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Chlamydia pneumoniae and Chronic Skin Wounds: A Focused Review

      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 inflammatory skin conditions analogous to its role in atherosclerosis is reviewed.

      Keywords

      Abbreviations

      CP
      Chlamydia pneumoniae
      Chronic skin ulcers have common characteristics that include intermittent or persistent inflammation, infection, and the failure to heal (
      • Lazarus G.S.
      • Cooper D.M.
      • Knighton D.R.
      • et al.
      Definitions and guidelines for assessment of wounds and evaluation of healing.
      ;
      • Mostow E.N.
      Diagnosis and classification of chronic wounds.
      ;
      • Philips T.J.
      Chronic cutaneous ulcers: etiology and epidemiology.
      ;
      • Margolis D.J.
      • Berlin J.A.
      • Strom B.L.
      Reliability and validity of the clinical interpretation of a healed chronic wound.
      ,
      • Margolis D.J.
      • Berlin J.A.
      • Strom B.L.
      Risk factors associated with the failure of a venous leg ulcer to heal.
      ;
      • Eaglstein W.H.
      • Falanga V.
      Chronic wounds.
      ). A nonhealing wound may be due to genetic, environmental, or idiopathic factors, or an imbalance of bacteria (
      • Robson M.C.
      Wound infection: a failure of wound healing caused by an imbalance of bacteria.
      ). Bacterial colonization of stage 2–4 skin ulcers is assumed to be present irrespective of etiology, so infection is a potential source of inflammation in all ulcers such as pressure sores. How and if such inflammation sustains the most common types of chronic skin ulcers is controversial and needs further clarification. The possible role(s) of Chlamydia pneumoniae in initiating or sustaining chronic skin conditions, including skin ulcers, has not been extensively investigated.
      • Abrams J.T.
      • Vonderheid E.C.
      • Kolbe S.
      • Appelt D.M.
      • Arking E.J.
      • Bailin B.J.
      Sezary T-cell activating factor is a Chlamydia pneumoniae-associated protein.
      claimed that 12 of 27 patients with cutaneous T cell lymphoma had a C. pneumoniae-associated protein that activated Sezary T cells.
      • Vannucci S.A.
      • Mitchell W.M.
      • Stratton C.W.
      • King Jr., L.E.
      Pyoderma gangrenosum and Chlamydia pneumoniae infection in a diabetic man: pathogenic role or coincidence?.
      detected C. pneumoniae serologically in a diabetic patient with pyoderma gangrenosum-like lesions that responded dramatically to antibiotics directed against Chlamydia.
      • Sams H.H.
      • Stratton C.W.
      • Mitchell W.M.
      • King Jr., L.E.
      : Culture and immunohistochemical identification of Chlamydia pneumoniae in ulcerative pyoderma gangrenosum.
      identified 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 Jr., L.E.
      • Bushman T.
      • Stratton C.W.
      • Mitchell W.M.
      Diabetic foot ulcers and Chlamydia pneumoniae. Innocent bystander of opportunistic pathogen?.
      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 inflammation.

      Chlamydia pneumoniae – “innocent bystander”, indicator of prior infection, or persistent intracellular pathogen?

      Numerous studies have detected the presence of C. pneumoniae in endovascular structures as well as peripheral blood mononuclear cells (
      • Maass M.
      • Bartels C.
      • Kruger S.
      • Krause E.
      • Engel P.M.
      • Dalhoff K.
      Endovascular presence of Chlamydia pneumoniae DNA is a generalized phenomenon in atherosclerotic vascular disease.
      ;
      • 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.
      ;
      • Blasi F.
      • Boman J.
      • Esposito G.
      • et al.
      Chlamydia pneumoniae DNA detection in peripheral blood mononuclear cells is predictive of vascular infection.
      ). The presence of viable C. pneumoniae in peripheral blood mononuclear cells suggests that C. pneumoniae may accompany these white blood cells to inflamed tissue sites and cause a secondary infection in the inflamed 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 S.M.
      • Roblin P.M.
      • Gaydos C.A.
      • et al.
      Failure to detect Chlamydia pneumoniae in coronary atheromas of patients undergoing atherectomy.
      ;
      • Lindholt J.S.
      • Ostergard L.
      • Henneberg E.W.
      • et al.
      Failure to demonstrate Chlamydia pneumoniae in symptomatic abdominal aneurysms by a nested polymerase chain reaction (PCR).
      ) to very high (>90%;
      • Jackson L.A.
      • Campbell L.A.
      • Schmidt R.A.
      • et al.
      Specificity of detection of Chlamydia pneumoniae in cardiovascular atheroma: evaluation of the innocent bystander hypothesis.
      ) in selected populations. Recent approaches focus on finding 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 F.
      • Boman J.
      • Esposito G.
      • et al.
      Chlamydia pneumoniae DNA detection in peripheral blood mononuclear cells is predictive of vascular infection.
      ;
      • Shor A.
      • Phillips J.I.
      Chlamydia pneumoniae and atherosclerosis.
      ;
      • Russell E.G.
      Evaluation of two serological tests for the diagnosis of chlamydial respiratory diseases.
      ). The data variability is due to the multiple difficulties in culturing C. pneumoniae (Maass and Dalhoff, 1995;
      • Maass M.
      • Harig U.
      Evaluation of culture conditions used for isolation of Chlamydia pneumoniae.
      ) and technical problems with immunohistochemical methods (
      • Taylor-Robinson D.
      • Thomas B.J.
      Chlamydia pneumoniae in arteries: the facts, their interpretation and future studies.
      ). Maximizing the detection of reaction product by antigen retrieval strategies using immunochemical methods may be necessary in some anti- C. pneumoniae antibody staining procedures in formalin-fixed tissues as fixation can denature antigenic epitopes of C. pneumoniae.

      Microbiologic features of chlamydia

      The genus, Chlamydophilia, Chlamydiaceae is rapidly expanding and now includes nine species, five 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 K.D.
      • Bush R.M.
      • Andersen A.A.
      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 five new species, and standards for the identification of organisms.
      ;
      • Kalman S.
      • Mitchell W.
      • Marathe R.
      • et al.
      Comparative genomes of C. trachomatis and C. pneumoniae.
      ). 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 K.D.
      • Bush R.M.
      • Andersen A.A.
      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 five new species, and standards for the identification of organisms.
      ). Chlamydia trachomatis induces conjunctivitis, keratitis and is the most common preventable cause of blindness worldwide (
      • Ward M.E.
      The immunobiology and immunopathology of chlamydial infections.
      ). These organisms also cause pelvic inflammatory disease, endometrosis, vaginitis, urethritis, and infertility. Chlamydia pneumoniae is a common pathogen in acute human upper and lower respiratory infections worldwide (
      • Ward M.E.
      The immunobiology and immunopathology of chlamydial infections.
      ).
      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 fission. 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 C.W.
      • Mitchell W.M.
      The pathogenesis of systemic Chlamydial infections: theoretical considerations of host cell energy depletion and its metabolic consequences.
      ); 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 J.
      • Winkler H.H.
      • Schwoppe C.
      • et al.
      Two nucleoside transport proteins in C. trachomatis, one for net nucleoside triphosphate uptake and the other for transport of energy.
      ;
      • Iliffe-Lee E.R.
      • McClarty G.
      Glucose metabolism in Chlamydia trachomatis: the “energy parasite” hypothesis revisited.
      ). The pathogenic significance of this recent finding 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 (
      • Ojcius D.M.
      • Degani H.
      • Mispelter J.
      • Dautry-Varsat A.
      Enhancement of ATP levels and glucose metabolism during infection by Chlamydia: NMR studies of living cells.
      ). Moreover, cells infected with C. pneumoniae have depleted energy and thus may be dysfunctional (
      • Shemer-Arni Y.
      • Lieberman D.
      Chlamydia pneumoniae-induced ciliostasis in ciliated bronchial epithelial cells.
      ).

      Pathobiology of chlamydial infections

      Chlamydia pneumoniae is a common cause of pneumonia in humans (
      • Grayston J.R.
      • Kuo C.C.
      • Wang S.P.
      • Altman J.
      A new Chlamydia psittaci strain, TWAR, isolated in acute respiratory tract infections.
      ) and is associated with inflammatory arthritis and atherosclerosis (see below). The antibody response to C. pneumoniae increases with age as measured in otherwise unselected human populations (
      • Grayston J.T.
      Infection caused by Chlamydia pneumoniae, strain TWAR.
      ). 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 S.C.
      • Stratton Mitchell W.
      Multiple sclerosis associated with Chlamydia pneumoniae infection of the CNS.
      ,
      • Sriram S.
      • Stratton C.W.
      • Yao S.
      • Ding L.
      • Bannan J.
      • Mitchell W.M.
      Chlamydia pneumoniae infection of the central nervous system in multiple sclerosis.
      ;
      • Stratton C.W.
      • Mitchell W.M.
      • Sriram S.
      Does Chlamydia pneumoniae play a role in the pathogenesis of multiple sclerosis?.
      ) and Reiter's disease (
      • Stratton C.W.
      Role of Chlamydia pneumoniae in connective tissue diseases.
      ;
      • Gerard H.C.
      • Wang G.F.
      • Balin B.J.
      • et al.
      Frequency of apolipoprotein E (APOE) allele types in patients with Chlamydia-associated arthritis and other arthritides.
      ). 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 inflammation and corneal scarring (
      • Beatty W.L.
      • Morrison R.P.
      • Byrne G.I.
      Persistent Chlamydiae from cell culture to a paradigm for chlamydial pathogenesis.
      ). Chlamydia trachomatis is detected in the quiescent phase of trachoma even in the absence of histologic and immunologic features of inflammation. 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 P.
      Chlamydial lipopolysaccharide.
      ;
      • Rund S.
      • Lindner B.
      • Brade H.
      • Holst O.
      Structural analysis of the lipopolysaccharide from Chlamydia trachomatis serotype L2.
      ). Chlamydia induce inflammation and clotting abnormalities, but no known chlamydial toxins have been positively identified likely to be responsible for their tissue injury-inducing effects.
      • Read T.D.
      • Brunham R.C.
      • Shen C.
      • et al.
      Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39.
      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 T.
      • Fischer E.R.
      • Scidmore M.A.
      • Rockeey D.R.
      • Heinzen R.A.
      Origins and functions of the chlamydial inclusion.
      ;
      • Braavoil P.M.
      • Hsia R-C.
      Type III secretion in Chlamydia.
      ;
      • Hueck C.J.
      Type III protein secretion systems in bacterial pathogens of animals and plants.
      ). Whether the in vivo activation of the immune system by Chlamydia in vivo is due to heat shock proteins (
      • Kol A.
      • Sukhove G.K.
      • Lichtman A.H.
      • Libby P.
      Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-alpha and matrix metalloproteinase expression.
      ;
      • LaVerda D.
      • Kalayoglu M.V.
      • Byrne G.I.
      Chlamydial heat shock proteins and disease pathology: new paradigms for old problems?.
      ), type III secreted proteins (see above,
      • Braavoil P.M.
      • Hsia R-C.
      Type III secretion in Chlamydia.
      ), or other T cell activation mechanisms (
      • Igieseme J.U.
      The molecular mechanism of T-cell control of Chlamydia in mice: role of nitric oxide.
      ) is unknown.

      Chlamydia pneumoniae and inflammation

      Signal transduction pathways are activated in endothelial cells following infection with C. pneumoniae (
      • Krull M.
      • Klucken A.C.
      • Wuppermann F.N.
      • et al.
      Signal transduction pathways activated in endothelial cells following infection with Chlamydia pneumoniae.
      ). Infection of human endothelial cells with C. pneumoniae stimulates transendothelial migration of neutrophils and monocytes (
      • Moazed T.C.
      • Kuo C.C.
      • Grayston J.L.
      • Campbell L.A.
      Evidence of systemic dissemination of Chlamydia pneumoniae via macrophages in the mouse.
      ;
      • Molestina R.E.
      • Miller R.D.
      • Ramirez J.A.
      • Summersgill J.T.
      Infection of human endothelial cells with Chlamydia pneumoniae stimulates transendothelial migration of neutrophils and monocytes.
      ). Chlamydia pneumoniae infection of vascular smooth muscle and endothelial cells activates NF-ĸB and induces tissue factor and plasminogen activator inhibitor 1 (PAI-1) expression (
      • Dechend R.
      • Maass M.
      • Gieffers J.
      • Dieta R.
      • Scheidereit C.
      • Leutz A.
      • Gulba D.C.
      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.
      ). Other pathogenic mechanisms that may be involved include chlamydial heat shock proteins (
      • Kol A.
      • Sukhove G.K.
      • Lichtman A.H.
      • Libby P.
      Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-alpha and matrix metalloproteinase expression.
      ;
      • LaVerda D.
      • Kalayoglu M.V.
      • Byrne G.I.
      Chlamydial heat shock proteins and disease pathology: new paradigms for old problems?.
      ) and Toll receptors that are involved in the innate immune responses (
      • 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.
      ). 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 G.
      • Boesen T.
      • Hjerno K.
      • et al.
      Molecular biology of Chlamydia pneumoniae surface proteins and their role in immunopathogenicity.
      ;
      • Mayr M.
      • Metzler B.
      • Kiechl S.
      • Willeit J.
      • Schett G.
      • Xu Q.
      • Wick G.
      Endothelial 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.
      ). Chlamydial heat shock protein 60, and potentially outer membrane proteins (
      • Christiansen G.
      • Boesen T.
      • Hjerno K.
      • et al.
      Molecular biology of Chlamydia pneumoniae surface proteins and their role in immunopathogenicity.
      ), are involved in the localization to and in human atheromas and regulates macrophage tumor necrosis factor-α and matrix metalloproteinase expression (
      • Kol A.
      • Sukhove G.K.
      • Lichtman A.H.
      • Libby P.
      Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-alpha and matrix metalloproteinase expression.
      ;
      • Christiansen G.
      • Boesen T.
      • Hjerno K.
      • et al.
      Molecular biology of Chlamydia pneumoniae surface proteins and their role in immunopathogenicity.
      ). A specific C. pneumoniae lipopolysaccharide, lipid A, induces macrophage foam cell formation (
      • Kalayoglu M.V.
      • Byrne G.I.A.
      Chlamydia pneumoniae component that induces macrophage foam cell formation is chlamydial lipopolysaccaride.
      ). 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 inflammation when no viable C. pneumoniae are present (
      • Wyrick P.B.
      • Knight S.T.
      • Paul T.R.
      • Rank R.G.
      • Barbie C.S.
      Persistent chlamydial envelope antigens in antibiotic-exposed infected cells trigger neutrophil chemotaxsis.
      ).

      Chlamydia pneumoniae, vascular inflammation, and atherosclerosis

      Atherosclerosis is now classified as an inflammatory disease (
      • Ross R.
      Atherosclerosis an inflammatory disease.
      ) so potentially it could be induced or modulated by an infectious agent. The association of C. pneumoniae with vascular disease was first demonstrated serologically in a Finnish population (
      • 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.
      ). Chlamydia pneumoniae was first identified as a respiratory pathogen by
      • Grayston J.R.
      • Kuo C.C.
      • Wang S.P.
      • Altman J.
      A new Chlamydia psittaci strain, TWAR, isolated in acute respiratory tract infections.
      , It was confirmed as a common cause of respiratory disease that was commonly detected in asymptomatic healthy adults (
      • Hyman C.L.
      • Roblin C.A.
      • Gaydos C.A.
      • Quinn T.C.
      • Schachter J.
      • Hammerschlag M.R.
      Prevalence of asymptomatic nasopharyngeal carriage of Chlamydia pneumoniae in subjectively healthy adults: assessment by polymerase chain reaction, enzyme immunoassay and culture.
      ). This organism was first directly associated with atherosclerotic lesions in South African patients (for a review see
      • Shor A.
      • Phillips J.I.
      Chlamydia pneumoniae and atherosclerosis.
      ). Recent evidence indicates that C. pneumoniae and potentially other microbes are associated with atherosclerosis in cardiovascular and cerebrovascular diseases (for reviews see
      • Danesh J.
      • Collins R.
      • Peto R.
      Chronic infections and coronary heart disease: is there a link?.
      ;
      • Cheng J.W.
      • Rivera N.G.
      Infection and atherosclerosis – focus on cytomegalovirus and Chlamydia pneumoniae.
      ;
      • Epstein S.E.
      • Zhou Y.F.
      • Zhu J.
      Infection and atherosclerosis: emerging mechanistic paradigms.
      ;
      • Chiu B.
      Multiple infections in carotid atherosclerotic plaques.
      ;
      • Grayston J.T.
      • Campbell L.A.
      The role of Chlamydia pneumoniae in atherosclerosis.
      ;
      • Leinonen M.
      • Saikku P.
      Interaction of Chlamydia pneumoniae infection with other risk factors of atherosclerosis.
      ;
      • 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.
      ). Convincing data that other infectious agents such as Mycoplasma pneumoniae and Helicobacter pylori may be involved are not available (
      • Blasi F.
      • Denti F.
      • Erba M.
      • et al.
      Detection of Chlamydia pneumoniae and not of Helicobacter pylori in atherosclerotic plaques of aortic aneurysms.
      ). 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 significant number of samples from human arterial tissues including peripheral vessels (
      • Shor A.
      • Phillips J.I.
      Chlamydia pneumoniae and atherosclerosis.
      ). Chlamydia pneumoniae infection is detectable in patients with cerebrovascular accidents (stroke) (
      • Cook P.J.
      • Honeybourne D.
      • Lip G.Y.
      • et al.
      Chlamydia pneumoniae antibody titers are significantly associated with acute stroke and transient cerebral ischemia. The West Birmingham Stroke Project.
      ) as well as Alzheimer's disease (
      • Balin B.J.
      • Gerard H.C.
      • Arking E.J.
      • et al.
      Identification and localization of Chlamydia pneumoniae in the Alzheimer's brain.
      ) and diabetic nephropathy (
      • Kanuchi M.
      • Kawano T.
      • Dohi K.
      Association of C. pneumoniae infection with diabetic nephropathy.
      ). 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 M.
      • Bartels C.
      • Kruger S.
      • Krause E.
      • Engel P.M.
      • Dalhoff K.
      Endovascular presence of Chlamydia pneumoniae DNA is a generalized phenomenon in atherosclerotic vascular disease.
      ). 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 R.F.
      • Bernhard V.M.
      Management of chronic ischemia of the lower extremities. Introduction and general considerations.
      ). 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 R.F.
      • Bernhard V.M.
      Management of chronic ischemia of the lower extremities. Introduction and general considerations.
      ). Atherosclerosis is generally assumed to be the cause of chronic ischemia of the lower extremities, including diabetic foot ulcers (
      • Chait A.
      • Bierman E.L.
      Pathogenesis of macrovascular disease in diabetes.
      ). There are very few 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 R.F.
      • Bernhard V.M.
      Management of chronic ischemia of the lower extremities. Introduction and general considerations.
      ).

      Is c. pneumoniae likely to be the only pathogen involved in atherosclerosis?

      Multiple infections of atherosclerotic plaques may be present (
      • Epstein S.E.
      • Zhou Y.F.
      • Zhu J.
      Infection and atherosclerosis: emerging mechanistic paradigms.
      ;
      • Chiu B.
      Multiple infections in carotid atherosclerotic plaques.
      ;
      • Zhu J.
      • Quyyumi A.A.
      • Norman J.E.
      • Csako G.
      • Waclawiw M.A.
      • Shearer G.M.
      • Epstein S.E.
      Effects of total pathogen burden on coronary artery risk and C-reactive protein levels.
      ). 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 J.
      • Quyyumi A.A.
      • Norman J.E.
      • Csako G.
      • Waclawiw M.A.
      • Shearer G.M.
      • Epstein S.E.
      Effects of total pathogen burden on coronary artery risk and C-reactive protein levels.
      ). 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 profile in a population of nurses (
      • Ricker P.M.
      • Hennekens C.H.
      • Buring J.E.
      • Rifai N.
      C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women.
      ).

      Detecting c. pneumoniae in chronic leg and diabetic foot ulcers

      The previous inability to detect these organisms may be due to the difficulty of recovering viable C. pneumoniae organisms that require special methods for their isolation and propagation (
      • Stephens R.S.
      Challenge of Chlamydia research.
      ). 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 C.C.
      • Grayston J.T.
      A sensitive cell line, HL cells, for isolation and propagation of Chlamydia pneumoniae strain TWAR.
      ). Chlamydia pneumoniae persistently infects and replicates in epithelium, endothelium, smooth muscle cells, macrophages (
      • Sriram S.
      • Stratton C.W.
      • Yao S-Y.
      • Tharp A.
      • Ding L.
      • Bannan J.D.
      • Mitchell W.M.
      Reply to Cann et al. Chlamydia, Rickettsia, and antibiotic treatment of multiple sclerosis.
      ), and neural tissue in vivo and in vitro (
      • Balin B.J.
      • Gerard H.C.
      • Arking E.J.
      • et al.
      Identification and localization of Chlamydia pneumoniae in the Alzheimer's brain.
      ;
      • Sriram S.C.
      • Stratton Mitchell W.
      Multiple sclerosis associated with Chlamydia pneumoniae infection of the CNS.
      ,
      • Sriram S.
      • Stratton C.W.
      • Yao S.
      • Ding L.
      • Bannan J.
      • Mitchell W.M.
      Chlamydia pneumoniae infection of the central nervous system in multiple sclerosis.
      ;
      • Stratton C.W.
      • Mitchell W.M.
      • Sriram S.
      Does Chlamydia pneumoniae play a role in the pathogenesis of multiple sclerosis?.
      ). The ability to isolate and propagate these organisms in HL cells (
      • Kuo C.C.
      • Grayston J.T.
      A sensitive cell line, HL cells, for isolation and propagation of Chlamydia pneumoniae strain TWAR.
      ;
      • Pruckler J.M.
      • Masse N.
      • Stevens V.A.
      • et al.
      Optimizing culture of Chlamydia pneumoniae by using multiple centrifugations.
      ;
      • Sriram S.
      • Stratton C.W.
      • Yao S-Y.
      • Tharp A.
      • Ding L.
      • Bannan J.D.
      • Mitchell W.M.
      Reply to Cann et al. Chlamydia, Rickettsia, and antibiotic treatment of multiple sclerosis.
      ) 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”), difficult to eradicate, and may be spread by contact with seemingly healthy humans (
      • Hyman C.L.
      • Roblin C.A.
      • Gaydos C.A.
      • Quinn T.C.
      • Schachter J.
      • Hammerschlag M.R.
      Prevalence of asymptomatic nasopharyngeal carriage of Chlamydia pneumoniae in subjectively healthy adults: assessment by polymerase chain reaction, enzyme immunoassay and culture.
      ). Alternatively and, we believe, most likely, C. pneumoniae in chronic skin ulcers may be initiated by parasitized mononuclear cells (
      • Airenne S.
      • Surcel H-M.
      • Alakarppa H.
      • Laitinen K.
      • Paavonen J.
      • Saikku P.
      • Laurila A.
      Chlamydia pneumoniae infection in human monocytes.
      ). In a mouse model, C. pneumoniae infectivity was demonstrated to be mediated by parasitized monocytes (
      • Moazed T.C.
      • Kuo C.C.
      • Grayston J.L.
      • Campbell L.A.
      Evidence of systemic dissemination of Chlamydia pneumoniae via macrophages in the mouse.
      ). 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 anti-LPS (
      • Abrams J.T.
      • Vonderheid E.C.
      • Kolbe S.
      • Appelt D.M.
      • Arking E.J.
      • Bailin B.J.
      Sezary T-cell activating factor is a Chlamydia pneumoniae-associated protein.
      ). These authors confirmed 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 J.T.
      • Vonderheid E.C.
      • Kolbe S.
      • Appelt D.M.
      • Arking E.J.
      • Bailin B.J.
      Sezary T-cell activating factor is a Chlamydia pneumoniae-associated protein.
      ). Recently, we detected C. pneumoniae serologically and by PCR in chronic cutaneous ulcers in a diabetic who responded dramatically to appropriate antibiotic therapy (
      • Vannucci S.A.
      • Mitchell W.M.
      • Stratton C.W.
      • King Jr., L.E.
      Pyoderma gangrenosum and Chlamydia pneumoniae infection in a diabetic man: pathogenic role or coincidence?.
      ). We cultured C. pneumoniae from chronic skin wounds in diabetic and nondiabetic patients such as those with pyoderma gangrenosum (
      • King Jr., L.E.
      • Bushman T.
      • Stratton C.W.
      • Mitchell W.M.
      Diabetic foot ulcers and Chlamydia pneumoniae. Innocent bystander of opportunistic pathogen?.
      ;
      • Sams H.H.
      • Stratton C.W.
      • Mitchell W.M.
      • King Jr., L.E.
      : Culture and immunohistochemical identification of Chlamydia pneumoniae in ulcerative pyoderma gangrenosum.
      ).

      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 conflicting data and opinions over the role of C. pneumoniae in atherosclerosis and coronary artery disease, it is important to first 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 definitive 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 J.
      • Quyyumi A.A.
      • Norman J.E.
      • Csako G.
      • Waclawiw M.A.
      • Shearer G.M.
      • Epstein S.E.
      Effects of total pathogen burden on coronary artery risk and C-reactive protein levels.
      ). 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 confirm 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 J.B.
      Bacterial infections and atherosclerosis.
      ;
      • Shor A.
      • Phillips J.I.
      Chlamydia pneumoniae and atherosclerosis.
      ). 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 (
      • Ojcius D.M.
      • Degani H.
      • Mispelter J.
      • Dautry-Varsat A.
      Enhancement of ATP levels and glucose metabolism during infection by Chlamydia: NMR studies of living cells.
      ). 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 D.M.
      • Degani H.
      • Mispelter J.
      • Dautry-Varsat A.
      Enhancement of ATP levels and glucose metabolism during infection by Chlamydia: NMR studies of living cells.
      ). 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 anti-C. pneumoniae serum IgG antibody titers in an ELISA (
      • Kanuchi M.
      • Kawano T.
      • Dohi K.
      Association of C. pneumoniae infection with diabetic nephropathy.
      ).

      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 R.
      Atherosclerosis an inflammatory disease.
      ). Animal models (
      • Moazed T.C.
      • Kuo C.
      • Grayston J.T.
      • Campbell L.A.
      Murine models of Chlamydia pneumoniae infection and atherosclerosis.
      ;
      • Saikku P.
      • Laitinen K.
      • Leinonen M.
      Animal models for Chlamydia pneumoniae infection.
      ;
      • Muhlestein J.B.
      • Anderson J.L.
      • Hammond E.H.
      • et al.
      Infection with Chlamydia pneumoniae accelerates the development of atherosclerosis and treatment with azithromycin prevents it in a rabbit model.
      ;
      • Fong I.W.
      • Chiu B.
      • Viira E.
      • et al.
      Can an antibiotic (macrolide) prevent Chlamydia pneumoniae-induced atherosclerosis in a rabbit model?.
      ) show that C. pneumoniae can induce atheromas and mimic inflammatory processes present in human atherosclerosis. Elevated C. pneumoniae antibody titers were associated with altered serum lipid profile in humans (
      • Laurila A.L.
      • Bloigu A.
      • Nayha S.
      • Hassi J.
      • Leinonen M.
      • Saikku P.
      Chlamydia pneumoniae antibodies associated with altered serum lipid profile.
      ). In vitro cellular oxidation of low-density lipoproteins (LDL) is induced by C. pneumoniae (
      • Kalayoglu M.V.
      • Hoerneman B.
      • LaVerda D.
      • Morrison S.G.
      • Morrison R.P.
      • Byrne G.I.
      Cellular oxidation of low-density lipoprotein by Chlamydia pneumoniae.
      ). The atherogenic effects of Chlamydia are claimed to be dependent on serum cholesterol and specific to C. pneumoniae (
      • Hu H.
      • Pierce G.N.
      • Zhong G.
      The atherogenic effects of Chlamydia are dependent on serum cholesterol and specific to Chlamydia pneumoniae.
      ).

      Therapy

      Published data to confirm the efficacy 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 J.
      • Fullgraf H.
      • Jahn J.
      • et al.
      Chlamydia pneumoniae infection in circulating monocytes is refractory to antibiotic treatment.
      ). 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 B.
      Multiple infections in carotid atherosclerotic plaques.
      ;
      • Zhu J.
      • Quyyumi A.A.
      • Norman J.E.
      • Csako G.
      • Waclawiw M.A.
      • Shearer G.M.
      • Epstein S.E.
      Effects of total pathogen burden on coronary artery risk and C-reactive protein levels.
      ). Chlamydia pneumoniae infected cells also may be more resistant to apoptosis than cells infected with other pathogens (
      • Geng Y.
      • Shane R.B.
      • Berencsi K.
      • et al.
      Chlamydia pneumoniae inhibits apoptosis in human peripheral blood mononuclear cells through induction of IL-10.
      ). Although chronic antibiotic therapy did alter the clinical course of multiple skin ulcers with serologically documented C. pneumoniae infection with (
      • Vannucci S.A.
      • Mitchell W.M.
      • Stratton C.W.
      • King Jr., L.E.
      Pyoderma gangrenosum and Chlamydia pneumoniae infection in a diabetic man: pathogenic role or coincidence?.
      ) and without diabetes (
      • Sriram S.
      • Stratton C.W.
      • Yao S-Y.
      • Tharp A.
      • Ding L.
      • Bannan J.D.
      • Mitchell W.M.
      Reply to Cann et al. Chlamydia, Rickettsia, and antibiotic treatment of multiple sclerosis.
      ), 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 D.J.
      • Kantor J.
      • Berlin J.A.
      Healing of diabetic neuropathic foot ulcers receiving standard treatment.
      so long-term, cost-effective therapy may be required. The efficacy and cost-effectiveness of specific regimens for chronic skin ulcers will likely be a productive research area.

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