In worlds, as mentioned earlier, defensins are released by scavenger cells and epithelial cells, such as in the tegument, respiratory, GI and GU piece of lands, which provide a first line of defense mechanism against occupying pathogens ( Harder et al. , 2007 ) . Human defensin concentrations are normally found to be at 40ng/ml in the blood but in the event of an infection, these concentrations can increase exponentially to 1000ng/ml in localized countries of infection ( Yang and Oppenheim, 2003 ) . Defensins perform multiple functions ( figure 1.8 ) with the most of import being the powerful antimicrobic activity exhibited against assorted occupying micro-organisms, such as Gram-negative bacteriums ( E.
coli ) , Gram-positive bacteriums ( S. aureus ) , mycobacteria ( M. fortuitum ) , certain Fungi ( C. albicans ) and enveloped viruses ( HSV ) ( Lehrer et al. , 1993 ) although at high concentrations defensins have been found to be cytotoxic to mammalian cells such as in lung epithelial tissue and tumor cell lines ( Ganz, 2003 ) . The antimicrobic activity displayed by I±- and I?-defensins is markedly reduced and potentially inhibited in the presence of serum, plasma proteins, extra bivalent cations such as Mg2+ and Ca2+ and ionic salts at their physiological concentrations ( i.e. 150mM of NaCl ) therefore accordingly optimum antimicrobic effects occur in phagosomes ( phagocytic vacuoles ) in scavenger cells and mucosal and epithelial surfaces such as the tegument, where ionic salt concentrations are low, as opposed to blood and extracellular fluid ( Yang and Oppenheim, 2003 ) .
The exact method by which defensins apply their microbicidal belongingss is non wholly understood, but it is widely accepted that defensin-induced microbic obliteration is due to the break of microbic cytoplasmatic membrane unity ( De Smet and Conreras, 2005 ) . To execute antimicrobic actions, the exposed cationic parts of defensins ab initio attract and later adhere electrostatically to the negatively charged microbic surfaces, which constitute of anionic molecules ( PAMPs ) such as the anionic medieties in LPS, negative phosphate groups and anionic lipoids on the outer membranes of Gram-negative bacteriums, and teichoic acids on the surface of Gram-positive membranes ( Brogden, 2005 ) . In the instance of Gram-negative bacteriums, to entree the interior cytoplasmatic membrane, defensins must foremost get the better of the outer membrane which is covered by bivalent Mg2+ and Ca2+ ions ( figure 1.3 ) ; as defensins have a higher affinity for LPS they are able to displace these cations with easiness, permeate the outer membrane and accordingly interact with the interior membrane ( Zasloff, 2002 ) . Defensins do non expose cytotoxic microbicidal activity to host and other mammalian cells ( unless at high concentrations ) as, in contrast to procaryotic membranes, the host ( eucaryotic ) plasma membranes are composed preponderantly of zwitterionic ( impersonal ) phospholipids such as sphingomyelin, and impersonal membrane-stabilising cholesterin molecules thereby cut downing defensin interactions ( Zasloff, 2002 ; Shai, 2002 ) .The amphipathic nature of defensins causes the hydrophobic and ( charged ) hydrophilic parts on the peptide to be spatially separated, hence leting the polar cationic hydrophilic parts of defensins to interact with anionic phospholipid caputs and H2O, and the hydrophobic parts to interact and adhere consequently to the phospholipid bilayer, with easiness ( Yang et al. , 2004 ; Shai, 2002 ) .
Once bound to microbic plasma membranes, defensins normally undergo conformational alterations which are energetically favorable, where defensin peptide monomers combine to organize oligomers and accordingly orientate, either parallel or perpendicular to the membrane, to infix into the lipid bilayer thereby taking to membrane permeablisation ( Diamond et al. , 2009 ) . It is by and large thought that defensin-induced membrane pervasion occurs due to formation of multimeric voltage-dependent ion-permeable pores. Though the exact mechanism has non yet been established, three theoretical theoretical accounts exist to explicate the pervasion of microbic cytoplasmatic membranes: barrel-stave ( figure 1.9-A ) , toroidal-pore ( figure 1.9-B ) and rug theoretical account ( figure 1.9-C ) ( Brogden, 2005 ) .
The barrel-stave theoretical account ( figure 1.9-A ) supports the position that the edge defensin peptides foremost assemble in a coiling construction on the pathogen outer membrane, orient sheer and accordingly infix in the lipid bilayer where the hydrophobic parts of defensins align with the lipid nucleus and hydrophilic parts face externally, interacting with H2O, therefore organizing a pore. Initially, a lower limit of two defensin monomers traverse the membrane, which initiate further enlisting of monomers, taking to an addition in pore size. The administration forms a pervasion tract, a stable transmembrane channel-like pore, which allows escape of intracellular H2O and electrolytes ( Oren and Shai, 1998 ; Shai, 2002 ) .
Harmonizing to the toroidal-pore theoretical account ( figure 1.9-B ) defensins ab initio insert similar to the barrel-stave theoretical account, in that the peptides accumulate and adsorb on the membrane surface and infix sheer into the membrane surface. Unlike the barrel-stave theoretical account, after the polar parts of the defensins align with the polar lipid caput groups, the defensin peptides induce a positive curvature in the membrane monolayers so that finally both phospholipid monolayers connect with each other in a uninterrupted crook, ensuing in an gap, named a toroidal-pore. As a consequence, the lms of the end point pore is non merely lined with defensin peptides but by polar phospholipid caput groups every bit good ( Brogden, 2005 ; Diamond et al. , 2009 ) .The rug theoretical account ( figure 1.9-C ) proposes that defensins aggregate either as monomers or oligomers on the microbic membrane surface, where they align parallel to the membrane positioning themselves so that the hydrophobic parts interact with the lipid nucleus and hydrophilic parts with the polar phospholipid caputs, accordingly organizing an extended carpet-like bed ( Shai, 2002 ) . Once a threshold concentration is reached ( i.
e. a peculiar concentration of defensin peptides accumulate upon the bilayer surface ) , the mark membrane destabilises, organizing big transient holes similar to toroidal-pores which cause the loss of cytoplasmatic contents. Ultimately defensins disintegrate the membrane curvature, similar to a detergent, finally taking to micelle formation ( Oren and Shai, 1998 ; Brogden, 2005 ) . Break of microbic membrane unity by defensins in either of the theoretical accounts finally leads to loss of the electrochemical gradient and membrane potency, break of cellular respiration and eventful escape of intracellular contents such as electrolytes, H2O and ATP, finally ensuing in osmolysis and cell decease ( Shai, 2002 ) .Surveies on E.coli have shown that one time internalised I±-defensins such human neutrophil peptide ( HNP ) 1 and 2 interfere with and suppress several intracellular procedures such as DNA, RNA and protein synthesis, therefore proposing the possibility that defensin-induced membrane pervasion is non the lone mechanism by which defensins are able to kill bugs, though farther surveies are necessary ( Brogden, 2005 ) .Figure 1.
8 – General belongingss of mammalian defensins. Human I±- and I?-defensins, apart from exhibiting powerful antimicrobic activity against many beings, are chemotactic to many cells of the innate ( monocytes, neutrophils ) and adaptative ( T cells, DCs ) immune system, cause mast cell degranulation and subsequent histamine release, fix lesions, promote cellular growing, interact with complement Cascadess and inhibit ACTH and associated glucocorticoid synthesis. Rabbit defensins entirely display opsonization belongingss ( Lehrer, 2004 ) .Figure 1.9 – The plausible mechanisms of defensin-induced microbic membrane break and eventful violent death.
The theoretical accounts that exist are the barrel-stave ( a ) and toroidal-pore ( B ) where pores are formed, and the rug ( degree Celsius ) theoretical account where membrane is disintegrated in a detergent-like mode, finally taking to micelle formation. Hydrophilic parts of defensin peptides are shown in ruddy ; hydrophobic parts are shown in blue ( Brogden, 2005 ) .Defensins are besides responsible in the upregulation of the innate immune response ; this upregulation is carried out by a assortment of mechanisms ( figure 1.10 ) . I±-defensins such as human neutrophil-derived peptides ( HNP ) 1 and 2 when released by azurophilic neutrophil granules are chemotactic to monocytes and macrophages and therefore enroll them to sites of infection and redness ( Lehrer et al.
, 1993 ) . I±-defensins besides enhance phagocytosis therefore easing the innate immune system in the fleet remotion of occupying pathogens ( Yang et al. , 2004 ) whilst I?-defensins, in peculiar human beta defensin ( HBD ) 2, promote migration of mast cells and bring on mast cell activation and eventful degranulation, doing the release of histamine and prostaglandin-D2 which addition vascular permeableness, thereby easing the inflow of neutrophils and other scavenger cells to inflammatory sites ( Yang et al. , 2001 ) . Defensins ( such as HNPs ) have besides been shown to excite epithelial cell production of IL-8, particularly in the lungs, which aboard mast cell merchandises such as histamine, are powerful neutrophil chemoattractants ; hence indirectly defensins advance farther accretion of neutrophils to sites of infection, where they in bend release extra defensins, which accordingly induce the coevals of more IL-8 resulting in a ‘positive-feedback cringle ‘ , magnifying the inflammatory innate immune response ( Yang et al. , 2001 ; Yang et al.
, 2002 ) . Furthermore it has been observed that human defensins such as HNP-1 have the ability to modulate the classical tract of the complement system by adhering to complement molecule C1q, thereby diminishing or increasing complement activation, an indispensable constituent of unconditioned unsusceptibility ( Yang et al. , 2004 ) .In add-on to upregulating unconditioned unsusceptibility, I±- and I?-defensins are besides chemotactic to assorted cells of the adaptative immune system such as immature DCs, CD4+ T cells and resting memory T cells and hence watchful and associate the adaptative immune response ( the 2nd line of defense mechanism ) to the innate immune response ( figure 1.10 ) .
Certain defensins such as HBD-1 to 3 bind straight to chemokine CCR6 receptor accordingly heightening enlisting of the aforesaid cells at sites of infection and redness, taking to the ripening of iDCs into antigen showing mDCs, which display MHC II composites on cell surfaces ready for antigen consumption, and induction of the effecter stage of the adaptative immune response. Defensins have besides been shown to excite DC ripening by heightening synthesis of potent DC ripening inducers such as IL-8 and TNF-I± and indirectly promote migration of mDCs to spleen and lymph nodes where they present antigens to naA?ve T cells, thereby easing and rushing the activation of the adaptative immune response ( figure 1.11 ) ( Yang et al. , 2002 ) . Defensins are able carry out their chemotactic belongingss at highly low nanomolar concentrations in the organic structure, and unlike antimicrobic activity, defensin-induced chemotaxis is non inhibited by ionic salts ( NaCl ) , bivalent cations ( Mg2+ and Ca2+ ) and serum ( Yang and Oppenheim, 2003 ) .At low concentrations defensins have besides been shown to be mitogenic to fibroblasts and excite DNA synthesis and subsequent proliferation in epithelial cells ( Murphy et al. , 1993 ) , particularly seen in the lungs, proposing that defensins take part strongly in airway tissue remodelling ( in certain inflammatory conditions ) ( Guani-Guerra et al.
, 2010 ) . Defensins have besides been shown to excite mucin cistron look in lung epithelial tissue, thereby advancing the synthesis of mucins which contribute to epithelial regeneration and aid in the clearance of occupying beings ( Diamond et al. , 2009 ) . Furthermore defensins besides play an of import function in wound fix as surveies have determined that I±-defensins such as HNP-1 to 3 stimulate epithelial cell migration ( in a time- and dose-dependent mode ) thereby increasing the rate of lesion closing ( Lehrer, 2004 ) .
Increased cellular migration and proliferation is besides induced by I?-defensins ( HBD-2, 3 and 4 ) in skin epithelial cells ( cuticular keratinocytes ) ( Harder et al. , 2007 ) . In vitro theoretical accounts have besides revealed that HNP-1 “ increases the look of pro-collagen messenger RNA and protein in cuticular fibroblast civilizations ” hence heightening cutaneal wound mending by increasing the deposition of extracellular matrix in injured tissues ( Steinstraesser et al.
, 2008 ) . In add-on, increased look of I?-defensins has been observed in injured tissues after injuring, yet in the cases of chronic lesions and Burnss, reduced degrees of defensins and other AMPs, and inordinate growing of bacteriums such as S. aureus has been seen ; therefore it has been suggested that I?-defensins such as HBD-2 and 3 are critical in wound fix as they are required to ease and speed up lesion closing, which is achieved by keeping a barrier against infection by cut downing growing and subsequent colonization of bacteriums and other disease-causing bugs ( Harder et al. , 2007 ; Radek and Gallo, 2007 ) .Additionally, human defensins such as HNP-2 are capable of suppressing fibrinolysis, by modifying tissue-type plasminogen activator ( TPA ) and plasminogen from adhering to fibrin and blood vessel endothelial cells. Defensins achieve this as a consequence of viing for fibrin binding sites and therefore suppressing TPA ( Risso, 2000 ) . TPA converts plasminogen to plasmin and accordingly causes the debasement of fibrin ; therefore suppression of fibrinolysis is thought to let the curdling cascade to successfully go on in infective and inflammatory state of affairss, thereby assisting to command microbic infections ( Eales, 2003, Risso, 2000 ) .Finally it has besides been shown in worlds, that certain defensins ( such as HNP-4 ) are adversaries to ACTH i.
e. inhibit adrenal steroid endocrine ACTH synthesis at nanomolar concentrations, by reversibly adhering to ACTH receptors and later barricading ACTH and associated steroid endocrines from adhering to the complementary receptors, thereby showing corticostatic activity. ACTH and associated steroid endocrines are exceptionally immunosuppressive therefore this repressive defensin activity is good as it enhances the initial innate immune response. Effective suppression of adrenal glucocorticoid synthesis has been observed when defensin degrees increase significantly ( to 100I?g/ml ) in infections ( Yang and Oppenheim, 2003 ) .
Figure 1.10 – Representation of the possible methods by which human defensins activate and link innate and adaptative unsusceptibility.
When host epithelial barrier is breached, occupying micro-organisms cause the production of defensins which affect the innate immune response in five possible ways ( as mentioned in diagram ) . This can ensue in the enlisting of T cells and activation of DCs in the adaptative immune response, straight by defensins themselves, or indirectly, by cytokines released in the innate response ( Yang et al. , 2004 ) .
11 – The possible mechanisms by which human defensins can act upon host adaptative immune response.
Defensins are produced by epithelial cells ( Ep ) and neutrophils ( N ) at sites of infection. Defensins promote enlisting of immature DCs ( iDC ) to infected tissues ( a ) and heighten antigen ( Ag ) consumption by iDCs ( B ) . They are besides able to do iDCs to maturate into mature DCs ( mDC ) by advancing synthesis of potent DC ripening inducers such as IL-8 and TNF-I± ( degree Celsius ) and facilitate enlisting of T cells ( vitamin D ) thereby rushing the activation of the adaptative immune response ( Adapted from Yang et al.
, 2002 ) .
1.5 Human Monocytic Mono Mac 6 ( MM6 ) cell line
To insulate monocytes from blood can be rather hard and can bring forth cells of changing conditions, hence utilizing cell lines in vitro which can mime belongingss of monocytes in vivo is utile to utilize as an experimental theoretical account for look intoing pathological conditions and detecting effects of many stimulations such as drugs, or in this undertaking, bacterial LPS ( Wright et al. , 1996 ) .
There are several monocyte-like cell lines available ; the selected cell line to be used in this survey is the human monocytic Mono Mac 6 ( MM6 ) cell line. This peculiar cell line was acquired from a German cell culturing company DSMZ-German Collection of Microorganisms and Cell Cultures and derived in 1985, from the peripheral blood of a 64-year old male patient enduring from acute monoblastic leukemia ( AML M5 ) ( Zeigler-Heitbrock et al. , 1988 ) . Alongside MM6, another ringer, Mono Mac 1 ( MM1 ) was isolated from the patients ‘ blood ( a month before the patient ‘s decease ) ; both were assigned, morphologically, cytochemically and immuno-biologically, to the monocyte line of descent.MM6 cell line, in comparing to the other human monocytic cell lines such as MM1, U937 and THP-1, is the merely cell line at present which exhibits phenotypic and functional belongingss of mature monocytes and hence provides a really suited in vitro theoretical account for mature monocytes ( Zeigler-Heitbrock et al. , 1988 ; Wright et al.
, 1996 ) .These belongingss include the look of LPS receptor CD14 on MM6 cells ‘ extracellular surface, ability to phagocytose antibody-coated red blood cells and mycobacteriums, responding with a panel of monoclonal antibodies specific for macrophages, antigen look in response to IFN-I? , let go ofing reactive O species ( Zeigler-Heitbrock et al. , 1988 ) and the production of an inflammatory response by releasing prostaglandins and cytokines such as IL-1I? , IL-6 and TNF-I± ( Zeigler-Heitbrock et al. , 1994 ) .
Other advantageous belongingss of human MM6 cell lines are that they are grown in suspension therefore do non necessitate a surface to turn upon and are later easy transferred and manipulated and that the MM6 cell line is a uninterrupted cell line hence supplying an limitless supply of homogeneous cells rapidly which would be utile in bring forthing dependable consequences ( Drexler et al. , 2000 ) .MM6 cells grow as individual or circular cells or as a little bunch of cells in suspension, though on occasion can adhere slackly to vessel walls ( MacLoed et al. , 1993 ) . Morphologically the cells have an approximative diameter of 16I?m, villous cellular surface, a big unit of ammunition or kidney shaped nucleus with a few nucleole, many cell organs such as chondriosomes, Golgi setup, unsmooth endoplasmic Reticulum every bit good as legion lysosomes, granules and cysts present in the cytol ( figure 1.12 ) ( Zeigler-Heitbrock et al. , 1988 ) .MM6 cells, though widely used as in vitro cell theoretical accounts, have non been used often as theoretical accounts in surveies look intoing the look of defensin cistrons.