**4.1 Cytokines implicated in brain death**

Increased blood levels of several cytokines, such as TNF-α, interleukin (IL)-6, IL-8, IL-1β, and IL-2R, have been observed after brain death [27, 28]. Cytokines derive mostly from T-cells and are classified into different groups according to their main function and T helper (Th) cell subtypes to which they are related.

The Th1-cell related cytokines are TNF-α, IL-1, IL-2, IL-12 and IFN-γ [29, 30]. They act early in the inflammatory cascade and stimulate and support the inflammation by mediating between different inflammatory pathways; they activate endothelial cells and cellular adhesion molecules, and contribute to T-cell maturation.

The Th2-cell-related cytokines IL-4, IL-5, IL-10 and IL-13 are not as significant and are considered to be anti-inflammatory when related to brain death and the early transplant period [19, 31, 32].

One of the most heavily implicated cytokines in brain death is IL-6, a member of Th-17 cell related mediators [33, 34]. Increased concentrations of IL-6 have been demonstrated both in plasma and organs of brain-dead donors, including the kidneys, lungs, liver and heart [34–36]. Higher values of IL - 6 are associated with worse transplantation outcomes and poorer survival of recipients [33, 37].

A significant increase in IL-8 values in the bronchoalveolar lavage fluid from brain-dead lung donors has been demonstrated and correlates with early graft dysfunction after lung transplant [38]. In addition, elevated IL-6 gene expression was observed in the preimplantation biopsies of patients who died within 30 days after lung transplant [39]. Furthermore, the levels of IL-1β and TNF-α were significantly higher in donor lungs rejected for transplantation compared to transplanted lungs [40]. The values of IL-6 and TNF-α in the myocardium of dysfunctional discarded donor hearts were higher than in transplanted donor hearts [41].

#### **4.2 Role of complement**

The complement cascade is an important part of the innate immune system and transplantation process. Activation of either the classic, alternative or lectin pathway of the complement system leads to the formation of a common terminal cell lytic complex or C5b-9, also known as membrane attack complex (MAC). MAC induces complement-mediated lysis of cells. Proteolytic complement fragments such as C5a, C3a and, to a lesser extent, C4a, further induce acute inflammation by activating mast cells, neutrophils and endothelial cells.

Studies have shown that all three pathway types are involved in systemic inflammation secondary to brain death [24]. Complement activation products have the ability to produce proinflammatory substances, including cytokines, and act as chemotactic factors for leukocytes.

In deceased brain donors, the increased complement plasma levels of C5b9 were higher than in the plasma of living organ donors. Higher levels of C5b9 in deceased brain-dead and deceased cardiac-dead donors were associated with worse tissue damage, a higher rate of acute and chronic rejection, and inferior function after transplantation [25].

Complement activation also results in the release of anaphylatoxins C3a and C5a, potent activators of T-cells. Brain-dead organ donors had higher values of C5a in plasma compared to living donors [26].
