention of vascular infection by such treatment may be problematic (Gieffers et al, 2001). Which antibiotic(s) to use in treating atherosclerosis is a moot point as multiple infectious agents may be involved and induce an unacceptably high pathogen burden (Chiu, 1999; Zhu et al, 2000). Chlamydia pneumoniae infected cells also may be more resistant to apoptosis than cells infected with other pathogens (Geng et al, 2000). Although chronic antibiotic therapy did alter the clinical course of multiple skin ulcers with serologically documented C. pneumoniae infection with (Vannuci et al, 2000) and without diabetes (Sams et al, 2001), it is not clear how many other patients would respond. Standard treatment of chronic venous ulcers or diabetic foot ulcers may not heal them within 12 wk (Margolis et al, 1999b), so long-term, cost-effective therapy may be required. The ef®cacy and cost-effectiveness of speci®c regimens for chronic skin ulcers will likely be a productive research area. REFERENCES Abrams JT, Vonderheid EC, Kolbe S, Appelt DM, Arking EJ, Bailin BJ: Sezary Tcell activating factor is a Chlamydia pneumoniae-associated protein. Clin Diagnostic Lab Immunol 6:895±905, 1999 Airenne S, Surcel H-M, Alakarppa H, Laitinen K, Paavonen J, Saikku P, Laurila A: Chlamydia pneumoniae infection in human monocytes. Infect Immun 67:1445± 1449, 1999 Balin BJ, Gerard HC, Arking EJ, et al: Identi®cation and localization of Chlamydia pneumoniae in the Alzheimer's brain. Med Microbiol Immunol 187:23±42, 1998 Bartels C, Maass M, Gregor B, et al. Association of serology with the endovascular presence of Chlamydia pneumoniae and cytomegalovirus in coronary artery and vein graft disease. Circ 101:137±141, 2000 Beatty WL, Morrison RP, Byrne GI: Persistent Chlamydiae from cell culture to a paradigm for chlamydial pathogenesis. Microbiol Rev 58:686±699, 1994 Blasi F, Denti F, Erba M, et al: Detection of Chlamydia pneumoniae and not of Helicobacter pylori in atherosclerotic plaques of aortic aneurysms. J Clin Microbiol 34:2766±2769, 1996 Blasi F, Boman J, Esposito G, et al: Chlamydia pneumoniae DNA detection in peripheral blood mononuclear cells is predictive of vascular infection. J Infect Dis 180:2074±2076, 1999 Boman J, Soderber S, Forsberg J, et al: High prevalence of Chlamydia pneumoniae DNA in peripheral blood mononuclear cells in patients with cardiovascular diseases and in middle-aged blood donors. J Infect Dis 178:274±277, 1998 Braavoil PM, Hsia R-C: Type III secretion in Chlamydia. Mol Microbiol 28:860±862, 1998 Chait A, Bierman EL: Pathogenesis of macrovascular disease in diabetes. In: CR Kahn, GC Weir, eds. Joslin's Diabetes Mellitus, 13th edn. Philadelphia: Lea & Febiger, 1994, pp 648±664 Cheng JW, Rivera NG: Infection and atherosclerosis ± focus on cytomegalovirus and Chlamydia pneumoniae. J Clin Pathol 51:793±797, 1998 Chiu B: Multiple infections in carotid atherosclerotic plaques. Am Heart J 138 (Suppl. 2):S534±S536, 1999 Christiansen G, Boesen T, Hjerno K, et al: Molecular biology of Chlamydia pneumoniae surface proteins and their role in immunopathogenicity. Am Heart J 138 (Suppl. 2):S491±S495, 1999 Cook PJ, Honeybourne D, Lip GY, et al: Chlamydia pneumoniae antibody titers are signi®cantly associated with acute stroke and transient cerebral ischemia. The West Birmingham Stroke Project. Stroke 29:404±410, 1998 Danesh J, Collins R, Peto R: Chronic infections and coronary heart disease: is there a link? Lancet 350:430±436, 1997 Dechend R, Maass M, Gieffers J, Dieta R, Scheidereit C, Leutz A, Gulba DC: Chlamydia pneumoniae infection of vascular smooth muscle and endothelial cells activates NF-kappa B and induces tissue factor and PAI-1 expression: a potential link to accelerated arteriosclerosis. Circ 100:1369±1373, 1999 Eaglstein WH, Falanga V: Chronic wounds. Surgl Clin NA 77:689±700, 1997 Epstein SE, Zhou YF, Zhu J: Infection and atherosclerosis: emerging mechanistic paradigms. Circ 100:20±28, 1999 Everett KD, Bush RM, Andersen AA: Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam.nov. & Simkaniaceae fam.nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and ®ve new species, and standards for the identi®cation of organisms. Int J System Bacteriol 49 (Suppl. 2):415±440, 1999 Fong IW, Chiu B, Viira E, et al: Can an antibiotic (macrolide) prevent Chlamydia pneumoniae-induced atherosclerosis in a rabbit model? Clin Diag Lab Immunol 6:891±894, 1999 Geng Y, Shane RB, Berencsi K, et al: Chlamydia pneumoniae inhibits apoptosis in
human peripheral blood mononuclear cells through induction of IL-10. J Immunol 164:5522±5529, 2000 Gerard HC, Wang GF, Balin BJ, et al: Frequency of apolipoprotein E (APOE) allele types in patients with Chlamydia-associated arthritis and other arthritides. Microb Pathogen 26:35±43, 1999 Gieffers J, Fullgraf H, Jahn J, et al: Chlamydia pneumoniae infection in circulating monocytes is refractory to antibiotic treatment. Circ 103:351±356, 2001 Grayston JT: Infection caused by Chlamydia pneumoniae, strain TWAR. Clin Infect Dis 15:757±761, 1992 Grayston JT, Campbell LA: The role of Chlamydia pneumoniae in atherosclerosis. Clin Infect Dis 28:993±994, 1999 Grayston JR, Kuo CC, Wang SP, Altman J: A new Chlamydia psittaci strain, TWAR, isolated in acute respiratory tract infections. N Eng J Med 315:161±168, 1986 Hackstadt T, Fischer ER, Scidmore MA, Rockeey DR, Heinzen RA: Origins and functions of the chlamydial inclusion. Trends Microbiol 5:288±293, 1997 Hu H, Pierce GN, Zhong G: The atherogenic effects of Chlamydia are dependent on serum cholesterol and speci®c to Chlamydia pneumoniae. J Clin Invest 103:747± 753, 1999 Hueck CJ: Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Molec Biol Rev 62:379±433, 1998 Hyman CL, Roblin CA, Gaydos CA, Quinn TC, Schachter J, Hammerschlag MR: Prevalence of asymptomatic nasopharyngeal carriage of Chlamydia pneumoniae in subjectively healthy adults: assessment by polymerase chain reaction, enzyme immunoassay and culture. Clin Infect Dis 20:1174±1178, 1995 Igieseme JU: The molecular mechanism of T-cell control of Chlamydia in mice: role of nitric oxide. Immunol 87:1±8, 1996 Iliffe-Lee ER, McClarty G: Glucose metabolism in Chlamydia trachomatis: the ``energy parasite'' hypothesis revisited. Mol Microbiol 33:177±187, 1999 Jackson LA, Campbell LA, Schmidt RA, et al: Speci®city of detection of Chlamydia pneumoniae in cardiovascular atheroma: evaluation of the innocent bystander hypothesis. Am J Pathol 150:1785±1790, 1997 Kalayoglu MV, Byrne GIA: Chlamydia pneumoniae component that induces macrophage foam cell formation is chlamydial lipopolysaccaride. Infect Immun 66:5067±5072, 1998 Kalayoglu MV, Hoerneman B, LaVerda D, Morrison SG, Morrison RP, Byrne GI: Cellular oxidation of low-density lipoprotein by Chlamydia pneumoniae. J Infect Dis 180:780±790, 1999 Kalman S, Mitchell W, Marathe R, et al: Comparative genomes of C. trachomatis and C. pneumoniae. Nat Genet 21:385±389, 1999 Kanuchi M, Kawano T, Dohi K: Association of C. pneumoniae infection with diabetic nephropathy. Diab Res Clin Pract 47:45±48, 2000 Kempczinski RF, Bernhard VM: Management of chronic ischemia of the lower extremities. Introduction and general considerations. In: RB Rutherford, ed. Vascular Surgery, 4th edn. Philadelphia: W.B. Saunders, 1995, pp 741±751 King LE Jr, Bushman T, Stratton CW, Mitchell WM: Diabetic foot ulcers and Chlamydia pneumoniae. Innocent bystander of opportunistic pathogen? Arch Dermatol 127:561±562, 2001 Kol A, Sukhove GK, Lichtman AH, Libby P: Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factoralpha and matrix metalloproteinase expression. Circ 198:300±307, 1998 Kosma P: Chlamydial lipopolysaccharide. Biochim Biophysica Acta 1455:387±402, 1999 Krull M, Klucken AC, Wuppermann FN, et al: Signal transduction pathways activated in endothelial cells following infection with Chlamydia pneumoniae. J Immuno 162:4834±4841, 1999 Kuo CC, Grayston JT: A sensitive cell line, HL cells, for isolation and propagation of Chlamydia pneumoniae strain TWAR. J Infect Dis 162:755±758, 1990 Laurila AL, Bloigu A, Nayha S, Hassi J, Leinonen M, Saikku P: Chlamydia pneumoniae antibodies associated with altered serum lipid pro®le. Int J Circumpolar Health 57 (Suppl. 1):329±332, 1998 LaVerda D, Kalayoglu MV, Byrne GI: Chlamydial heat shock proteins and disease pathology: new paradigms for old problems? Infect Dis Obstet Gynecol 7:64±71, 1999 Lazarus GS, Cooper DM, Knighton DR, et al: De®nitions and guidelines for assessment of wounds and evaluation of healing. Arch Dermatol 130:489±493, 1994 Leinonen M, Saikku P: Interaction of Chlamydia pneumoniae infection with other risk factors of atherosclerosis. Am Heart J 138 (Suppl. 2):S504±S506, 1999 Lindholt JS, Ostergard L, Henneberg EW, et al: Failure to demonstrate Chlamydia pneumoniae in symptomatic abdominal aneurysms by a nested polymerase chain reaction (PCR). Eur J Vasc Endovasc Surg 15:161±164, 1998 Maass M, Dalhoff K: Comparison of sample preparation methods for detection of Chlamydia pneumoniae in broncheoalveolar lavage ¯uid by PCR. J Clin Microbiol 32:2616±2619, 1994 Maass M, Harig U: Evaluation of culture conditions used for isolation of Chlamydia pneumoniae. Am J Clin Pathol 103:141±148, 1995 Maass M, Bartels C, Kruger, S, Krause E, Engel PM, Dalhoff K: Endovascular presence of Chlamydia pneumoniae DNA is a generalized phenomenon in atherosclerotic vascular disease. Atherosclerosis 140 (Suppl. 1):S25±S30, 1998 Margolis DJ, Berlin JA, Strom BL: Reliability and validity of the clinical interpretation of a healed chronic wound. Wound Repair Regeneration 4:335± 338, 1996 Margolis DJ, Berlin JA, Strom BL: Risk factors associated with the failure of a venous leg ulcer to heal. Arch Dermatol 135:920±926, 1999a Margolis DJ, Kantor J, Berlin JA: Healing of diabetic neuropathic foot ulcers receiving standard treatment. Diabetes Care 22:692±695, 1999b Mayr M, Metzler B, Kiechl S, Willeit J, Schett G, Xu Q, Wick G: Endothelial
VOL. 6, NO. 3 DECEMBER 2001
cytotoxicity mediated by serum antibodies to heat shock proteins of Escherichia coli and Chlamydia pneumoniae: immune reactions to heat shock proteins as a possible link between infection and atherosclerosis. Circ 99:1560±1566, 1999 Moazed TC, Kuo C, Grayston JT, Campbell LA: Murine models of Chlamydia pneumoniae infection and atherosclerosis. J Infect Dis 175:883±890, 1997 Moazed TC, Kuo CC, Grayston JL, Campbell LA: Evidence of systemic dissemination of Chlamydia pneumoniae via macrophages in the mouse. J Infect Dis 177:1322±1325, 1998 Molestina RE, Miller RD, Ramirez JA, Summersgill JT: Infection of human endothelial cells with Chlamydia pneumoniae stimulates transendothelial migration of neutrophils and monocytes. Infect Immun 67:1323±1330, 1999 Mostow EN: Diagnosis and classi®cation of chronic wounds. Clin Dermatol 12:3±11, 1994 Muhlestein JB: Bacterial infections and atherosclerosis. J Invest Med 46:396±402, 1998 Muhlestein JB, Anderson JL, Hammond EH, et al: Infection with Chlamydia pneumoniae accelerates the development of atherosclerosis and treatment with azithromycin prevents it in a rabbit model. Circ 97:633±636, 1998 Muzio M, Polentanrutti, Bosisio D, et al. Toll-like receptors a growing family of immune receptors that are differentially expressed and regulated by different leukocytes. J Leukoc Biol 67:450±456, 2000 Ojcius DM, Degani H, Mispelter J, Dautry-Varsat A: Enhancement of ATP levels and glucose metabolism during infection by Chlamydia: NMR studies of living cells. J Biol Chem 273:7052±7058, 1998 Philips TJ: Chronic cutaneous ulcers: etiology and epidemiology. J Invest Dermatol 102 (Suppl.):38S±41S, 1994 Pruckler JM, Masse N, Stevens VA, et al: Optimizing culture of Chlamydia pneumoniae by using multiple centrifugations. J Clin Microbiol 37:3399±3401, 1999 Read TD, Brunham RC, Shen C, et al: Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39. Nucl Acids Res 28:1397±1406, 2000 Ricker PM, Hennekens CH, Buring JE, Rifai N: C-reactive protein and other markers of in¯ammation in the prediction of cardiovascular disease in women. N Eng J Med 342:836±843, 2000 Robson MC: Wound infection: a failure of wound healing caused by an imbalance of bacteria. Surg Clin NA 77:637±650, 1997 Ross R: Atherosclerosis an in¯ammatory disease. N Engl J Med 340:115±126, 1999 Rund S, Lindner B, Brade H, Holst O: Structural analysis of the lipopolysaccharide from Chlamydia trachomatis serotype L2. J Biol Chem 274:16819±16824, 1999 Russell EG: Evaluation of two serological tests for the diagnosis of chlamydial respiratory diseases. Pathol 31:403±405, 1999 Saikku P, Laitinen K, Leinonen M: Animal models for Chlamydia pneumoniae infection. Atherosclerosis 140 (Suppl. 1):S17±S19, 1998 Saikku P, Leinonen M, Mattila K, et al: Serological evidence of an association of a novel Chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction. Lancet 2:983±986, 1988
CHLAMYDIA PNEUMONIAE AND CHRONIC SKIN WOUNDS
Sams
237
HH, Stratton CW, Mitchell WM, King LE Jr: Culture and immunohistochemical identi®cation of Chlamydia pneumoniae in ulcerative pyoderma gangrenosum. J Am Acad Dermatol, 2001, in press Shemer-Arni Y, Lieberman D: Chlamydia pneumoniae-induced ciliostasis in ciliated bronchial epithelial cells. J Infect Dis 171:1274±1278, 1995 Shor A, Phillips JI: Chlamydia pneumoniae and atherosclerosis. J Am Med Assoc 282:2071±2073, 1999 Sriram SC, Stratton Mitchell W: Multiple sclerosis associated with Chlamydia pneumoniae infection of the CNS. Neurol 50:571±572, 1998 Sriram S, Stratton CW, Yao S, Ding L, Bannan J, Mitchell WM: Chlamydia pneumoniae infection of the central nervous system in multiple sclerosis. Ann Neurol 46:6±14, 1999 Sriram S, Stratton CW, Yao S-Y, Tharp A, Ding L, Bannan JD, Mitchell WM: Reply to Cann et al. Chlamydia, Rickettsia, and antibiotic treatment of multiple sclerosis. Ann Neurol 47:409±411, 2000 Stephens RS: Challenge of Chlamydia research. Infect Agents Dis 6:279±293, 1992 Stratton CW: Role of Chlamydia pneumoniae in connective tissue diseases. AFC 2 (Suppl. 2):S31±S38, 1998 Stratton CW, Mitchell WM: The pathogenesis of systemic Chlamydial infections: theoretical considerations of host cell energy depletion and its metabolic consequences. Antimicrobics Infectious Dis Newsletter 16:33±38, 1997 Stratton CW, Mitchell WM, Sriram S: Does Chlamydia pneumoniae play a role in the pathogenesis of multiple sclerosis? J Med Microbiol 49:1±3, 2000 Taylor-Robinson D, Thomas BJ: Chlamydia pneumoniae in arteries: the facts, their interpretation and future studies. J Clin Pathol 51:793±797, 1998 Tjaden J, Winkler HH, Schwoppe C, et al: Two nucleoside transport proteins in C. trachomatis, one for net nucleoside triphosphate uptake and the other for transport of energy. J Bacteriol 181:1196±1202, 1999 Vannucci SA, Mitchell WM, Stratton CW, King LE Jr: Pyoderma gangrenosum and Chlamydia pneumoniae infection in a diabetic man: pathogenic role or coincidence? J Am Acad Dermatol 42:295±297, 2000 Ward ME: The immunobiology and immunopathology of chlamydial infections. APMIS 103:769±796, 1995 Weiss SM, Roblin PM, Gaydos CA, et al: Failure to detect Chlamydia pneumoniae in coronary atheromas of patients undergoing atherectomy. J Infect Dis 173:957± 962, 1996 Wyrick PB, Knight ST, Paul TR, Rank RG, Barbie CS: Persistent chlamydial envelope antigens in antibiotic-exposed infected cells trigger neutrophil chemotaxsis. J Infect Dis 179:954±966, 1999 Zhu J, Quyyumi AA, Norman JE, Csako G, Waclawiw MA, Shearer GM, Epstein SE: Effects of total pathogen burden on coronary artery risk and C-reactive protein levels. Am J Cardiol 85:140±146, 2000
Departments of *Medicine (Dermatology) and ²Pathology, and *Nashville Veterans Administration Medical Centers, Nashville, Tennessee, U.S.A.
The genus, Chlamydophilia, as obligate intracellular pathogens, induce chronic scarring in humans. Chlamydia pneumoniae, a common cause of pneumonia, infects endothelial cells and circulating macrophages. Evidence that C. pneumoniae is an opportunistic pathogen in chronic skin ulcers and
other in¯ammatory skin conditions analogous to its role in atherosclerosis is reviewed. Key words: Chlamydia/chronic skin ulcers/diabetes mellitus/vasculopathy. Journal of Investigative Dermatology Symposium Proceedings 6:233±237, 2001
C
CHLAMYDIA PNEUMONIAE ± ``INNOCENT BYSTANDER'', INDICATOR OF PRIOR INFECTION, OR PERSISTENT INTRACELLULAR PATHOGEN?
hronic skin ulcers have common characteristics that include intermittent or persistent in¯ammation, infection, and the failure to heal (Lazarus et al, 1994; Mostow, 1994; Phillips, 1994; Margolis, 1996, 1999a; Eaglstein et al, 1997). A nonhealing wound may be due to genetic, environmental, or idiopathic factors, or an imbalance of bacteria (Robson, 1997). Bacterial colonization of stage 2±4 skin ulcers is assumed to be present irrespective of etiology, so infection is a potential source of in¯ammation in all ulcers such as pressure sores. How and if such in¯ammation sustains the most common types of chronic skin ulcers is controversial and needs further clari®cation. The possible role(s) of Chlamydia pneumoniae in initiating or sustaining chronic skin conditions, including skin ulcers, has not been extensively investigated. Abrams et al (1999) claimed that 12 of 27 patients with cutaneous T cell lymphoma had a C. pneumoniae-associated protein that activated Sezary T cells. Vannucci et al (2000) detected C. pneumoniae serologically in a diabetic patient with pyoderma gangrenosum-like lesions that responded dramatically to antibiotics directed against Chlamydia. Sams et al (2001) identi®ed by serologic, immunohistochemical, and culture methods C. pneumoniae in a patient with pyoderma gangrenosum that responded to prolonged antichlamydial antibiotic therapy with decreases in anti-C. pneumoniae antibody titers. Serologic evidence of C. pneumoniae was retrospectively detected by PCR and anti-C. pneumoniae IgG and IgM methods in 13 of 20 patients with a clinical diagnosis of pyoderma gangrenosum (King et al, unpublished observations). King et al (2001) also cultured C. pneumoniae from foot ulcer specimens from four of nine diabetic patients. We therefore propose that C. pneumoniae is present in chronic skin conditions both as an ``innocent bystander'' and as an opportunistic pathogen capable of maintaining in¯ammation.
Numerous studies have detected the presence of C. pneumoniae in endovascular structures as well as peripheral blood mononuclear cells (Maass et al, 1998; Boman et al, 1998; Blasi et al, 1999). The presence of viable C. pneumoniae in peripheral blood mononuclear cells suggests that C. pneumoniae may accompany these white blood cells to in¯amed tissue sites and cause a secondary infection in the in¯amed tissue; however, these studies do not in and of themselves prove a direct pathogenic role for C. pneumoniae in atherosclerosis, stroke, or other vasculopathies. For example, the prevalence rate of C. pneumoniae in cardiovascular atheroma differing human populations varies from very low (0%; Weiss et al, 1996; Lindholt et al, 1998) to very high (>90%; Jackson et al, 1997) in selected populations. Recent approaches focus on ®nding reliable prognostic tests such as immunoreactive proteins and lipopolysaccharide (LPS) or PCR detectable DNA and RNA from C. pneumoniae that would be indicative of active infection that is pathogenetically highly relevant (Blasi et al, 1999; Shor and Phillips, 1999; Russell, 1999). The data variability is due to the multiple dif®culties in culturing C. pneumoniae (Maass and Dalhoff, 1995; Maass and Harig, 1995) and technical problems with immunohistochemical methods (Taylor-Robinson and Thomas, 1998). Maximizing the detection of reaction product by antigen retrieval strategies using immunochemical methods may be necessary in some antiC. pneumoniae antibody staining procedures in formalin-®xed tissues as ®xation can denature antigenic epitopes of C. pneumoniae. MICROBIOLOGIC FEATURES OF CHLAMYDIA The genus, Chlamydophilia, Chlamydiaceae is rapidly expanding and now includes nine species, ®ve of which were recently been added in 1999 (see below). The various Chlamydia species are readily distinguished by analysis of signature sequences in the 16S and 23S ribosomal genes (Everett et al, 1999; Kalman et al, 1999). Two species of Chlamydia are pathogenic in humans (C. pneumoniae, C. trachomatis) and seven are primarily pathogenic in other vertebrates (four species of C. psittacci, C. pecorum, C. muradarum sp. nov, and C. suis sp. nov) (Everett et al, 1999). Chlamydia trachomatis induces conjunctivitis, keratitis and is the most common preventable cause of blindness worldwide (Ward, 1995). These organisms also cause
Manuscript received February 14, 2001; revised May 18, 2001; accepted for publication June 8, 2001. Reprint requests to: Dr. Lloyd E. King Jr, Division of Dermatology, 3900 The Vanderbilt Clinic, Nashville, TN 37232. Email: [email protected] Abbreviation: CP, Chlamydia pneumoniae. 1087-0024/01/$15.00
´ Copyright # 2001 by The Society for Investigative Dermatology, Inc. 233
234
KING ET AL
pelvic in¯ammatory disease, endometrosis, vaginitis, urethritis, and infertility. Chlamydia pneumoniae is a common pathogen in acute human upper and lower respiratory infections worldwide (Ward, 1995). As a genus the Chlamydia are obligate intracellular microorganisms that cause chronic and relapsing diseases in humans and other animals. Chlamydia have a unique biphasic life cycle with functionally and morphologically distinct dimorphic forms. The extracellular form, the elementary body, is infectious but metabolically inactive. After endocytosis, elementary bodies differentiate into the metabolic active reticulate body that replicates by binary ®ssion. Chlamydiae have been thought to be intracellular ATP scavengers as they contain nucleotide transport proteins and lack many enzymes of the electron transport chain necessary for de novo ATP biosynthesis (Stratton and Mitchell, 1997); however, recent data indicate that enough glucose-catabolizing enzymes are present in Chlamydia that have the functional capacity to produce some of their own ATP and reducing power (Tjaden et al, 1999; Iliffe-Lee and McClarty, 1999). The pathogenic signi®cance of this recent ®nding is unclear as Chlamydiae are known to stimulate glucose transport into the host cell to compensate for the extra energy load on the infected cell (Ojicius et al, 1998). Moreover, cells infected with C. pneumoniae have depleted energy and thus may be dysfunctional (Shemer-Arni and Lieberman, 1995). PATHOBIOLOGY OF CHLAMYDIAL INFECTIONS Chlamydia pneumoniae is a common cause of pneumonia in humans (Grayston et al, 1986) and is associated with in¯ammatory arthritis and atherosclerosis (see below). The antibody response to C. pneumoniae increases with age as measured in otherwise unselected human populations (Grayston, 1992). Since its discovery just over a decade ago, C. pneumoniae has been associated with a number of chronic diseases including some presumed to be autoimmune, such as multiple sclerosis (Sriram et al, 1998, 1999; Stratton et al, 2000) and Reiter's disease (Stratton, 1998; Gerard et al, 1999). Chlamydial infections including those caused by C. pneumoniae are remarkable because of their persistence in tissues. This feature has been best studied in humans with trachoma that has periodic exacerbations and remissions with progressive conjunctival in¯ammation and corneal scarring (Beatty et al, 1994). Chlamydia trachomatis is detected in the quiescent phase of trachoma even in the absence of histologic and immunologic features of in¯ammation. The mechanism(s) of chlamydial-induced tissue injury is unclear but likely is relatively unique compared with gram(+) and gram(±) bacteria. Its lipopolysaccharide, lipid A, has unique structural features that induce only minimal endotoxin effects (Kosma, 1999; Rund et al, 1999). Chlamydia induce in¯ammation and clotting abnormalities, but no known chlamydial toxins have been positively identi®ed likely to be responsible for their tissue injury-inducing effects. Read et al (2000) reported a potential type III secreted toxin with homology to the Shiga toxin produced by E. coli 0157:H7. Chlamydia have type III secretion mechanisms that enable it to secrete and inject pathogenicity proteins into the cytosol of eukaryotic host cells, although the identity of the proteins secreted into the cytosol are unknown (Hackstadt et al, 1997; Braavoil and Hsia, 1998; Hueck, 1998). Whether the in vivo activation of the immune system by Chlamydia in vivo is due to heat shock proteins (Kol et al, 1998; LaVerda et al, 1999), type III secreted proteins (see above, Braavoil and Hsia, 1998), or other T cell activation mechanisms (Igieseme, 1996) is unknown. CHLAMYDIA PNEUMONIAE AND INFLAMMATION Signal transduction pathways are activated in endothelial cells following infection with C. pneumoniae (Krull et al, 1999). Infection of human endothelial cells with C. pneumoniae stimulates transendothelial migration of neutrophils and monocytes (Moazed et al, 1998; Molestina et al, 1999). Chlamydia pneumoniae infection of vascular smooth muscle and endothelial cells activates NF-kB and induces tissue factor and plasminogen activator inhibitor 1 (PAI-1)
JID SYMPOSIUM PROCEEDINGS
expression (Dechend et al, 1999). Other pathogenic mechanisms that may be involved include chlamydial heat shock proteins (Kol et al, 1998; LaVerda et al, 1999) and Toll receptors that are involved in the innate immune responses (Muzio et al, 2000). Endothelial cytotoxicity immune reactions mediated by serum antibodies to heat shock proteins of Escherichia coli and C. pneumoniae and other outer membrane proteins, were proposed as a possible link between infection and atherosclerosis (Christiansen et al, 1999; Mayr et al, 1999). Chlamydial heat shock protein 60, and potentially outer membrane proteins (Christiansen et al 1999), are involved in the localization to and in human atheromas and regulates macrophage tumor necrosis factor-a and matrix metalloproteinase expression (Kol et al, 1998; Christiansen et al, 1999). A speci®c C. pneumoniae lipopolysaccharide, lipid A, induces macrophage foam cell formation (Kalayoglu and Byrne, 1998). A plausible explanation for the chronic and relapsing nature of C. pneumoniae infection despite antibiotic therapy was lacking until recently. Chlamydia pneumoniae antigens remain accessible to immunocytes for at least 4 wk and are capable of sustaining in¯ammation when no viable C. pneumoniae are present (Wyrick et al, 1999). CHLAMYDIA PNEUMONIAE, VASCULAR INFLAMMATION, AND ATHEROSCLEROSIS Atherosclerosis is now classi®ed as an in¯ammatory disease (Ross, 1999) so potentially it could be induced or modulated by an infectious agent. The association of C. pneumoniae with vascular disease was ®rst demonstrated serologically in a Finnish population (Saikku et al, 1988). Chlamydia pneumoniae was ®rst identi®ed as a respiratory pathogen by Grayston et al (1986), It was con®rmed as a common cause of respiratory disease that was commonly detected in asymptomatic healthy adults (Hyman et al, 1995). This organism was ®rst directly associated with atherosclerotic lesions in South African patients (for a review see Shor and Phillips, 1999). Recent evidence indicates that C. pneumoniae and potentially other microbes are associated with atherosclerosis in cardiovascular and cerebrovascular diseases (for reviews see Danesh et al, 1997; Cheng et al, 1998; Epstein et al, 1999; Chiu, 1999; Grayston and Campbell, 1999; Leinonen and Saikku, 1999; Bartels et al, 2000). Convincing data that other infectious agents such as Mycoplasma pneumoniae and Helicobacter pylori may be involved are not available (Blasi et al, 1996). In contrast, culture, serologic, immunocytochemical, polymerase chain reaction (PCR), in situ hybridization, and transmission electron microscopy data combined with epidemiologic data show that C. pneumoniae is detected in a statistically signi®cant number of samples from human arterial tissues including peripheral vessels (Shor and Phillips, 1999). Chlamydia pneumoniae infection is detectable in patients with cerebrovascular accidents (stroke) (Cook et al, 1998) as well as Alzheimer's disease (Balin et al, 1998) and diabetic nephropathy (Kanuchi et al, 2000). Therefore, C. pneumoniae is likely to be widely prevalent in the vasculature. PATHOGENETIC SIGNIFICANCE OF ATHEROSCLEROSIS AND C. PNEUMONIAE IN DIABETICS It has been claimed that the endovascular presence of C. pneumoniae DNA is a generalized phenomenon in atherosclerotic vascular disease (Maass et al, 1998). These data are intriguing as atherosclerosis is the underlying cause of chronic limb ischemia and often is the cause of chronic ulcers in many patients. The progression of atherosclerosis is accelerated by the coexistence of hypertension, lipoprotein abnormalities, tobacco addiction, and diabetes mellitus (Kempczinski and Bernhard, 1995). Although atherosclerosis is not qualitatively different in diabetics, it appears at an earlier age and progresses more rapidly. Peripheral vascular disease (PVD) in diabetics most commonly affects the tibial, popliteal, and profunda femoris arteries rather than the aorta and iliac arteries (Kempczinski and Bernhard, 1995). Atherosclerosis is generally assumed to be the cause of chronic ischemia of the lower extremities, including diabetic foot ulcers (Chait and Bierman, 1994). There are very few
VOL. 6, NO. 3 DECEMBER 2001
collateral vessels in the lower leg so even nonsegmental occlusion of the distal and proximal tibial arteries may result in gangrene or severe ischemia requiring arterial reconstruction (Kempczinski and Bernhard, 1995). IS C. PNEUMONIAE LIKELY TO BE THE ONLY PATHOGEN INVOLVED IN ATHEROSCLEROSIS? Multiple infections of atherosclerotic plaques may be present (Epstein et al, 1999; Chiu, 1999; Zhu et al, 2000). Serum levels of high-sensitivity C reactive protein (hs-CRP), an acute phase reactant, correlated well with the total pathogen burden or number of chronic endogenous pathogens and coronary artery risk (Zhu et al, 2000). In a much larger study, the levels of hs-CRP were as accurate a predictor of coronary artery disease in a long-term study of the coronary risk factors as cholesterol and lipid pro®le in a population of nurses (Ricker et al, 2000). DETECTING C. PNEUMONIAE IN CHRONIC LEG AND DIABETIC FOOT ULCERS The previous inability to detect these organisms may be due to the dif®culty of recovering viable C. pneumoniae organisms that require special methods for their isolation and propagation (Stephens, 1992). Studies on C. pneumoniae were aided by the discovery of a human lung cancer cell line, HL, that allowed isolation and propagation of the TWAR strain of C. pneumoniae (Kuo and Grayston, 1990). Chlamydia pneumoniae persistently infects and replicates in epithelium, endothelium, smooth muscle cells, macrophages (Sriram et al, 2000), and neural tissue in vivo and in vitro (Balin et al, 1998; Sriram et al, 1998, 1999; Stratton et al, 2000). The ability to isolate and propagate these organisms in HL cells (Kuo and Grayston, 1990; Pruckler et al, 1999; Sriram et al, 2000) requires additional centrifugation steps and a 7 d culture time, resulting in a 500±5000-fold increase in the number of detectable inclusion-forming units. As punch or excisional biopsies are relatively contraindicated in patients with evolving or resolving diabetic foot ulcers, routine curettage samples of diabetic foot ulcers are especially helpful as a standard source for culture. SOURCE OF C. PNEUMONIAE IN CHRONIC SKIN ULCERS If C. pneumoniae is to be considered as a source of infection for chronic skin ulcers, then the origin of the C. pneumoniae infection is germane. Chlamydia pneumoniae is common in the environment (``innocent bystander''), dif®cult to eradicate, and may be spread by contact with seemingly healthy humans (Hyman et al, 1995). Alternatively and, we believe, most likely, C. pneumoniae in chronic skin ulcers may be initiated by parasitized mononuclear cells (Airenne et al, 1999). In a mouse model, C. pneumoniae infectivity was demonstrated to be mediated by parasitized monocytes (Moazed et al, 1998). A possibility that has not been previously considered is that extracellular elementary bodies may be noncovalently bound or attached to circulating RBC via interaction with membrane molecules such as heparin. IS A SPECIFIC SKIN TYPE CELL THE SOURCE OF C. PNEUMONIAE IN CHRONIC SKIN ULCERS? Chlamydia pneumoniae was detected in human keratinocytes, endothelial cells, and histiocytes that contained intracellular inclusions stained with anti-MOMP (outer membrane protein) and antiLPS (Abrams et al, 1999). These authors con®rmed the presence of C. pneumoniae DNA and RNA in skin by PCR and RT-PCR and productively infected keratinocytes in vitro with C. pneumoniae (Abrams et al, 1999). Recently, we detected C. pneumoniae serologically and by PCR in chronic cutaneous ulcers in a diabetic who responded dramatically to appropriate antibiotic therapy (Vanucci et al, 2000). We cultured C. pneumoniae from chronic
CHLAMYDIA PNEUMONIAE AND CHRONIC SKIN WOUNDS
235
skin wounds in diabetic and nondiabetic patients such as those with pyoderma gangrenosum (King et al, 2001; Sams et al, 2001). DOES C. PNEUMONIAE INFECTION PREDIPOSE PATIENTS TO DEVELOP CHRONIC SKIN ULCERS? This is the fundamental question that underlies the debate over whether C. pneumoniae is an ``innocent bystander'' or an opportunistic pathogen (see reviews cited above). As demonstrated by the con¯icting data and opinions over the role of C. pneumoniae in atherosclerosis and coronary artery disease, it is important to ®rst document that an active infection by C. pneumoniae is unequivocally present at the site of tissue damage. Similarly, the ability to document the evolving pathologic responses or nonresponses to C. pneumoniae is critical to interpretation of epidemiologic data. Studies on the vasculopathy of coronary arteries, the aorta, and the abdominal aorta are severely limited by their accessibility for de®nitive biopsies and long-term follow-up. Epstein Barr virus, Cytomegalovirus, Herpes simplex type I and II, and potentially other members of the herpes virus family as well as Hepatitis A virus were proposed as opportunistic agents or copathogens for chronic vasculopathies (Zhu et al, 2000). Antibody titers, PCR titers, and culture results do not always directly correlate especially with patients receiving high doses of immunosuppresants (Sams and King, unpublished data). No published data are currently available to document the presence of chronic C. pneumoniae skin infections in diabetics or other causes of chronic leg ulcers so more study is required to evaluate this possibility. CHLAMYDIA PNEUMONIAE, SKIN INFECTIONS, OR ULCERATIONS AND DIABETES No direct data are available to con®rm that there is an increase in C. pneumoniae skin infections in the chronic leg ulcer population; however, there are known associations between DM and atherosclerosis as well as between C. pneumoniae and atherosclerosis (Muhlestein, 1998; Shor and Phillips, 1999). Indirect evidence suggests that increased blood glucose due to DM may help sustain persistent C. pneumoniae infections to compensate for the extra energy load on infected cells (Ojicius et al, 1998). Chlamydial infections increase glucose consumption, lactate production, glutamate synthesis, glycogen accumulation, and an associated increase expression of the glucose transporter, GLUT-1, in vitro (Ojcius et al, 1998). Conversely, diabetics are assumed to be very susceptible to infections so that it is not unreasonable to assume that C. pneumoniae are among the unusual and/or opportunistic pathogens that might be detected in some diabetics with chronic ulcers. There may be an association, moreover, between chronic C. pneumoniae infections and diabetic nephropathy based upon antiC. pneumoniae serum IgG antibody titers in an ELISA (Kanuchi et al, 2000). CHLAMYDIA PNEUMONIAE, SERUM LIPIDS, DM, AND ATHEROSCLEROSIS Abnormal cholesterol and lipoproteins are major risk factors associated with atherosclerosis in diabetic as well as and nondiabetic patients. Atherosclerotic plaques contain foam cells and evidence of altered lipid composition (Ross, 1999). Animal models (Moazed et al, 1997; Saikku et al, 1998; Muhlestein et al, 1998; Fong et al, 1999) show that C. pneumoniae can induce atheromas and mimic in¯ammatory processes present in human atherosclerosis. Elevated C. pneumoniae antibody titers were associated with altered serum lipid pro®le in humans (Laurila et al, 1998). In vitro cellular oxidation of low-density lipoproteins (LDL) is induced by C. pneumoniae (Kalayoglu et al, 1999). The atherogenic effects of Chlamydia are claimed to be dependent on serum cholesterol and speci®c to C. pneumoniae (Hu et al, 1999).
236
KING ET AL
JID SYMPOSIUM PROCEEDINGS
THERAPY Published data to con®rm the ef®cacy of chronic antibiotic therapy in the treatment of coronary artery disease to eliminate chronic infection are inconclusive. Chlamydia pneumoniae use monocytes as a transport for systemic dissemination and enter a persistent state not covered by an otherwise effective antichlamydial treatment, so prevention of vascular infection by such treatment may be problematic (Gieffers et al, 2001). Which antibiotic(s) to use in treating atherosclerosis is a moot point as multiple infectious agents may be involved and induce an unacceptably high pathogen burden (Chiu, 1999; Zhu et al, 2000). Chlamydia pneumoniae infected cells also may be more resistant to apoptosis than cells infected with other pathogens (Geng et al, 2000). Although chronic antibiotic therapy did alter the clinical course of multiple skin ulcers with serologically documented C. pneumoniae infection with (Vannuci et al, 2000) and without diabetes (Sams et al, 2001), it is not clear how many other patients would respond. Standard treatment of chronic venous ulcers or diabetic foot ulcers may not heal them within 12 wk (Margolis et al, 1999b), so long-term, cost-effective therapy may be required. The ef®cacy and cost-effectiveness of speci®c regimens for chronic skin ulcers will likely be a productive research area. REFERENCES Abrams JT, Vonderheid EC, Kolbe S, Appelt DM, Arking EJ, Bailin BJ: Sezary Tcell activating factor is a Chlamydia pneumoniae-associated protein. Clin Diagnostic Lab Immunol 6:895±905, 1999 Airenne S, Surcel H-M, Alakarppa H, Laitinen K, Paavonen J, Saikku P, Laurila A: Chlamydia pneumoniae infection in human monocytes. Infect Immun 67:1445± 1449, 1999 Balin BJ, Gerard HC, Arking EJ, et al: Identi®cation and localization of Chlamydia pneumoniae in the Alzheimer's brain. Med Microbiol Immunol 187:23±42, 1998 Bartels C, Maass M, Gregor B, et al. Association of serology with the endovascular presence of Chlamydia pneumoniae and cytomegalovirus in coronary artery and vein graft disease. Circ 101:137±141, 2000 Beatty WL, Morrison RP, Byrne GI: Persistent Chlamydiae from cell culture to a paradigm for chlamydial pathogenesis. Microbiol Rev 58:686±699, 1994 Blasi F, Denti F, Erba M, et al: Detection of Chlamydia pneumoniae and not of Helicobacter pylori in atherosclerotic plaques of aortic aneurysms. J Clin Microbiol 34:2766±2769, 1996 Blasi F, Boman J, Esposito G, et al: Chlamydia pneumoniae DNA detection in peripheral blood mononuclear cells is predictive of vascular infection. J Infect Dis 180:2074±2076, 1999 Boman J, Soderber S, Forsberg J, et al: High prevalence of Chlamydia pneumoniae DNA in peripheral blood mononuclear cells in patients with cardiovascular diseases and in middle-aged blood donors. J Infect Dis 178:274±277, 1998 Braavoil PM, Hsia R-C: Type III secretion in Chlamydia. Mol Microbiol 28:860±862, 1998 Chait A, Bierman EL: Pathogenesis of macrovascular disease in diabetes. In: CR Kahn, GC Weir, eds. Joslin's Diabetes Mellitus, 13th edn. Philadelphia: Lea & Febiger, 1994, pp 648±664 Cheng JW, Rivera NG: Infection and atherosclerosis ± focus on cytomegalovirus and Chlamydia pneumoniae. J Clin Pathol 51:793±797, 1998 Chiu B: Multiple infections in carotid atherosclerotic plaques. Am Heart J 138 (Suppl. 2):S534±S536, 1999 Christiansen G, Boesen T, Hjerno K, et al: Molecular biology of Chlamydia pneumoniae surface proteins and their role in immunopathogenicity. Am Heart J 138 (Suppl. 2):S491±S495, 1999 Cook PJ, Honeybourne D, Lip GY, et al: Chlamydia pneumoniae antibody titers are signi®cantly associated with acute stroke and transient cerebral ischemia. The West Birmingham Stroke Project. Stroke 29:404±410, 1998 Danesh J, Collins R, Peto R: Chronic infections and coronary heart disease: is there a link? Lancet 350:430±436, 1997 Dechend R, Maass M, Gieffers J, Dieta R, Scheidereit C, Leutz A, Gulba DC: Chlamydia pneumoniae infection of vascular smooth muscle and endothelial cells activates NF-kappa B and induces tissue factor and PAI-1 expression: a potential link to accelerated arteriosclerosis. Circ 100:1369±1373, 1999 Eaglstein WH, Falanga V: Chronic wounds. Surgl Clin NA 77:689±700, 1997 Epstein SE, Zhou YF, Zhu J: Infection and atherosclerosis: emerging mechanistic paradigms. Circ 100:20±28, 1999 Everett KD, Bush RM, Andersen AA: Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam.nov. & Simkaniaceae fam.nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and ®ve new species, and standards for the identi®cation of organisms. Int J System Bacteriol 49 (Suppl. 2):415±440, 1999 Fong IW, Chiu B, Viira E, et al: Can an antibiotic (macrolide) prevent Chlamydia pneumoniae-induced atherosclerosis in a rabbit model? Clin Diag Lab Immunol 6:891±894, 1999 Geng Y, Shane RB, Berencsi K, et al: Chlamydia pneumoniae inhibits apoptosis in
human peripheral blood mononuclear cells through induction of IL-10. J Immunol 164:5522±5529, 2000 Gerard HC, Wang GF, Balin BJ, et al: Frequency of apolipoprotein E (APOE) allele types in patients with Chlamydia-associated arthritis and other arthritides. Microb Pathogen 26:35±43, 1999 Gieffers J, Fullgraf H, Jahn J, et al: Chlamydia pneumoniae infection in circulating monocytes is refractory to antibiotic treatment. Circ 103:351±356, 2001 Grayston JT: Infection caused by Chlamydia pneumoniae, strain TWAR. Clin Infect Dis 15:757±761, 1992 Grayston JT, Campbell LA: The role of Chlamydia pneumoniae in atherosclerosis. Clin Infect Dis 28:993±994, 1999 Grayston JR, Kuo CC, Wang SP, Altman J: A new Chlamydia psittaci strain, TWAR, isolated in acute respiratory tract infections. N Eng J Med 315:161±168, 1986 Hackstadt T, Fischer ER, Scidmore MA, Rockeey DR, Heinzen RA: Origins and functions of the chlamydial inclusion. Trends Microbiol 5:288±293, 1997 Hu H, Pierce GN, Zhong G: The atherogenic effects of Chlamydia are dependent on serum cholesterol and speci®c to Chlamydia pneumoniae. J Clin Invest 103:747± 753, 1999 Hueck CJ: Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Molec Biol Rev 62:379±433, 1998 Hyman CL, Roblin CA, Gaydos CA, Quinn TC, Schachter J, Hammerschlag MR: Prevalence of asymptomatic nasopharyngeal carriage of Chlamydia pneumoniae in subjectively healthy adults: assessment by polymerase chain reaction, enzyme immunoassay and culture. Clin Infect Dis 20:1174±1178, 1995 Igieseme JU: The molecular mechanism of T-cell control of Chlamydia in mice: role of nitric oxide. Immunol 87:1±8, 1996 Iliffe-Lee ER, McClarty G: Glucose metabolism in Chlamydia trachomatis: the ``energy parasite'' hypothesis revisited. Mol Microbiol 33:177±187, 1999 Jackson LA, Campbell LA, Schmidt RA, et al: Speci®city of detection of Chlamydia pneumoniae in cardiovascular atheroma: evaluation of the innocent bystander hypothesis. Am J Pathol 150:1785±1790, 1997 Kalayoglu MV, Byrne GIA: Chlamydia pneumoniae component that induces macrophage foam cell formation is chlamydial lipopolysaccaride. Infect Immun 66:5067±5072, 1998 Kalayoglu MV, Hoerneman B, LaVerda D, Morrison SG, Morrison RP, Byrne GI: Cellular oxidation of low-density lipoprotein by Chlamydia pneumoniae. J Infect Dis 180:780±790, 1999 Kalman S, Mitchell W, Marathe R, et al: Comparative genomes of C. trachomatis and C. pneumoniae. Nat Genet 21:385±389, 1999 Kanuchi M, Kawano T, Dohi K: Association of C. pneumoniae infection with diabetic nephropathy. Diab Res Clin Pract 47:45±48, 2000 Kempczinski RF, Bernhard VM: Management of chronic ischemia of the lower extremities. Introduction and general considerations. In: RB Rutherford, ed. Vascular Surgery, 4th edn. Philadelphia: W.B. Saunders, 1995, pp 741±751 King LE Jr, Bushman T, Stratton CW, Mitchell WM: Diabetic foot ulcers and Chlamydia pneumoniae. Innocent bystander of opportunistic pathogen? Arch Dermatol 127:561±562, 2001 Kol A, Sukhove GK, Lichtman AH, Libby P: Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factoralpha and matrix metalloproteinase expression. Circ 198:300±307, 1998 Kosma P: Chlamydial lipopolysaccharide. Biochim Biophysica Acta 1455:387±402, 1999 Krull M, Klucken AC, Wuppermann FN, et al: Signal transduction pathways activated in endothelial cells following infection with Chlamydia pneumoniae. J Immuno 162:4834±4841, 1999 Kuo CC, Grayston JT: A sensitive cell line, HL cells, for isolation and propagation of Chlamydia pneumoniae strain TWAR. J Infect Dis 162:755±758, 1990 Laurila AL, Bloigu A, Nayha S, Hassi J, Leinonen M, Saikku P: Chlamydia pneumoniae antibodies associated with altered serum lipid pro®le. Int J Circumpolar Health 57 (Suppl. 1):329±332, 1998 LaVerda D, Kalayoglu MV, Byrne GI: Chlamydial heat shock proteins and disease pathology: new paradigms for old problems? Infect Dis Obstet Gynecol 7:64±71, 1999 Lazarus GS, Cooper DM, Knighton DR, et al: De®nitions and guidelines for assessment of wounds and evaluation of healing. Arch Dermatol 130:489±493, 1994 Leinonen M, Saikku P: Interaction of Chlamydia pneumoniae infection with other risk factors of atherosclerosis. Am Heart J 138 (Suppl. 2):S504±S506, 1999 Lindholt JS, Ostergard L, Henneberg EW, et al: Failure to demonstrate Chlamydia pneumoniae in symptomatic abdominal aneurysms by a nested polymerase chain reaction (PCR). Eur J Vasc Endovasc Surg 15:161±164, 1998 Maass M, Dalhoff K: Comparison of sample preparation methods for detection of Chlamydia pneumoniae in broncheoalveolar lavage ¯uid by PCR. J Clin Microbiol 32:2616±2619, 1994 Maass M, Harig U: Evaluation of culture conditions used for isolation of Chlamydia pneumoniae. Am J Clin Pathol 103:141±148, 1995 Maass M, Bartels C, Kruger, S, Krause E, Engel PM, Dalhoff K: Endovascular presence of Chlamydia pneumoniae DNA is a generalized phenomenon in atherosclerotic vascular disease. Atherosclerosis 140 (Suppl. 1):S25±S30, 1998 Margolis DJ, Berlin JA, Strom BL: Reliability and validity of the clinical interpretation of a healed chronic wound. Wound Repair Regeneration 4:335± 338, 1996 Margolis DJ, Berlin JA, Strom BL: Risk factors associated with the failure of a venous leg ulcer to heal. Arch Dermatol 135:920±926, 1999a Margolis DJ, Kantor J, Berlin JA: Healing of diabetic neuropathic foot ulcers receiving standard treatment. Diabetes Care 22:692±695, 1999b Mayr M, Metzler B, Kiechl S, Willeit J, Schett G, Xu Q, Wick G: Endothelial
VOL. 6, NO. 3 DECEMBER 2001
cytotoxicity mediated by serum antibodies to heat shock proteins of Escherichia coli and Chlamydia pneumoniae: immune reactions to heat shock proteins as a possible link between infection and atherosclerosis. Circ 99:1560±1566, 1999 Moazed TC, Kuo C, Grayston JT, Campbell LA: Murine models of Chlamydia pneumoniae infection and atherosclerosis. J Infect Dis 175:883±890, 1997 Moazed TC, Kuo CC, Grayston JL, Campbell LA: Evidence of systemic dissemination of Chlamydia pneumoniae via macrophages in the mouse. J Infect Dis 177:1322±1325, 1998 Molestina RE, Miller RD, Ramirez JA, Summersgill JT: Infection of human endothelial cells with Chlamydia pneumoniae stimulates transendothelial migration of neutrophils and monocytes. Infect Immun 67:1323±1330, 1999 Mostow EN: Diagnosis and classi®cation of chronic wounds. Clin Dermatol 12:3±11, 1994 Muhlestein JB: Bacterial infections and atherosclerosis. J Invest Med 46:396±402, 1998 Muhlestein JB, Anderson JL, Hammond EH, et al: Infection with Chlamydia pneumoniae accelerates the development of atherosclerosis and treatment with azithromycin prevents it in a rabbit model. Circ 97:633±636, 1998 Muzio M, Polentanrutti, Bosisio D, et al. Toll-like receptors a growing family of immune receptors that are differentially expressed and regulated by different leukocytes. J Leukoc Biol 67:450±456, 2000 Ojcius DM, Degani H, Mispelter J, Dautry-Varsat A: Enhancement of ATP levels and glucose metabolism during infection by Chlamydia: NMR studies of living cells. J Biol Chem 273:7052±7058, 1998 Philips TJ: Chronic cutaneous ulcers: etiology and epidemiology. J Invest Dermatol 102 (Suppl.):38S±41S, 1994 Pruckler JM, Masse N, Stevens VA, et al: Optimizing culture of Chlamydia pneumoniae by using multiple centrifugations. J Clin Microbiol 37:3399±3401, 1999 Read TD, Brunham RC, Shen C, et al: Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39. Nucl Acids Res 28:1397±1406, 2000 Ricker PM, Hennekens CH, Buring JE, Rifai N: C-reactive protein and other markers of in¯ammation in the prediction of cardiovascular disease in women. N Eng J Med 342:836±843, 2000 Robson MC: Wound infection: a failure of wound healing caused by an imbalance of bacteria. Surg Clin NA 77:637±650, 1997 Ross R: Atherosclerosis an in¯ammatory disease. N Engl J Med 340:115±126, 1999 Rund S, Lindner B, Brade H, Holst O: Structural analysis of the lipopolysaccharide from Chlamydia trachomatis serotype L2. J Biol Chem 274:16819±16824, 1999 Russell EG: Evaluation of two serological tests for the diagnosis of chlamydial respiratory diseases. Pathol 31:403±405, 1999 Saikku P, Laitinen K, Leinonen M: Animal models for Chlamydia pneumoniae infection. Atherosclerosis 140 (Suppl. 1):S17±S19, 1998 Saikku P, Leinonen M, Mattila K, et al: Serological evidence of an association of a novel Chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction. Lancet 2:983±986, 1988
CHLAMYDIA PNEUMONIAE AND CHRONIC SKIN WOUNDS
Sams
237
HH, Stratton CW, Mitchell WM, King LE Jr: Culture and immunohistochemical identi®cation of Chlamydia pneumoniae in ulcerative pyoderma gangrenosum. J Am Acad Dermatol, 2001, in press Shemer-Arni Y, Lieberman D: Chlamydia pneumoniae-induced ciliostasis in ciliated bronchial epithelial cells. J Infect Dis 171:1274±1278, 1995 Shor A, Phillips JI: Chlamydia pneumoniae and atherosclerosis. J Am Med Assoc 282:2071±2073, 1999 Sriram SC, Stratton Mitchell W: Multiple sclerosis associated with Chlamydia pneumoniae infection of the CNS. Neurol 50:571±572, 1998 Sriram S, Stratton CW, Yao S, Ding L, Bannan J, Mitchell WM: Chlamydia pneumoniae infection of the central nervous system in multiple sclerosis. Ann Neurol 46:6±14, 1999 Sriram S, Stratton CW, Yao S-Y, Tharp A, Ding L, Bannan JD, Mitchell WM: Reply to Cann et al. Chlamydia, Rickettsia, and antibiotic treatment of multiple sclerosis. Ann Neurol 47:409±411, 2000 Stephens RS: Challenge of Chlamydia research. Infect Agents Dis 6:279±293, 1992 Stratton CW: Role of Chlamydia pneumoniae in connective tissue diseases. AFC 2 (Suppl. 2):S31±S38, 1998 Stratton CW, Mitchell WM: The pathogenesis of systemic Chlamydial infections: theoretical considerations of host cell energy depletion and its metabolic consequences. Antimicrobics Infectious Dis Newsletter 16:33±38, 1997 Stratton CW, Mitchell WM, Sriram S: Does Chlamydia pneumoniae play a role in the pathogenesis of multiple sclerosis? J Med Microbiol 49:1±3, 2000 Taylor-Robinson D, Thomas BJ: Chlamydia pneumoniae in arteries: the facts, their interpretation and future studies. J Clin Pathol 51:793±797, 1998 Tjaden J, Winkler HH, Schwoppe C, et al: Two nucleoside transport proteins in C. trachomatis, one for net nucleoside triphosphate uptake and the other for transport of energy. J Bacteriol 181:1196±1202, 1999 Vannucci SA, Mitchell WM, Stratton CW, King LE Jr: Pyoderma gangrenosum and Chlamydia pneumoniae infection in a diabetic man: pathogenic role or coincidence? J Am Acad Dermatol 42:295±297, 2000 Ward ME: The immunobiology and immunopathology of chlamydial infections. APMIS 103:769±796, 1995 Weiss SM, Roblin PM, Gaydos CA, et al: Failure to detect Chlamydia pneumoniae in coronary atheromas of patients undergoing atherectomy. J Infect Dis 173:957± 962, 1996 Wyrick PB, Knight ST, Paul TR, Rank RG, Barbie CS: Persistent chlamydial envelope antigens in antibiotic-exposed infected cells trigger neutrophil chemotaxsis. J Infect Dis 179:954±966, 1999 Zhu J, Quyyumi AA, Norman JE, Csako G, Waclawiw MA, Shearer GM, Epstein SE: Effects of total pathogen burden on coronary artery risk and C-reactive protein levels. Am J Cardiol 85:140±146, 2000