Matrix Metalloproteinases in Central Nervous System Disease

CNS injury triggers a host immune response that generates inflammatory cytokines that increase BBB permeability and mediates the recruitment of peripheral immune cells. matrix metalloproteinases (MMPs) have been implicated in many CNS diseases (Kieseier, Seifert et al. 1999; Hashimoto, Wen et al. 2003). During injury, resident CNS cells and peripheral infiltrating leukocytes can secrete cytokines and MMPs which mediate inflammation by the acute opening of the BBB, demyelination and axonal injury, and cell death (Rosenberg 1995; Kieseier, Seifert et al. 1999; Yong, Power et al. 2001). MMPs and tissue inhibitor of matrix metalloproteinases (TIMPs) have been implicated in MS and EAE and MMP genetic polymorphisms are associated with risk and clinical course of MS (Dasilva and Yong 2008; Mirowska-Guzel, Gromadzka et al. 2009; Alexander, Harris et al. 2010). MMP-12-null mice induced with MOG_35-55 peptide EAE exhibit a more severe disease course than wildtype controls (Weaver, Goncalves da Silva et al. 2005). During EAE disease onset and prior to clinical symptoms, MMP-12 is highly expressed and secreted by a subpopulation of monocytoid Iba-1-reactive cells, resident microglia and infiltrating macrophages (Dasilva and Yong 2008). MMP-12 expression and activity continues into the early phase but is lost following peak clinical disease (Dasilva and Yong 2008).

During early CNS disease, astrocytes become reactive and respond in astrogliosis, and mediate many pathogenic mechanisms. It has been shown that astrocytes increase MMP-13 expression in a time-dependant manner in various diseases (Brinckerhoff, Rutter et al. 2000; Stickens, Behonick et al. 2004; Lu, Yu et al. 2009). Furthermore, astrocyte-derived MMP-13 perturbs the continuity and mediates the destruction of the ZO-1 protein which leads to increased BBB permeability in hypoxic brain injury (Lu, Yu et al. 2009). In addition to MMP-13, reactive astrocytes also increase their expression of MMP-9 in various CNS injury models (Bauer, Burgers et al. 2010; Wang, Hsieh et al. 2010) It has been shown that MMP-9 mediates increased BBB permeability via gap formation and tight junction protein (occludin and ZO-1) discontinuity (Bauer, Burgers et al. 2010). Treatment with an MMP inhibitor reduced vascular leakage and attenuated TJ disorganization (Bauer, Burgers et al. 2010). BBB hyperpermeability allows CNS infiltration of leukocytes which secrete a variety of cytokines and factors, among them is leukocyte-derived MMP-9, which exerts its proinflammatory actions by promoting leukocyte recruitment and migration in CNS parenchyma (Gidday, Gasche et al. 2005; Zozulya, Reinke et al. 2007).

Inflammatory cytokines and signaling factors play an important role in the regulation and activity of MMPs. Tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1 β), two key factors implicated in MS and EAE, are closely associated with the disruption of the BBB (Sharief and Thompson 1992) and TNF-α, IL-1β, and platelet activating factor (PAF) are implicated in MMP production, specifically MMP-9, 12 and 13 (Birkedal-Hansen, Moore et al. 1993; Lee, Shin et al. 2003). Transforming growth factor (TGF)-ß mediates increased in vitro endothelial cell layer permeability by inducing MMP-9 expression which leads to reduced occludin levels in TJs (Behzadian, Wang et al. 2001). In many CNS injury models including EAE, BBB hyperpermeability is dependent on vascular endothelial growth factor (VEGF) which mediates changes in TJ protein expression and rearrangement (Proescholdt, Jacobson et al. 2002; Schoch, Fischer et al. 2002; Sasaki, Lankford et al. 2010). VEGF increases endothelial permeability through direct activation of MMP-9 and inhibition of VEGF not only blocks vascular leakage but also attenuates MMP-9 activity (Bauer, Burgers et al. 2010).

Alexander, J. S., M. K. Harris, et al. (2010). "Alterations in serum MMP-8, MMP-9, IL-12p40 and IL-23 in multiple sclerosis patients treated with interferon-beta1b." Mult Scler 16(7): 801-9.

Bauer, A. T., H. F. Burgers, et al. (2010). "Matrix metalloproteinase-9 mediates hypoxia-induced vascular leakage in the brain via tight junction rearrangement." J Cereb Blood Flow Metab 30(4): 837-48.

Behzadian, M. A., X. L. Wang, et al. (2001). "TGF-beta increases retinal endothelial cell permeability by increasing MMP-9: possible role of glial cells in endothelial barrier function." Invest Ophthalmol Vis Sci 42(3): 853-9.

Birkedal-Hansen, H., W. G. Moore, et al. (1993). "Matrix metalloproteinases: a review." Crit Rev Oral Biol Med 4(2): 197-250.

Brinckerhoff, C. E., J. L. Rutter, et al. (2000). "Interstitial collagenases as markers of tumor progression." Clin Cancer Res 6(12): 4823-30.

Dasilva, A. G. and V. W. Yong (2008). "Expression and regulation of matrix metalloproteinase-12 in experimental autoimmune encephalomyelitis and by bone marrow derived macrophages in vitro." J Neuroimmunol 199(1-2): 24-34.

Gidday, J. M., Y. G. Gasche, et al. (2005). "Leukocyte-derived matrix metalloproteinase-9 mediates blood-brain barrier breakdown and is proinflammatory after transient focal cerebral ischemia." Am J Physiol Heart Circ Physiol 289(2): H558-68.

Hashimoto, T., G. Wen, et al. (2003). "Abnormal expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in brain arteriovenous malformations." Stroke 34(4): 925-31.

Kieseier, B. C., T. Seifert, et al. (1999). "Matrix metalloproteinases in inflammatory demyelination: targets for treatment." Neurology 53(1): 20-5.

Lee, W. J., C. Y. Shin, et al. (2003). "Induction of matrix metalloproteinase-9 (MMP-9) in lipopolysaccharide-stimulated primary astrocytes is mediated by extracellular signal-regulated protein kinase 1/2 (Erk1/2)." Glia 41(1): 15-24.

Lu, D. Y., W. H. Yu, et al. (2009). "Hypoxia-induced matrix metalloproteinase-13 expression in astrocytes enhances permeability of brain endothelial cells." J Cell Physiol 220(1): 163-73.

Mirowska-Guzel, D., G. Gromadzka, et al. (2009). "Association of MMP1, MMP3, MMP9, and MMP12 polymorphisms with risk and clinical course of multiple sclerosis in a Polish population." J Neuroimmunol 214(1-2): 113-7.

Proescholdt, M. A., S. Jacobson, et al. (2002). "Vascular endothelial growth factor is expressed in multiple sclerosis plaques and can induce inflammatory lesions in experimental allergic encephalomyelitis rats." J Neuropathol Exp Neurol 61(10): 914-25.

Rosenberg, G. A. (1995). "Matrix metalloproteinases in brain injury." J Neurotrauma 12(5): 833-42.

Sasaki, M., K. L. Lankford, et al. (2010). "Focal experimental autoimmune encephalomyelitis in the lewis rat induced by immunization with myelin oligodendrocyte glycoprotein and intraspinal injection of vascular endothelial growth factor." Glia.

Schoch, H. J., S. Fischer, et al. (2002). "Hypoxia-induced vascular endothelial growth factor expression causes vascular leakage in the brain." Brain 125(Pt 11): 2549-57.

Sharief, M. K. and E. J. Thompson (1992). "In vivo relationship of tumor necrosis factor-alpha to blood-brain barrier damage in patients with active multiple sclerosis." J Neuroimmunol 38(1-2): 27-33.

Stickens, D., D. J. Behonick, et al. (2004). "Altered endochondral bone development in matrix metalloproteinase 13-deficient mice." Development 131(23): 5883-95.

Wang, H. H., H. L. Hsieh, et al. (2010). "Endothelin-1 enhances cell migration via matrix metalloproteinase-9 up-regulation in brain astrocytes." J Neurochem 113(5): 1133-49.

Weaver, A., A. Goncalves da Silva, et al. (2005). "An elevated matrix metalloproteinase (MMP) in an animal model of multiple sclerosis is protective by affecting Th1/Th2 polarization." FASEB J 19(12): 1668-70.

Yong, V. W., C. Power, et al. (2001). "Metalloproteinases in biology and pathology of the nervous system." Nat Rev Neurosci 2(7): 502-11.

Zozulya, A. L., E. Reinke, et al. (2007). "Dendritic cell transmigration through brain microvessel endothelium is regulated by MIP-1alpha chemokine and matrix metalloproteinases." J Immunol 178(1): 520-9.

New Research into Ischemic Stroke Treatment

A stroke (also called a cerebrovascular accident or CVA) occurs due to reduced blood supply to the brain that results in loss of brain functions. This reduction can be due to ischemia caused by a thrombosis or embolism or due to a hemorrhage,which is the leakage of blood into tissues; hence the terms ischemic and hemorraghic stroke. Ischemic strokes are treated with thrombolysis which serves to dissolve the clot. The current preferred treatment for ischemic strokes is a drug called rtPA (recombinant tissue plasminogen activator). As with most stroke drugs, rtPA must be administered within the first few hours of a stroke or the risks of treatment outweigh the benefits. A potential risk involves a sudden rise in blood pressure due to the dissolving of the clot, which can subsequently lead to blood vessel rupture and bleeding into the brain.

Less than 10% of stroke victims will make it to the hospital early enough to be treated with rtPA. The rest are given drugs that reduce the possibility of futher clot formation but do not dissolve the initial clot. A reason for this includes the diagnosis of the type of stroke by brain scans. Administration of rtPA, or other "clot busters", in hemorrhagic stroke will lead to increased bleeding into the brain.

New research shows that tPA (tissue plasminogen activator) can be released by neurons themselves. In a postulated hypothesis, tPA in small quantities can bind to NMDA receptors. Normally, NMDA receptors allow the influx of sodium and calcium, the latter mechanism being important for learning and memory. But damaged neurons, for example, during stroke, release tPA in large quantities. High levels of tPA can cause neighboring neurons to die, by NMDA-mediated excitotoxicity, and can even damage the blood-brain barrier.

Mechanistically, it is possible to prevent the association of tPA with NMDA receptors by antibody neutralization. In mouse stroke model studies, injection of anti-tPA antibodies resulted in reduced stroke-inflicted brain damage both on its own or in combination with administered rtPA both following stroke and 6 hours later. Using the antibody, not only did the researchers see a decreased level of brain damage but also the antibody seems to work beyond the current critical time window.

Newscientist article: http://www.newscientist.com/article/mg20727682.500-antibody-cuts-brain-damage-in-strokes.html

Alzheimer’s disease and the reversal of pathology and behavior in human APP transgenic mice by mutation of Asp664

Galvan V, Gorostiza OF, Banwait S, Ataie M, Logvinova AV, Sitaraman S, Carlson E, Sagi SA, Chevallier N, Jin K, Greenberg DA, Bredesen DE. Reversal of Alzheimer's-like pathology and behavior in human APP transgenic mice by mutation of Asp664. Proc Natl Acad Sci USA 2007 Apr 17; 104(16): 6876.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder of the central nervous system (CNS) and is associated with memory loss, behavior and personality changes, and cognitive decline. The neuropathophysiology of AD is accompanied by three major structural changes: extracellular deposition of β-amyloid (Aβ), intracellular deposits called neurofibrillary tangles (NFT) consisting of hyperphosphorylated tau proteins, and selective neuronal loss, cholinergic deficits and astrogliosis (astrocyte proliferation) in the cerebral cortex, hippocampus, and other brain regions essential for memory and cognitive ability (1).

There are two hypotheses that postulate the molecular mechanisms of this disease. The cholinergic hypothesis suggests a dysfunctional cholinergic system is sufficient to cause memory loss as demonstrated animal models analogous to AD-related dementia. The brains of AD patients show selective degeneration of cholinergic neurons of the basal forebrain (nucleus basalis of Meynert) resulting in a substantial decline in choline acetyltransferase (ChAT) and acetyl cholinesterase (AChase). These enzymes are responsible for the production and regulation of acetylcholine (ACh) (a neurotransmitter) and its loss is associated with cognitive decline (2).

The amyloid cascade hypothesis states that the pathophysiology of AD is the result of abnormal processing of the amyloid precursor protein (APP), which is a membrane protein expressed during cell stress (3). Genetic studies have implicated four genes in the APP proteolytic cascade: APP gene (gene product is substrate) (21q21.2) (4), presenilin 1 (PSEN1) (14q24.3) and presenilin 2 (PSEN2) (1q31-q42) (5, 6), and apolipoprotein E (ApoE) (19q13.2) (7). Mutations in these genes are linked to the autosomal dominant or familial early onset AD (FAD) (8). The production of Aβ is dependant on the activities of two enzymes: β-secretase and γ-secretase. Depending on the type of APP mutation, either β- or γ-secretase activity is altered. PSEN proteins (PS) have been proposed to function in the APP processing pathway. PS1 is hypothesized to be either an essential cofactor or is itself γ-secretase (9). PS have also been proposed to function in apoptosis. Mutated PS expression or PS overexpression leads to increased sensitivity to apoptosis in transfected cells (10). PS mutations destabilize Ca2+ homeostasis (11) and may play a direct role in Fas-mediated apoptosis (12).

ApoE is a 34kDa lipid transport protein that has also been implicated in the pathogenesis of AD (13). It has also been shown to repair nerve cells in response to oxidative damage by reducing secondary glutamate (primary CNS neurotransmitter) excitotoxicity (14). ApoE exists in three allelic variants: E2, E3, and E 4. Biochemical tests have shown that ApoE can directly interact with Aβ. ApoE3 has been reported to protect neurons against Aβ neurotoxicity, through association and internalization of Aβ (15). However, the ApoE4 allele is associated with anticipation of AD as it binds more rapidly to Aβ and leads to its increased deposition.

Tau (MAPT gene), a microtubule associated protein, has multiple phosphorylation sites and binds tubulin molecules and facilitates microtubule assembly and stability (16). In AD, tau is hyperphosphorylated and this impairs its ability to perform its function and undermines microtubule stability (17).

The CNS is especially vulnerable to oxidative stress due to the brain's high oxygen consumption, abundance of lipid content, and relative absence of antioxidants compared to other tissues (18). This oxidation may come from the reaction of peroxynitrite, a powerful oxidant from the reaction of O2- and NO. This occurs from the overstimulation of neurons during cell stress when they are unable to uptake glutamate (CNS neurotransmitter) and allows the accumulation of intracellular calcium. Excess calcium activates neuronal NO synthases, enzymes that form NO from L-arginine, which leads to excitotoxicity.

Astrocytes and microglia are also involved in causing damage to the AD brain. In AD, the number of astrocytes is increased (astrogliosis) and the expression of phospholipase A2 is upregulated leading to arachidonic acid/prostaglandins inflammatory products. Activated microglial cells are also abundant in the AD brain and produce a number of neurotoxic compounds: superoxide anions, glutamate, and NO (19). They upregulate MHC expression and secrete cytokines and chemokines, including interleukin-1 (which increases APP expression), TNF-α, and interferon-γ (both involved in the cellular immune attack).

Recent evidence suggests that APP may be cleaved intracytoplasmically at Asp664 by caspases (activated during apoptosis) (20, 21) to release a cytotoxic carboxy-terminal peptide: APP-C31 (21, 22). This finding suggests a model where Aβ oligomerizes to APP leading to cytosolic cleavage and initiation of synaptic and neuronal damage (23). However, it is unknown whether APP intracytoplasmic cleavage in vivo results in AD pathogenesis. Galvan et al. (2005) generated transgenic mice expressing human APP transgene with the familial AD-associated Swedish and Indiana mutations (24, 25) except that the cytosolic Asp was mutated to Ala. These mice were designated as PDAPP(D664A).

The authors incubated 3 month-old (mo) brain tissue samples with an antibody that binds the C-terminal APP cleavage peptide but not full length APP (20, 22). Immunoreactivity in PDAPP(D664A) mice was substantially lower than PDAPP mice suggesting that Asp664 mutation prevented hAPP C-terminal cleavage in vivo. The authors also examined Aβ production and deposition, hippocampal presynaptic density and dentate gyrus (DG) volume, astrogliosis, and cognitive and behavioral abnormalities.

Immunoprecipitation and western blot analysis for Aβ revealed that Asp664 mutation had no demonstrable effect on Aβ production in vivo. ELISA quantification revealed that mouse lines with APP overexpression and D664A mutation still had high levels of Aβ. Plaque formation was also unaffected by Asp664 mutation as shown by Aβ antibody (4G8) immunoreactivity and positive thioflavin-S results.

Synaptophysin is a synaptic vesicle glycoprotein involved in synaptic transmission. Its reduction in the hippocampus and prefrontal cortex is correlated with cognitive decline (26). PDAPP mice show decreased numbers of hippocampal-synaptophysin-immunoreactive presynaptic densities (HSPDs). However, the PDAPP(D664A) mice have HSPD levels indistinguishable from nontransgenic (wildtype) mice suggesting that Asp664 mutation blocks hippocampal synaptic loss.

DG volume in 3 mo transgenic mice were determined using digital 3D reconstruction of Nissl-stained sections (IMARIS 3D (Bitplane)) and by manual Calvieri analysis. DG volume reduction, as observed in PDAPP mice, was not seen in PDAPP(D664A) mice indicating that Asp664 mutation can rescue DG neuronal loss.

Neuronal loss allows astrocyte activation, infiltration, and proliferation (27). Hippocampal samples of 12 mo transgenic mice were incubated with antibodies specific for an astrocyte marker: glial fibrillary acidic protein (GFAP). A significant increase in GFAP immunoreactivity was observed in PDAPP mice but not in PDAPP(D664A) or nontransgenic mice. Thus, Asp664 mutation prevents astrogliosis.

AD patients, and animal models, exhibit many cognitive deficits and behavioral abnormalities (1). Learning and spatial memory, which are hippocampus-dependant, were evaluated using the Morris water maze (MWM) test (28). Performance of PDAPP mice was considerably impaired compared to PDAPP(D664A) and nontransgenic mice. Behavioral abnormalities, such as neophobia (29) where the exploration time of novel object or region is reduced, have also been described in AD transgenic models. Neophobia is associated with decreased glucose utilization in the entorhinal cortex. This behavior pattern was detected in some PDAPP mice but nonexistent in PDAPP(D664A) mice. These results demonstrate that learning, spatial memory, and behavior problems associated with AD can be rescued by ASP664 mutation.

Although Aβ production and deposition remain unchanged, all consequential pathophysiological features of AD are improved in PDAPP(D664A) mice. This shows that Asp664 is critical for the downstream pathological consequences of Aβ and it is likely to be either cleavage or protein interaction site. The results obtained by Galvan et al. (2005) lends support to the new proposed model of AD, where Aβ binds and oligomerizes APP leading to cleavage at Asp664 and neurotoxicity (23).


References:

(1)Lesli RA. Imaging Alzheimer’s disease in vivo: not so implaque-able anymore. Trends Neurosci. 2002; 25(5):232-233.
(2)Bowen DM et al. Classical neurotransmitters in Alzheimer’s disease. In: Terry RD (ed) Aging and the Brain. Raven Press, New York 1988; 115-128.
(3)Checler F et al. Alzheimer’s and Prion diseases: distinct pathologies, common proteolytic denominators. Trends Neurosci. 2002; 25(12):616-620.
(4)Citron M et al. Mutations of the β-amyloid precrusor protein in familial Alzheimer’s disease increase β-protein production. Nature 1992; 360: 672-674.
(5)Levy-Lahad E et al. Candidate gene for the chromosome I familial Alzheimer’s disease locus. Science 1995; 269:973-977
(6)Sherrington R et al. Cloning of a gene bearing missense mutations in early-onset familial Alzheimer’s disease. Nature 1995; 375: 754-760.
(7)Poirier J et al. Apolipoprotein E polymorphism and Alzheimer’s disease. Lancet 1993; 342: 697-699.
(8)Selkoe DJ et al. Translating cell biology into therapeutic advances in Alzheimer’s disease. Nature 1999; 399: A23-31.
(9)Kimberly WT et al. The transmembrane asparatases in presenilin 1 and 2 are obligatory for gamma-secretase activity and amyloid-beta protein generation. J. Biol. Chem. 2000; 275: 3173-3178.
(10)Wolozin B et al. Regulation of apoptosis by presenilin 1. Neurobiol. Aging 1998; 19: 523-527.
(11)Buxbaum JD et al. Calsenilin: a calcium-binding protein that interacts with the presenilins and regulates the level of a presenilin fragment. Nature Med. 1998; 4: 1177-1181.
(12)Drouet B et al. Molecular basis of Alzheimer’s disease. Cell Mol. Life Sci. 2000; 57: 705-715.
(13)Marques MA et al. Apolipoprotein E-mediated neurotoxicity as a therapeutic target for Alzheimer’s disease. J. Mol. Neurosci. 2003; 20: 327-337.
(14)Lee Y et al. Apolipoprotein E protects against oxidative stress in mixed neuronal-glial cell cultures by reducing glutamate toxicity. Neurochem. Int. 2004; 44: 107-18.
(15)Jordan J et al. Isoform specific effect of apolipoprotein E on cell survival and β-amyloid-induced toxicity in rat hippocampal pyramidal neuronal cultures. J. Neurosci. 1998; 18: 195-204.
(16)Guela C et al. Aging renders the brain vulnerable to amyloid β-protein neurotoxicity. Nature Med. 1998; 4: 827-831.
(17)Frank RA et al. Biological markers for therapeutic trails in Alzheimer’s disease proceedings of the biological markers working group; NIA on neuroimaging in Alzheimer’s disease. Neurobiol. Aging 2003; 24: 521=536.
(18)Reiter RJ. Oxidative processes and antioxidative defense mechanisms in the aging brain. FASEB J. 1995; 9: 526-533.
(19)Brown GC, Bal-Price A. Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol. Neurobiol. 2003; 27: 325-355.
(20)Gervais, F. G., Xu, D., Robertson, G. S., Vaillancourt, J. P., Zhu, Y., Huang, J., LeBlanc, A., Smith, D., Rigby, M., Shearman, M. S., et al. Involvement of Caspases in Proteolytic Cleavage of Alzheimer’s Amyloid-β Precursor Protein and Amyloidogenic Aβ Peptide Formation Cell 1999; 97: 395–406.
(21)Lu, D. C., Rabizadeh, S., Chandra, S., Shayya, R. F., Ellerby, L. M., Ye, X., Salvesen, G. S., Koo, E. H. & Bredesen, D. E. A second cytotoxic proteolytic peptide derived from amyloid β-protein precursor. Nat. Med.200; 6: 397–404.
(22)Galvan, V., Chen, S., Lu, D., Logvinova, A., Goldsmith, P., Koo, E. H. & Bredesen, D. E. Caspase cleavage of members of the amyloid precursor family of proteins. J. Neurochem.2002; 82: 283–294.
(23)Lu, D. C., Shaked, G. M., Masliah, E., Bredesen, D. E. & Koo, E. H. Amyloid βprotein toxicity mediated by the formation of amyloid-β protein precursor complexes. Ann. Neurol. 2003; 54: 781–789.
(24)Hsia, A. Y., Masliah, E., McConlogue, L., Yu, G.-Q., Tatsuno, G., Hu, K., Kholodenko, D., Malenka, R. C., Nicoll, R. A. & Mucke, L. Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models. Proc. Natl. Acad. Sci. USA 1999; 96: 3228–3233.
(25)Mucke, L., Masliah, E., Yu, G. Q., Mallory, M., Rockenstein, E. M., Tatsuno, G., Hu, K., Kholodenko, D., Johnson-Wood, K. & McConlogue, L. High-Level Neuronal Expression of Aβ1-42 in Wild-Type Human Amyloid Protein Precursor Transgenic Mice: Synaptotoxicity without Plaque Formation. J. Neurosci. 2000; 20: 4050–4058.
(26)Terry, R. D., Masliah, E., Salmon, D. P., Butters, N., DeTeresa, R., Hill, R., Hansen, L. A. & Katzman, R. Physical basis of cognitive alterations in alzheimer's disease: Synapse loss is the major correlate of cognitive impairment. Ann. Neurol.1991; 30, 572–580.
(27)Streit, W. J. Microglia and Alzheimer's disease pathogenesis. J. Neurosci. Res. 2004; 77: 1–8.
(28)Morris, R. Developments of a water-maze procedure for studying spatial learning in the rat. J. Neurosci. Methods 1984; 11: 47–60.
(29)Hsiao, K. K., Borchelt, D. R., Olson, K., Johannsdottir, R., Kitt, C., Yunis, W., Xu, S., Eckman, C., Younkin, S., Price, D., et al. Age-related CNS disorder and early death in transgenic FVB/N mice overexpressing Alzheimer amyloid precursor proteins. Neuron 1995; 15: 1203–1218.

Intervention Strategies for Invasive Candidiasis Infections in Surgical Intensive Care Units of Resource-limited Countries

Invasive fungal infections are major sources of morbidity and mortality among critically ill patients in surgical ICU and have emerged as an important public health problem especially in third-world nations. Over the last decade, fungal sepsis has continued to increase rapidly despite the use of antifungal agents and mortality remains high due to delays in proper diagnosis and administration of appropriate antifungal therapy (Meerssemann et al 2004). The majority of nosocomial fungal infections are due to Candida species. Candida are frequent commensal yeasts of the human gastrointestinal, respiratory, reproductive tracts and the skin. Candida colonization is an important prerequisite to invasive disease and sepsis. A significant proportion of invasive candidiasis occurs in the surgical ICU and rates of Candida colonization increase with length of stay and is exacerbated in critically ill patients with impaired host defenses (Hajjeh et al 2004). The transition of Candida from a harmless commensal fungus to an invasive opportunistic pathogen requires the disruption of anatomical barriers and translocation of pathogen to circulation (Spellberg 2008). Candida systemic infection is associated with several risk factors including broad-spectrum antibiotics, corticosteroids, central venous lines, malnutrition, hemodialysis, mechanical ventilation, renal failure, and venous/urinary catheterization, which are fairly common following major surgery (Bendel et al 2002). Candidemia in surgical patients in the ICU may also occur through the horizontal transmission of Candida by hospital staff through the insertion of central venous or urinary catheters. Significant efforts must be made to develop proper risk stratification strategies to guide antifungal therapy and reduce candidemia-related mortalities in resource-poor nations in an efficient and cost-effective manner.

The diagnosis of severe candidiasis in the third world is frequently based on overall clinical status with few laboratory tests, which are done using cultures of sputum, blood, urine, or stool. However, the reliance on laboratory tests to confirm a diagnosis of invasion can be time consuming and misleading. In fact, approximately only half of patients with invasive candidiasis have positive blood cultures (Solomkin et al 1982). This may be due to difficulty of Candida to grow in culture or by interference from a coincident bacterial infection from the patient. This has significant implications in resource-poor countries as sample cultures are usually the only means of confirming a candidemia diagnosis. Using nonculture markers of candidiasis, such as serum (1,3)-β-ᴅ-glucan (BG) (polysaccharide cell wall components), ᴅ-aribinitol, enolase, and mannan, can be used for a more definitive confirmation of candidemia. For example, serum BG levels exceeding 60 pg/ml positively identifies 80% of patients with invasive fungal disease (Ostrosky-Zeichner et al 2005). Using non-culture diagnostic tools are promising in terms of rapidly identifying candidemia in resource-limited settings. More experimental methods of detecting candidemia from patient samples include using Candida species specific polymerase chain reaction (PCR) or peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) (McMullan et al 2008). PNA-FISH can be performed on Candida blood culture growths and provide rapid determination of species infection and facilitate specific antifungal treatment selection. Although the latter two diagnostic tools still require cultures, they are promising bench-to-bedside tools that will facilitate early detection of invasive candidiasis. Promoting the use of standard and experimental diagnostic aids in resource-poor countries can be more cost-effective as early detection and preventative measures will decrease length of ICU stay.

The concept of early presumptive therapy (EPT) is a strategy that can be used to assess the risk of developing invasive candidemia and the treatment of high-risk patients who exhibit signs and symptoms of the disease even in the absence of positive cultures. Critically ill surgical patients at risk for Candida colonization have an increased risk of developing invasive disease and prevention of colonization improves patient outcomes. However, not all cases of colonization results in invasive candidiasis. Therefore, clinical evaluation becomes a critical and cost-effective strategy in resource-limited settings to assess patients who would benefit from EPT before invasive candidemia development. Unfortunately, there are no established diagnostic tests that reliably distinguish infection from colonization. The overgrowth and invasion of Candida in the gastrointestinal tract corresponds with colonization of other multiple sites. Candiduria, presence and increased growth of Candida in the bladder, can be used as an indicator of disseminated candidiasis, where the presence of candidemia without candiduria is very unlikely (Stone et al 1974). A positive test result for candiduria can be used as a risk factor to initiate prophylaxis with antifungal agents. An important idea to consider is that the urinary system is not the only colonization site that can lead to invasive disease. Investigation into Candida colonization of multiple sites, such as peritoneal cavity or respiratory tract, can provide an indication of high-risk of invasive/disseminated disease for the initiation of EPT. Thereby, early therapy with low doses of antifungal agent(s) may prevent subsequent severe disseminated candidemia and will be more resource-effective in poor nations.

The main intervention for established fungal infections in the ICU involves antifungal therapy. Current key antifungal agents include amphotericin B (AmpB), fluconazole, voriconazole, and the echinocandins. Other agents can include lipid-amphotericin B formulations and 5-flucytosine, however, in resource-limited settings, usually only AmpB and fluconazole are available. When selecting an antifungal therapy, we must consider the drug's activity, toxicity, pharmacological kinetic and dynamic attributes, multi-drug interactions, possibility of resistance development, and affordability. Particularly for critically ill patients in resource-limited settings, these choices become vitally important and new strategies must be adopted to improve survival.

AmpB is widely used against invasive fungal infections due to their broad activity spectrum, clinical efficacy, availability and affordability. However, AmpB is associated with considerable nephrotoxicty and high doses are unsuitable for post-operative patients, especially during renal failure and hemodialysis. Lipid-AmpB formulations which have lower toxic effects may be used but this represents a higher cost per treatment and may not be the most viable option in poor countries. An alternate treatment involves AmpB and fluconazole combination therapy. This allows using lower doses of AmpB in combination with a safer, affordable and effective antifungal to reduce toxicity in critically ill patients with invasive candidiasis. Fluconazole dosage can be adjusted for patients with renal dysfunction and undergoing hemodialysis. However, fluconazole is not without its own caveats. Certain species of Candida (glabrata and krusei) have acquired fluconazole resistance. Although these species comprise less than one-third of all Candida, it raises the emerging issue and need for new therapies especially in resource-poor nations.

Voriconazole is commonly used for invasive aspergillosis but is effective for patients with invasive candidiasis (Kullberg et al 2005). Voriconazole is active against a wide spectrum of fungi and is commonly used against Candida that have gained resistance to fluconazole. Critically ill patients can have variability in drug bioavailability dependant on route of administration and it is generally advisable to administer initial therapy intravenously. However, intravenous voriconazole requires cyclodextrin, which is highly nephrotoxic thereby limiting its use in critically ill patients. Furthermore, unlike the benefits from AmpB, voriconazole and fluconazole combination therapy may lead to azole resistance and adverse drug-drug interactions.

The echinocandins are an increasingly used first-line therapy for critically ill patients with invasive candidiasis in the ICU of developed countries (Sobel et al 2007). Several properties make echinocandins attractive for treatment of invasive fungal infections: broad activity spectrum against Candida, lack of azole cross-resistance which allows the use of combination therapies, high clinical efficacy and low risk for adverse drug-drug interactions. Echinocandins are mainly metabolized by the liver and is unaffected by renal dysfunction. However, echinocandins confer a higher cost and is not readily available in developing nations.

Inappropriate antibiotic treatments and antibiotic misuse have been a major contributing factor in the emergence of resistance bacteria. Furthermore, inappropriate antibiotic treatment is one of the major contributing factors in the development of invasive candidemia in critically ill surgical patients. Antibacterial agents lead to the suppression of commensal intestinal flora, which can inhibit Candida growth and prevent its adherence to mucosal cells (Stone et al 1974). Critically ill surgical patients are prone to measures that can disrupt the normal intestinal barrier such as physical disruption of anatomical barriers due to surgery, intestinal ischemia, bowel obstruction, immunosuppression, and malnutrition. An immunosuppressed state can lead to an increased risk of invasion by low virulent pathogens such as Candida. The gut-associated lymphoid tissue (GALT) in the human gastrointestinal tract is a mucosal innate defense system that protects against invasion by pathogens. Host defenses in the GALT have been associated with innate phagocytic cells, which protect against dissemination, mediated by a specific γδ-T cell subset population, which prevent colonization of pathogens (Hajjeh et al 2004). Immunosuppression is a common measure following surgery and increases the patient's risk of developing invasive fungal disease. Malnutrition is another major concern for patients in third-world countries and can alter the commensal intestinal flora into a state that promotes Candida overgrowth and colonization. The implementation of a pro-biotic nutrition plan for all patients following surgical procedures could limit the risk of developing disseminated candiasis and replace the commensal microbes responsible for the inhibition of Candida growth and colonization.

A final concern to address in terms of public health is the development and spread of nosocomial candidemia. Candida colonization of hospital workers' hands may facilitate horizontal transmission of the pathogen particularly if they are involved in post-surgical care of the patients. For instance, the handling of central venous or urinary catheters by a nurse carrying Candida can serve as an increased risk for candidemia in surgical patients and this risk may only be elevated with prolonged stays in the ICU. This problem is especially relevant in resource-poor nations as poor sanitation habits and lack of proper health care techniques due to perhaps the disruption of resources through military conflict, lack of sanitary food/water, or overcrowding in hospitals can lead to increased risks of nosocomial infections. The education of third-world health care workers on the subject and establishing the practice of hygiene techniques will dramatically reduce the risk of nosocomial infections (including fungal infections) in the ICU. Perhaps health care workers can be instructed to wear personal protective equipment (gloves, face mask, etc) when administering care, discontinue the reuse of catheter tubes to cut costs, and not to overcrowd critically ill patients in the ICU. Adoption of simple sanitation practices will work to greatly reduce transmission of nosocomial fungal infections.

The incidences of candidemia in surgical patients in resource-poor nations is increasing at an alarming rate given the difficulty in diagnosis, emerging antifungal resistance, and the high mortality and morbidity that is associated with invasive candidiasis. Further research that aims at understanding the epidemiology of invasive fungal infections can provide insights into the development of risk stratification strategies to minimize disease in critically ill surgical patients in the ICU. A more stringent clinical assessment of critically ill surgical patients must be adopted to identify fungal infections, even in moderately ill patients who are still at risk of developing invasive, disseminated fungal infections. Clinical thresholds must prophylaxis and initiation of antifungal treatment must be lowered, since traditional methods of multiple blood cultures or positive biopsies before treatment are too late to prevent serious illness and death. More research is needed to develop diagnostic tools that can distinguish between infection from colonization and invasion. New intervention strategies must be adopted where the misuse of antibiotics are limited and more definitive laboratory tests used to identify Candida species infection to initiate the surgical patient on EPT using low doses of minimally less toxic antifungals to prevent life-threatening invasive disease. A novel idea involves the ability to alter the gastrointestinal microbiome to facilitate an environment that restricts growth and colonization of Candida. Overall, the best intervention strategies for invasive candidiasis in surgical patients in resource-limited settings would involve early detection of invasive fungal infections, prevention of its spread in the ICU, and prophylactic/pre-emptive therapy that would be more efficient and resource-effective by decreasing the need for costly higher efficacy antifungals, risk of antifungal resistance and length of ICU stay.

References:

Bendel CM, Wiesner SM, Garni RM, Cebelinski E, Wells CL. Cecal colonization and systemic spread of Candida albicans in mice treated with antibiotics and dexamethasone. Pediatr Res.2002;51(3):290-295.

Hajjeh RA,Sofair AN,Harrison LH, et al. Incidence of Bloodstream infections due to Candida species and in vitro Susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. J Clin Microbiol. 2004;42(4):1519-1527

Kullberg BJ, Sobel JD, Ruhnke M, et al. Voriconazole versus a regimen of amphotericin B followed by fluconazole for candidaemiainnon-neutropenicpatients: a randomized non-inferiority trial. Lancet.2005;366(9495):1435-1442

McMullan R, Metwally L, Coyle PV et al. A prospective clinical trial of a real time polymerase chain reaction assay for the diagnosis of candidemia in nonneutropenic critically ill adults.Clin Infect Dis.2008;46(6):890-896.

Meersseman W, Vandecasreele SJ, Wilmer A, Verbeken E, Peetermans WE, Van Wijngaerden E. (2004) Invasive aspergillosis in critically ill patients without malignancy. Am J Respir Crit Care Med. 170(6):621-625.

Ostrosky-Zeichner L, Alexander BD, Kett DH, et al. Multicenter clinical evaluation of the (1->3) beta-D-glucan assay as an aid to diagnosis of fungal infections in humans. Clin Infect Dis. 2005;41(5):654-659.

Sobel JD, Revankar SG. Echinocandins - first-choice or first-line therapy for invasive candidiasis? N Engl J Med. 2007; 356(24): 2525-2526

Solomkin ,J.S., Flohr, A.M., Simmons, R.L.: Indications for therapy for fungemia in postoperative patients. Arch. Surg. 117:1272,1982

Spellberg B. Novel insights into disseminated candidiasis: pathogenesis research and clinical experience converge. PLoS Pathog. 2008;4(2):e38.

Stone, H.H., Kolb, L.D., Currie, C.A., Geheber, C.E., Cuzzell, J.Z.:Candida sepsis: pathogenesis and principles of treatment. Ann. Surg.179:697,1974

Commentary: Link found between infectious disease and IQ

Christopher Eppig and colleagues at the University of New Mexico, Albuquerque recently reported that "a country's disease burden is strongly linked to the average IQ of its population". You can read the article here at NewScientist: http://www.newscientist.com/article/mg20727670.301-link-found-between-infectious-disease-and-iq.html) Now, being a researcher, I am always suspicious of articles (especially clinical or translational medicine articles) that make such bold statements while only showing a small table or graph that seems to support the hypothesis, so I decided to take a look at the original article. Sure enough, Christopher Eppig is pubmed-able and I was able to read his paper (PMID: 20591860).

Basically, Eppig et al. state that cognitive ability or intelligence is related to the intensity of infectious diseases on the basis that a developing human brain requires a certain metabolic cost and fighting an infection decreases the ability of the body to provide sufficient nutrition. In human newborns, the metabolic demand of the developing central nervous system is great, approximately 90% of the body metabolic demand is supplied to the brain (Holliday 1986) and nutrition is vital to mental development, and malnutrition can lead to decreased brain mass, head size, and lower psychometric intelligence (Lynn 1990, 1993). It is well realized that deficient metabolic supply will diminish the ability of the brain to develop properly, especially at a developmental timepoint, where the brain is undergoing a high level of structural and functional assembly. Eppig et al. offers the parasite-stress hypothesis where parasitic infections can energetically affect the body in four ways: i) loss of tissue that must be replaced at a biological cost to the host, ii) malabsorption of nutrients through the gastrointestinal tract, iii) hijacking of host cellular machinery at high biological cost to the host, and iv) activation of the immune system to fight the infection. Now this doesn't seem like a very far-fetched idea: energetic costs associated with fighting an infection, especially a chronic infection, would severly diminish the level of metabolic supply for other aspects, such as brain development during early age.

However, this hypothesis fails into taking account that the brain is a very plastic organ. Assembly and reassembly on a molecular and cellular level of the brain (and central nervous system for that matter) is continous and does not just end in preadolescence. In fact, it is well known that the brain continues its development well through adolescence and into adulthood (Cunningham et al. 2002; Sowell et al. 1999; Casey et al. 2005). Furthermore, Gage 2002 shows that neurogenesis, the process by which neurons are generated. is evident even in the adult brain. Also, most infections persist only for a short while and not for years, so long as the body is able to fight and clear the infection. Its seems unlikely that the metabolic demand to fight infection would be diverted away from brain development for such an extended period of time to have devastating effects on cognitive ability. And in children with severe infections, where the pathogen persists and the disease is chronic, have more to worry about than just retarded brain development.

Of course, I am ignoring the inconsistency of using IQ tests as an assessment of intelligence and cognitive ability. And on a social standpoint, in my opinion, the lack of access to education and the hardships associated with living in resource-poor countries provides more of an association for a lower IQ score. Futhermore, what about children born in infectious "high risk" countries who then migrate to "developed" countries during adolescence or adulthood? How do their IQ scores compare to control populations?

In conclusion, it seems that although parasitic infections are a harsh reality for children living in resource-poor countries, its association with lower intelligence and cognitive ability is unclear, and is likely not a major contributor.