**3.4 Cytokines as biomarkers**

*Clinical Management of Shock - The Science and Art of Physiological Restoration*

likelihood of a fatal outcome.

cal marker for sepsis.

later cases of sepsis.

**3.3 Haptoglobin**

**3.2 Nitric oxide**

In the same year, Wang et al. [41] demonstrated that the decrease in sRAGE levels in mice results in improved sepsis-induced lung damage, thus decreasing mortality in this condition. Another study by Narvaez-Rivera et al. [36] demonstrated in 2015 that sRAGE's level in the plasma is high in patients with communityacquired pneumonia associated with sepsis and is also an independent factor for the

Further studies are still needed to elucidate the mechanism of action of sRAGE in sepsis and septic shock; however, this receptor appears to be a promising biologi-

Nitric oxide (NO) is a highly reactive soluble gas that is endogenously synthesized

In the same year, Martin et al. reported an association between NOS2 and NOS3 with sepsis. In the study, the authors suggest that this association could be related to the high level of NO in the blood plasma, which could consequently induce a failure

Haptoglobin (Hp) is a protein whose main biological function is to bind free hemoglobin (Hb) and to prevent the loss of iron and subsequent kidney damage following intravascular hemolysis. When red cells are lysed, Hb binds to circulating Hp forming the Hp-Hb complex, which is then degraded by the reticuloendothelial system [46]. Although it is recognized that Hp is predominantly synthesized in the liver, studies reveal the expression of this protein also in other parts of the body, such as the lung, kidneys, heart, spleen, thymus, and brain [47–49]. There are reports that Hp levels are influenced by the acute inflammatory process and that such protein

It is known that some patients with sepsis present deformity of hemoglobins, thus causing lysis of these cells and releasing them into the circulation [53]. In this context, the decrease in blood levels of Hp has already been described as a factor linked to increased mortality in patients diagnosed with sepsis, and, in animal models of sepsis, Hp supplementation has been shown to be able to decrease biomarkers

Even with this result, prospective and randomized studies are still needed to better elucidate the potential protective effects of endogenous and exogenous haptoglobin against the deleterious effects of free hemoglobin in septic patients.

More recently, the critical care department of the First People's Hospital in Chun'an (China) demonstrated that monitoring changes in NO serum and amyloid A levels can be an efficient tool for defining patient prognosis and, when compared with CRP, would present better clinical results [45]. Despite the positive results on the use of nitric oxide as a biological marker for sepsis, many studies are still needed

of hemodynamics and increase the mortality of septic patients [44].

exerts an important antimicrobial and antioxidant function [50–52].

to fully understand its role in this condition.

of acute systemic inflammation [54, 55].

by the three isoforms of the nitric oxide synthase enzyme (NOS), neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3). These molecules are known to be highly involved in cardiovascular homeostasis, so recent research has focused on its action on sepsis-induced heart disease [42]. In 2014, Nardi et al. [43] showed an increased NOS1 expression in vascular tissues in the sepsis condition, suggesting that this molecule could be a way to justify vascular dysfunction induced by the disease. This study suggests that the inhibition of this isoform may be an alternative to restore the effectiveness of vasopressors in

**16**

Sepsis is characterized by two phases: a period of hyperinflammation, where the innate immune system is overactivated leading to production of proinflammatory cytokines such as TNF, IL-1b, IL-6, and IL8, and another period of immunosuppression where both adaptive and innate immunity are acting [56]. Clinical trials in septic patients showed an increase in the above-mentioned pro-inflammatory cytokines [57, 58].

However, contradictory results have been obtained for TNF and IL-1b. For example, the treatment of septic patients with anti-TNF antibodies did not affect the clinical outcome of patients [59]. In addition, these cytokines are not altered only in sepsis, after surgery or in autoimmune disease they are also altered and therefore are not specific.

On the other hand, studies have reported that IL-6 shows great promise as a biomarker [60–63]. Like TNF and IL-1b, IL-6 is not altered only in sepsis; nevertheless, several studies have shown its importance in the prognosis of sepsis presenting strong correlations with patient mortality [62, 63]. These results were also shown in an animal model of acute septic peritonitis (CLP) [61]. In this way, IL6 levels can show which patients may develop severe sepsis, and this reflects on possible interventions. Like IL6, IL8 has also been mentioned as a prognostic biomarker in septic patients especially in the early stages of the disease [64].

In the immunosuppression stage, IL-10 plays a key role in development of CARS trying to reduce hyperinflammation [65]. Therefore, studies have shown that high levels of IL-10 are correlated with a worse outcome and death [65–67]. In neonatal sepsis, IL-10 also proved to be an accurate biomarker. **Figure 1** illustrates the model proposed by van der Poll and van Deventer [68], emphasizing that the development of CARS still occurs in the pro-inflammatory phase of the disease. Thus, the mortality observed in the early stages of sepsis may be related to the hyper-inflammatory phase, and the late-stage deaths are related to the immunosuppressive phase as well as to secondary infections [56].

**Figure 1.** *Stages of sepsis and cytokine profile.*

Accordingly, the cytokine profile in the septic patient could provide information about the stage of the disease and the patient's prognosis, contributing to a better intervention. In addition, we currently have a multiplex assay that simultaneously measures multiple cytokines with small plasma samples; however, this information should be interpreted with caution since the dosages of some cytokines in septic patients appear unclear in the literature needing to be standardized. Once standardized, the multiplex assay may be useful in the clinic.

#### **3.5 Biomarker combinations**

As previously seen, no biomarker has 100% sensitivity or specificity capable of predicting the clinical outcome for the patient with sepsis. Studies have shown that combining biomarkers may facilitate diagnosis and predict the outcome more faithfully.

As above mentioned, Guo demonstrated that the combination among lactate, CRP and BNP, or NT-proBNP has greater specificity for prognosis than isolated lactate dosage, being 100 and 69.23%, respectively [15]. Yu and colleagues [45] also showed that combining NO with SAA is an important tool to improve the prognosis of septic patients.

Clinical scores are not effective in early identification of infection in critically ill patients; however, combining these scores with biomarkers allows an early and accurate identification of sepsis. For example, Yoo and co-workers found that combining Modified Early Warning Score (MEWS), a tool for monitoring sepsis, with blood lactate levels was efficient for early identification of the disease [69]. The same was found by Bozza et al. [70] and Oberholzer et al. [71] when they combined the levels of MPC-1 or IL-6 with APACHE II, respectively, and found greater accuracy in the prognosis of the patients.

Other biomarkers aforementioned are also more accurate when combined. Han et al. showed that the association with CRP and PCT is an important tool to differentiate bacterial sepsis from other possible types of infection in critically ill patients [72]. Angeletti et al. [73] also showed that the combination among PCT, Tumor Necrosis Factor-α (TNF-α), and the adrenomedullin hormone fragment may help in the prior diagnosis and prognosis of septic patients, thus optimizing treatment of patients.

As mentioned above, IL-6, IL10, and IL-8 cytokines may also be altered in septic patients. In this way, combining cytokine dosage with another biomarker may improve the diagnosis. For example, it has been shown that combining CRP dosage with IL8 and IL2 was useful in the diagnosis of neonatal sepsis [74]. Another study with adult septic patients measured TNF, IL6, and IL10 and demonstrated that combining IL6 (pro-inflammatory) with IL10 (anti-inflammatory) cytokines was useful in establishing the prognosis. Moreover, high levels of IL-6 and IL-10 were related to high patient mortality [75].

Another combination has been demonstrated by Wong and colleagues who showed that interleukin-27 (IL-27) when combined with PCT can improve the diagnostic accuracy in septic patients when compared to each biomarker alone [76]. In 2012, Andaluz-Ojeda et al. [66] using the multiplex assay demonstrated that combining pro—IL-6 and IL-8—and anti-inflammatory cytokine—IL10 and MCP-1—levels was more predictive than analyzing each cytokine separately. Furthermore, high levels of these cytokines were positively correlated with the patient's mortality rate.
