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Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5421_Библиотеки_им_академика_М_И_Перельмана

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observed that there was an elevation in levels of TNF- in Female T2DM cases asα
compared to Male T2DM Cases which are in agreement with the findings of Insha
et al., [9, 10, 33, 35]. There are many research studies on this subject which dem-
onstrated that levels of markers of inflammatory reactions increased with the
decrease in insulin sensitivity depending on the severity of T2DM [36, 37]. In this
study both Male and Female sexes have confirmed the importance of inflammatory
mediators in the pathogenesis of T2DM. The levels of TNF- rise significantly inα
both sexes compared to control group showing correlation with glycemic profile
and Insulin sensitivity thus, being considered an independent predictor of risk of
developing T2DM [34].
This study experimentally determined that only pro-inflammatory cytokine
TNF- can leads to pathogenesis of T2DM while other inflammatory cytokinesα
shows negative and weak correlation with T2DM. This research study showed
vibrant changes in concentrations of pro-inflammatory cytokines, in T2DM. Our
findings are in concurrence with the results of [32], which showed serum expres-
sion of candidate mediators (TNF- ) are elevated in T2DM cases which are inde-α
pendent of physical activity and other risk factors [38]. It is suggest that TNF- is anα
important predictor for the development of T2DM for Male and female, in both
rural and urban populations.
Interestingly, results of our study showed a high degree of correlation between
these promising cytokines (TNF, WBC) in T2DM in comparison to healthy controls.
The results are statically significant. In this case control study, we found in our–
T2DM cases there were significantly higher concentration of TNF- as compared toα
those of controls which may be the possible cause of low grade inflammation and
predisposes subjects to the T2DM or towards its complications. These assertions aggress with the findings of AL-Shukaili, et al. [39]. Furthermore our experimental finding provides evidence that the pattern and variation of these cytokines (TNF- ,α
and WBC) are important in the pathogenesis of T2DM [32]. Significant correlation
of TNF- inflammatory mediator in T2DM cases with glycemic profile and insulinα
sensitivity leads to pathogenesis of diseases in this ethnic population [32]. These
findings are in agreement with the fact that inflammatory reactions depends on
group of cytokines rather than a single one. The reports of inflammation has a role
in pathogenesis of T2DM has been elucidated in several studies in different
populations.
Figure 7.
Correlation of TNF- with insulin sensitivity in cases.α
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Inflammatory mediators Cases Controls
HBA1c Fasting glucose HBA1c Fasting glucose
Males n = 81 Females n = 79 Males n = 81 Females n = 79 Males n = 80 Females n = 80 Males n = 80 Females n = 80
TNF- *p = 0.02α
*r = 0.89
p = 0.003
r = 0.883
p = 0.004
r = 0.459
p = 0.005
r = 0.546
*p = 0.035
*r = 0.388
p = 0.011
r = 0.368
p = 0.013
r = 0.260
p = 0.063
r = 0.211
Table 6.
Pearson correlation coefficients of inflammatory mediators with glycemic profile, sex-wise.
Inflammatory mediators Insulin HOMA-IR Insulin HOMA-IR
Males n = 81 Females n = 79 Males n = 81 Females n = 79 Males n = 80 Females n = 80 Males n = 80 Females n = 80
TNF- *p = 0.008α
*r = 0.478
p = 0.009
r = 0.368
p = 0.008
r = 0.374
p = 0.004
r = 0.388
*p = 0.012
*r = 0.016
p = 0.011
r = 0.019
p = 0.111
r = 0.099
p = 0.008
r = 0.319
Table 7.
Pearson correlation coefficients of inflammatory mediators with insulin sensitivity (sex-wise).
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5. Conclusion
The study findings confirms that TNF- , plays a positive role in the pathogenesisα
of T2DM and can act as early prediction biomarkers which can prevent T2DM in
this population. Further studies on the wider range of inflammatory mediators in
association with other biochemical, immunoassay and hematological parameters are
needed to establish role of inflammatory markers as early prediction biomarkers
which can prevent T2DM.
6. Highlights of chapter
1.Inflammation is initiated by trauma or injury, infection, and hence effects
cascades of numerous cytokines and white blood cells. The low grade
inflammation triggers inflammatory cells like neutrophils, macrophages and
monocytes in blood stream and also expresses the pro-inflammatory cytokines
like Tumor necrosis factor-alpha, and interleukin-6.
2.The liver cells synthesize acute-phase proteins under the stimulus of some cytokines, which flow through the bloodstream, reach the site of
inflammation, and eradicate the pathogens through opsonization and eliciting
the complement pathways.
3.The variations in the serum concentrations of TNF- leads to pathogenesis ofα
T2DM.
4.Diagnostic routine tests are sometimes invasive. To augment the modern diagnostics in patient care, the employment of noninvasive biomarkers are
needed.
5.Molecular biological tools have modernized the field of the biomarkers. For the development of biomarkers, genomics and proteomics, pathophysiology of a
disease are needed to understand, the most available technique is correlating
serologic markers with clinical parameters.
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Chapter 6
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Mass Spectrometry (Imaging) for Detection and Identification of Cyclic AMPs: Focus on Human Neutrophil Peptides (HNPs)
ElineBerghmans and GeertBaggerman
Abstract
Antimicrobial peptides (AMPs) are known best for their role in innate immunity against bacteria, viruses, parasites and fungi. However, not only are they showing increasing promise as potential antimicrobial drug candidates, recently, it has been reported that certain AMPs also show a cytotoxic effect against cancer cells. Their possible antitumor effect could make AMPs interesting candidate cancer biomarkers and a possible lead for new anticancer therapy. Due to their cyclic structure, detection and identification of AMPs is challenging, however, mass spectrometry (imaging; MSI) has been shown as a powerful tool for visualization and identification of (unknown) cyclic AMPs. In this Chapter, we will discuss how mass spectrometry (imaging), com- bined with the use of electron-transfer dissociation (ETD) as fragmentation technique, can be used as a reliable method to identify AMPs in their native cyclic state. Using this approach, we have previously detected and identified human neutrophil peptides (HNPs) as important AMPs in cancer, of which a detailed bacterial, viral and cancer­related overview will be presented.
Keywords: human neutrophil peptide 1, 2 and 3 (HNP1-3), proteomics, mass spectrometry (imaging), immunomodulatory function, non-small cell lung cancer (NSCLC)
. Introduction
In the beginning of last century, the first antimicrobial protein, lysozyme, was reported. A few years later, the best known antimicrobial compound, called penicillin, was discovered, which made research into natural antimicrobial proteins/peptides (AMPs) a very important research domain for therapeutic molecules that can be used against bacterial infections [1]. AMPs are naturally occurring small proteins (or peptides) in different organisms and are produced by many tissues and different cell types, acting as host defense molecules against bacteria, but with some also showing a fast antifungal, antiviral, antiparasitic and antitumor response [1–3].
The largest part of AMPs consists of antibacterial peptides with an inhibitory activity towards bacteria, both Gram-positive and Gram-negative [2]. Studies have revealed that AMPs exhibit an overall positive charge, allowing electrostatic inter­actions with negatively charged phospholipid groups in the bacterial membrane.
Insights on Antimicrobial Peptides
By this attribute, pores can be formed by AMPs to disrupt the membrane integrity. Some AMPs are able to cross the lipid bilayer, followed by disruption of intracel­lular functions such as blocking enzyme activity or inhibition of protein synthesis, both resulting in bacterial cell lysis [1]. For this reason, AMPs are often referred to ‘natural antibiotics’.
AMP activity is not restricted to antimicrobial mechanisms, also AMP activity against parasites has been observed: a few AMPs are reported as antimalarial peptides and can possibly serve as new future drug targets against the malaria parasite. For example, cecropins have been shown to block the development of oocysts into sporozo­ites, while dermaseptins (and some derivatives) have been found to be able to permea­bilize the host cell membrane [4].
Furthermore, a subset of AMPs have shown antifungal characteristics against some fungi commonly found in food and agriculture, but also against the common Aspergillus and Candida albicans infections [2]. These antifungal peptides can inter­act with fungal membranes to form pores, comparable to the AMP mechanism in bacteria, but they can also act by targeting the specific fungus cell wall or by acting as nucleic acid inhibitors through direct binding to nucleic acids [5]. A smaller part of AMPs also exhibit antiviral activity, by acting through different mechanisms. A first mechanism includes inhibition of virus attachment and cell membrane fusion. As an example, during the recent COVID-19 pandemic, the antiviral peptide (AVP) EK1C4 has been found to be very effective against S-mediated membrane fusion of the viral particles, thus inhibiting entry of the virus and thereby infection [6]. Another example of inhibition of virus attachment is demonstrated by derma­septins, which possibly affect the lipid bilayer to alter the fusogenic properties of herpes simplex virus [7]. The virus for host cell infection can also be impeded by the direct action of certain AMPs, such as indolicidin, against enveloped virions, causing membrane instability by destruction of the virus envelope [8]. Combined, there is great potential for future therapeutic development of AVPs for both preven­tion as treatment of infection [6]. A small number of AMPs are believed to be active as anticancer peptides (ACP). It has been suggested that they specifically target the membrane of cancer cells through interaction with phospholipids, mainly phos­phatidylserine, present at the outer leaflet of the cancer cell membrane in higher amounts compared to normal cells. Moreover, the ACP LTX-315 has demonstrated both cytolytic and immunogenic properties towards cancer cells, as LTX-315 induces tumor antigen and danger-associated molecular patterns (DAMPs) release, triggering an immune response towards the cancer cells [9].
AMPs are considered key components of the innate immune system, as shortly after an infection, these are promptly synthesized to neutralize a wide variety of pathogens, but through another mechanism compared to that of cytokines or phagocytes [10]. High concentrations of AMPs are usually required to exert an optimal pathogen killing activity, but in vivo, lower concentrations of AMPs are reported, in this case possibly acting as potent immune regulators, also leading to pathogen killing but rather through an indirect mechanism [11]. Besides permeabi­lizing lipid membranes and bacterial walls, their primary role as antimicrobicidal agents, other targets of AMPs are thus reported. Recently, some AMPs are found to also modulate immune responses in vertebrates, through chemotactic activity, attraction, activation and differentiation of leukocytes and monocytes, influencing Toll-like receptor (TLR) recognition and through secretion of proinflammatory cytokines and chemokines, although their underlying mechanisms have not been fully characterized yet [1].
The best studied AMPs include the human defensins and cathelicidins and both have been shown to be chemotactic: defensins (hBD) recruit (memory) β Tcells and immature dendritic cells through their chemotactic activity, suggesting
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Mass Spectrometry (Imaging) for Detection and Identification of Cyclic AMPs: Focus on Human… DOI: http://dx.doi.org/10.5772/ TexLi.99251I
they promote cellular immune responses via interactions with the G protein­coupled receptor CCR6 [11]. Another example of direct AMP chemotaxis includes cathelicidin LL-37, an AMP that has been proven to be chemotactic for neutrophils, monocytes and T cells, but not dendritic cells [11]. Additionally, an indirect chemotactic effect is possible by AMPs through inducing the release of pro-inflammatory cytokines and chemokines, to further refine and activate the innate, and eventually the adaptive, immune response [ ]. In synergy with 1, 11 particular immune mediators, LL-37 has been shown to enhance IL-6 and IL-10 cytokine production, even as the production of macrophage chemoattractant proteins (MCP-1 and MCP-3) chemokines, resulting in an strengthened (innate) immune response [12]. Toll-like receptors (TLR) are key players in innate immu­nity by recognizing microbe-associated molecular patterns (MAMPs). TLR activation leads to secretion of AMPs, but some AMPs, including cathelicidins, can modulate TLR-mediated inflammatory responses by strongly reducing LPS-induced TLR activation, mostly by inhibiting TLR4 [ ]. Lastly, AMPs, 11, 13 e.g. cathelicidins and defensins, also exert a regenerative function by affecting β wound healing, by stimulating migration, proliferation and tube formation of endothelial cells, through a cascade of activated pathways [11].
Overall, AMPs are important key players in host protection. Due to increasing antibiotic resistance, several AMPs have good potential therapeutic purposes, rang­ing from antimicrobial, anti-inflammatory and immunomodulatory properties. Also co-administration of AMPs with existing therapies can have good clinical outcomes [14]. Recently, the ACP LTX-315 which has been described earlier, demonstrated in phase I human clinical studies to be an effective drug, due to its immunostimulatory effect resulting in tumor necrosis [9]. Currently, a phase I clinical trial for transder­mally accessible tumors is ongoing to evaluate the efficacy of LTX-315 monotherapy or in combination with immune checkpoint inhibitor immunotherapy [9]. Still, some limitations for the therapeutic use of AMPs need to be resolved: high proteo­lytic degradation of AMPs (i.e. susceptibility to proteases) is commonly observed, unpredicted toxicity is known to occur, chemical synthesis is costly and delivery of AMP targets to the site of infection can be very difficult [9, , 14 15]. As an example, LL-37 has proven to be very effective against Ebola virus infection, but its use as therapeutic molecule is limited as LL-37 is rapidly degraded and can lose its activity under certain conditions. These limitations were overcome with the design of an engineered LL-37 which prevents cell entry of the virus. The therapeutic outcome of these AMPs in animal models is ongoing, possibly combined with other small molecules that interfere with viral replication or together with virus-neutralizing antibodies [16].
AMPs mostly consist of 10 to 60 amino acids, including mainly basic and hydrophobic residues, resulting in positively charged molecules [1 2, ]. They can be classified, based on their structure, into four categories; 1) linear extension struc­ture, 2) -helical AMPs, 3) AMPs consisting of -strands stabilized by disulfide α β bonds and 4) both -helical and -sheet structures [ ]. Due to their cationic properα β 2 - ties, AMPs are easily detectable by mass spectrometric analysis. In addition, struc­tural information of AMPs can be obtained by tandem mass spectrometry in which fragmentation spectra are obtained. Based upon this, the corresponding amino acid sequence can be determined and the precursor ion can be identified [17]. Even if the AMPs have a cyclic nature, due to their cysteine bond formation, mass spectrometry can be used as an identification tool, although a specific approach is needed [18–21]. The added benefit of using proteomic approaches to study AMPs is the fact that the majority of AMPs are post- or co-translational proteolytically processed from their large polyprotein precursor, resulting in the release of the active AMP. This is important as it allows for the identification of the active AMP in physiological
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