**5. On SAAs' functions and their identification by blocking the invariant epitopes of SAA**

### **5.1. The multifunctional SAA family**

Various functions of SAA have been reported, including the systemic and local elevation of SAA in inflammations in an APR due to the systemic and local cytokine increase. SAA is involved in very many functions as being an opsonin of Gram-negative bacteria, a chemoattractant, an inducer of chemokines and cytokines, a stimulator of angiogenesis, important in cholesterol transport and a modulator in the migration of white blood cells. SAA acts concentration dependently on polymorphonuclear cells and the degradation of SAA (by matrix degradation enzymes?), which can release the AA 1–76 fragment and can thereby induce the fatal AA amyloidosis in humans and animals. Other fragments of SAA and other APPs may, in vivo, influence this still not understood complex network of the SAA family, which has been reviewed in [1, 2, 43, 44]. Here, some of these vital functions of SAA have been identified by blocking these functions by way of monoclonal AA/SAA antibodies. At the same time, the SAA binding motives have been localized at the surface of SAA (see **Figure 5**). Alternatively, these ligands for the SAA binding motives can, in part, be blocked with the respective synthetic peptides of SAA [44, 45].

of SAA (or its fragments) during high fever, which is blocking SAA's return to the reversible binding to HDL. (This febrile temperature that induced the aggregation of AA-antigenic proteins has also been noticed in vitro and documented in **Figure 3** at 38°C and 40°C). The possibly unfavorable consequences of these aggregates in humans or animals are unknown today.

The Invariant Peptide Clusters of Serum Amyloid A Are Humoral Checkpoints for Vital Innate Functions…

The anti-inflammatory potential of SAA on neutrophils [33] has been confirmed for SAA at reported serum concentrations [46]. Oxidative burst, migration and the neutrophil myeloperoxidase release were also inhibited. SAA peptides (aa 1–14, 15–101 and 83–104) also contributed to this inhibitory effect. However, at higher concentrations of more than 50 μg/ml, SAA

increased above that. Thus, SAA plays a dual role, it downregulates inflammatory processes

SAA functions can be identified by SAA-generic antibodies [33, 34, 46] but they can also be blocked by synthetic peptides of SAA [45]. This was shown through the use of a 14mer synthetic peptide (aa 29–42) of SAA. This peptide inhibited the binding of T lymphocytes and mouse M4 melanoma cells to adhesive glycoproteins of the extra cellular matrix. This SAA 14mer peptide contained the laminin-like (aa 29–33) and fibronectin-like (aa 13–15) domains of the extracellular matrix. Finally, by extending these data of the 14mer SAA peptide, by comparing to the binding of our generic antibodies mc21 and mc29, it is to be said that these antibodies bind to a similar peptide of SAA, which is the largest invariable peptide (no. 7 of coil 2) as shown in **Figure 5**.

Phagocytosis was examined on fixed bacteria by normal and stimulated blood monocytes at the SAA concentration that were inhibitory to human neutrophil activation [33]. There was no

Human platelet adhesion was shown to immobilize SAA and the mechanism of binding was examined [35]. Among the many receptors on platelets, the receptors for laminin and fibronectin were chosen to be examined because SAA has laminin-like and fibronectin-like motives in its sequence. Immobilized SAA binds platelets as do fibronectin and, to a lesser degree, fibrinogen. This binding of SAA to platelets was completely abolished by anti-SAA (mc29), which binds to the laminin-like motive on SAA (aa 29–33) that is part of the mc29-binding peptide. Also, a 29–42-containing peptide could inhibit the binding of platelets to SAA. In addition, an antibody against an integrin receptor also inhibited the binding as well the RGD-containing peptide GRGDSP. Also, the anti-SAA (mc29) did not inhibit the RGD-dependent binding motive to a significant extent, thus indicating that the overlap of two amino acids (aa 39–40) of the peptide

difference in phagocytosis in the presence or in the absence of SAA [47].

in lower concentration, but, during the full APR, the action of SAA can be promoting.

release was inhibited up to 0.1 μg/ml, but the O2

release was

83

http://dx.doi.org/10.5772/intechopen.91983

**5.4. Anti-inflammatory potential of SAA on neutrophils**

**5.5. SAA functions inhibited by synthetic peptides**

**5.7. Platelets and binding motives similar to SAA**

was stimulating. In addition, O2

**5.6. Phagocytosis**

### **5.2. HDL binding site**

The HDL binding site of SAA was identified as the peptide aa 5–17 with the monoclonal mc1 (see Section 4). The presumptive estimate by Turnell et al. [41] was aa 1–11.

### **5.3. Human neutrophils**

Strong binding of isolated, acute-phase human SAA (and recombinant SAA2, not presented) were shown with human neutrophils [33] assuming the existence of an SAA receptor, which may have regulatory functions [1, 2]. The FMLP-induced oxidative burst of normal human neutrophils could be reduced, concentration dependently, by SAA at concentrations of 0.1 μg/ml and 1.0 μg/ml. This inhibitory reduction of SAA could be blocked by the monoclonal antibody mc29 (see **Table 1** and **Figure 5**), which binds to the synthetic peptide aa 28–40 of SAA, thus proving that this blocked area is responsible for this inhibitory effect of SAA [33]. This was the first time that a function of SAA was blocked by a monoclonal AA antibody. Moreover, at the same time, the responsible peptide of SAA was identified, which was the invariant peptide no. 7 of coil 2. The monoclonal antibody mc29 used probably also blocks the laminin-like domain (aa 29–33) and may also be participating partially with the RGD-like domain (aa 39–41). In addition, human neutrophils were exposed to full human APS at different temperatures [34]. At 41°C, the inhibition of the oxidative burst was much stronger than at 37°C, indicating the role of SAA freed from HDL and in its active state (see Section 3.4; see **Figures 3** and **4**). However, when the acute-phase serum was preheated to 41°C for 15 min and assayed at 37°C, the SAA-containing serum did not return to the 37°C value, but stayed with the increased 41°C inhibiting effect at 37°C. This indicated an irreversible structural change of SAA (or its fragments) during high fever, which is blocking SAA's return to the reversible binding to HDL. (This febrile temperature that induced the aggregation of AA-antigenic proteins has also been noticed in vitro and documented in **Figure 3** at 38°C and 40°C). The possibly unfavorable consequences of these aggregates in humans or animals are unknown today.

### **5.4. Anti-inflammatory potential of SAA on neutrophils**

The anti-inflammatory potential of SAA on neutrophils [33] has been confirmed for SAA at reported serum concentrations [46]. Oxidative burst, migration and the neutrophil myeloperoxidase release were also inhibited. SAA peptides (aa 1–14, 15–101 and 83–104) also contributed to this inhibitory effect. However, at higher concentrations of more than 50 μg/ml, SAA was stimulating. In addition, O2 release was inhibited up to 0.1 μg/ml, but the O2 release was increased above that. Thus, SAA plays a dual role, it downregulates inflammatory processes in lower concentration, but, during the full APR, the action of SAA can be promoting.

### **5.5. SAA functions inhibited by synthetic peptides**

SAA functions can be identified by SAA-generic antibodies [33, 34, 46] but they can also be blocked by synthetic peptides of SAA [45]. This was shown through the use of a 14mer synthetic peptide (aa 29–42) of SAA. This peptide inhibited the binding of T lymphocytes and mouse M4 melanoma cells to adhesive glycoproteins of the extra cellular matrix. This SAA 14mer peptide contained the laminin-like (aa 29–33) and fibronectin-like (aa 13–15) domains of the extracellular matrix. Finally, by extending these data of the 14mer SAA peptide, by comparing to the binding of our generic antibodies mc21 and mc29, it is to be said that these antibodies bind to a similar peptide of SAA, which is the largest invariable peptide (no. 7 of coil 2) as shown in **Figure 5**.

### **5.6. Phagocytosis**

**5. On SAAs' functions and their identification by blocking the** 

Various functions of SAA have been reported, including the systemic and local elevation of SAA in inflammations in an APR due to the systemic and local cytokine increase. SAA is involved in very many functions as being an opsonin of Gram-negative bacteria, a chemoattractant, an inducer of chemokines and cytokines, a stimulator of angiogenesis, important in cholesterol transport and a modulator in the migration of white blood cells. SAA acts concentration dependently on polymorphonuclear cells and the degradation of SAA (by matrix degradation enzymes?), which can release the AA 1–76 fragment and can thereby induce the fatal AA amyloidosis in humans and animals. Other fragments of SAA and other APPs may, in vivo, influence this still not understood complex network of the SAA family, which has been reviewed in [1, 2, 43, 44]. Here, some of these vital functions of SAA have been identified by blocking these functions by way of monoclonal AA/SAA antibodies. At the same time, the SAA binding motives have been localized at the surface of SAA (see **Figure 5**). Alternatively, these ligands for the SAA binding motives can, in part, be blocked with the respective synthetic peptides of

The HDL binding site of SAA was identified as the peptide aa 5–17 with the monoclonal mc1

Strong binding of isolated, acute-phase human SAA (and recombinant SAA2, not presented) were shown with human neutrophils [33] assuming the existence of an SAA receptor, which may have regulatory functions [1, 2]. The FMLP-induced oxidative burst of normal human neutrophils could be reduced, concentration dependently, by SAA at concentrations of 0.1 μg/ml and 1.0 μg/ml. This inhibitory reduction of SAA could be blocked by the monoclonal antibody mc29 (see **Table 1** and **Figure 5**), which binds to the synthetic peptide aa 28–40 of SAA, thus proving that this blocked area is responsible for this inhibitory effect of SAA [33]. This was the first time that a function of SAA was blocked by a monoclonal AA antibody. Moreover, at the same time, the responsible peptide of SAA was identified, which was the invariant peptide no. 7 of coil 2. The monoclonal antibody mc29 used probably also blocks the laminin-like domain (aa 29–33) and may also be participating partially with the RGD-like domain (aa 39–41). In addition, human neutrophils were exposed to full human APS at different temperatures [34]. At 41°C, the inhibition of the oxidative burst was much stronger than at 37°C, indicating the role of SAA freed from HDL and in its active state (see Section 3.4; see **Figures 3** and **4**). However, when the acute-phase serum was preheated to 41°C for 15 min and assayed at 37°C, the SAA-containing serum did not return to the 37°C value, but stayed with the increased 41°C inhibiting effect at 37°C. This indicated an irreversible structural change

(see Section 4). The presumptive estimate by Turnell et al. [41] was aa 1–11.

**invariant epitopes of SAA**

82 Infectious Process and Sepsis

SAA [44, 45].

**5.2. HDL binding site**

**5.3. Human neutrophils**

**5.1. The multifunctional SAA family**

Phagocytosis was examined on fixed bacteria by normal and stimulated blood monocytes at the SAA concentration that were inhibitory to human neutrophil activation [33]. There was no difference in phagocytosis in the presence or in the absence of SAA [47].

### **5.7. Platelets and binding motives similar to SAA**

Human platelet adhesion was shown to immobilize SAA and the mechanism of binding was examined [35]. Among the many receptors on platelets, the receptors for laminin and fibronectin were chosen to be examined because SAA has laminin-like and fibronectin-like motives in its sequence. Immobilized SAA binds platelets as do fibronectin and, to a lesser degree, fibrinogen. This binding of SAA to platelets was completely abolished by anti-SAA (mc29), which binds to the laminin-like motive on SAA (aa 29–33) that is part of the mc29-binding peptide. Also, a 29–42-containing peptide could inhibit the binding of platelets to SAA. In addition, an antibody against an integrin receptor also inhibited the binding as well the RGD-containing peptide GRGDSP. Also, the anti-SAA (mc29) did not inhibit the RGD-dependent binding motive to a significant extent, thus indicating that the overlap of two amino acids (aa 39–40) of the peptide (see **Figure 5**) did not lead to an efficient paratope subsite of mc29 for the method applied. Finally, all controls were in line with the conclusion that SAA was binding to platelets via the laminin-like and fibronectin-like motives. Since the related binding motives are not chemically identical with laminin or fibronectin, they could have a lower affinity, which may be exerted differently at lower concentrations as compared with higher concentrations, i.e., during the APR.

a functional relationship between these two systems. The authors suggest that SAA produced in the malignant tissue may contribute to increased matrix degeneration and tumor spread [36].

The Invariant Peptide Clusters of Serum Amyloid A Are Humoral Checkpoints for Vital Innate Functions…

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85

Increased levels of SAA were reported in a wide range of malignancies, as well as another unspecific tumor marker, with an increase in metastatic tumors and regression when therapy is successful [26]. Here, the presence of SAA in serum (with CRP and CA-123) and expressed locally in tissues was examined and compared with different stages of tumor growth. Compared were normal ovarian tissues, benign, borderline, carcinoma and metastatic tissues of patients using immunohistochemistry with monoclonal antibodies against AA (mc1 and mc29, see **Table 1** and **Figure 5**) and in situ hybridization. In some patients (and in cell line OVCR-3), RT-PCR was applied, and SAA1 and SAA4 were detected. The result shows a continuous increase of SAA (CRP and CA-125) in serum during the gradual increase of the malignant nature of the ovarian tissue proliferation. In addition, and most important, the SAA expression in tissue increases, in the same manner, with a steep increase in the SAAsynthesizing cells from the normal cells, without (or with only a trace of) SAA, over the borderline tumors with weak expression to the maximal expression of the distinct carcinomas and metastases. Therefore, it is likely that the serum level of SAA in these malignancies may,

The data show that the quantity of local intracellular expression of SAA correlates directly with the grade of malignancy of the ovarian epithelial neoplasias and runs in parallel with the serum value of SAA, CRP and CA-125. Therefore, SAA may have a role in ovarian tumor-

Ovarian reproduction includes a kind of inflammatory process [28]. Therefore, the cellular expression and localization of SAA in all stages of follicular development was examined in in vitro fertilization (IVF) patients applying nonradioactive in situ hybridization and immunochemistry with the monoclonal anti-AA (mc1 and mc29) antibodies. In parallel, SAA of follicular fluids and SAA in serum were examined using micro-ELISA. Expression of SAA mRNA was found in all follicular cells (granulosa, thecal and luteal) of all stages of development, from primordial, primary and secondary follicles to corpora lutea and even in oocytes. The concentration of SAA in serum and in the matched follicular fluid was very closely associated

 = 0.80), although both values could vary considerably by a factor of ca. 30× for blood SAA and by 100× for the follicular fluid. In addition, elevated follicular SAA values have a strong correlation with the patients' body mass index. Values over 30 are associated with a reduced pregnancy rate. Taken together, SAA is locally produced by all follicular cells and is a constituent of the follicular fluid. Therefore, it has a role in ovarian development and in the rate of pregnancy, which is reduced when SAA values are too high in overweight female patients with a BMI of over 30. Finally, since human ovarian epithelial tissues reproduce SAA during reproduction (see above), the neoplastic degenerated cells in ovarian carcinoma continue their SAA synthesis [26].

**5.10. Gradual increase of SAA while progressing to malignancy in ovarian** 

**epithelial tumors**

igenesis [27].

(R2

in part, originate from the ovarian tumor itself.

**5.11. SAA in the female reproductive system**

Thus, SAA may play a role in inhibiting and modulating platelet adhesion at vascular injury sites by sharing platelet receptors with other platelet-adhesive proteins. In addition, depending on the kind of disease, the window between bleeding and thrombosis may sometimes be very narrow; how can it be widened? Finally, systemic and local thrombosis are not rare, which are life-threatening sequels of many conditions. These are related to arteriosclerosis, heart conditions, nutrition-related ailments, deranged lipid metabolism, smoking and other drugs, cancer, injuries, bacterial infections and sepsis, mostly in a more advanced age as well as in cases of vascular injury. The role of platelets is central in these and many other diseases, and the concentration-dependent role of SAA and its antidotes (humanized monoclonals and others) in vivo needs to be explored and then further developed.

### **5.8. Presence of SAA in human cancer and other cells**

Intracellular SAA of colon tissue with cancer of progressing stages of anaplasia was examined on formalin-fixed paraffin sections from 26 patients with colon cancer (after SAA plasma levels were shown by others to be elevated in carcinomas, assuming that the elevated SAA is of hepatic origin) [26]. SAA was detected immunohistochemically by using the monoclonal antibodies mc1 and mc29 (specificity, see **Figure 5** and **Table 1**). On normal cells, no reaction or only traces were detected. However, stronger reactions were found in carcinoma cells. The staining intensity increased gradually from dysplasia to the stage of malignant neoplasia. The metastases also showed the presence of SAA, but weaker. In addition, cells, other than colon cells in these sections, also showed the presence of SAA as lymphoid cells of the intestinal wall, inflammatory cells, ganglion cells and endothelial cells. The presence of SAA has been confirmed by in situ hybridization and reverse transcriptase polymerase chain reaction (RT-PCR). The genes of SAA1 and SAA4 in the colon carcinomas were activated. Although the role of SAA in colon carcinoma is unknown, the close association of the increasing grade of malignancy with the increased SAA synthesis may indicate a role of SAA in tumorigenesis. SAA can serve as an adhesive ligand for tumor-cell homing; it induces inflammation, which may be neoplastic. It also induces migration and can be involved in metastasis, or it can be inhibitory to attachment [26].

### **5.9. Protein SAA enhances plasminogen activation and may contribute to tumor spread**

The colon carcinoma cell line HT-29 showed plasminogen activity (PA) enhanced by SAA measured with a chromogenic substrate. This activity could be inhibited using monoclonal antibodies against SAA (mc1 + mc29). The cell line also produced endogenous SAA1 by itself, which could be augmented by exogenous SAA and also by cytokines IL-1b and IL-6. This activity was also inhibited in part by the monoclonal antibodies against SAA [36]. The concomitant overexpression and co-localization of SAA and PA in colon cancer cells raises the possibility of a functional relationship between these two systems. The authors suggest that SAA produced in the malignant tissue may contribute to increased matrix degeneration and tumor spread [36].

### **5.10. Gradual increase of SAA while progressing to malignancy in ovarian epithelial tumors**

Increased levels of SAA were reported in a wide range of malignancies, as well as another unspecific tumor marker, with an increase in metastatic tumors and regression when therapy is successful [26]. Here, the presence of SAA in serum (with CRP and CA-123) and expressed locally in tissues was examined and compared with different stages of tumor growth. Compared were normal ovarian tissues, benign, borderline, carcinoma and metastatic tissues of patients using immunohistochemistry with monoclonal antibodies against AA (mc1 and mc29, see **Table 1** and **Figure 5**) and in situ hybridization. In some patients (and in cell line OVCR-3), RT-PCR was applied, and SAA1 and SAA4 were detected. The result shows a continuous increase of SAA (CRP and CA-125) in serum during the gradual increase of the malignant nature of the ovarian tissue proliferation. In addition, and most important, the SAA expression in tissue increases, in the same manner, with a steep increase in the SAAsynthesizing cells from the normal cells, without (or with only a trace of) SAA, over the borderline tumors with weak expression to the maximal expression of the distinct carcinomas and metastases. Therefore, it is likely that the serum level of SAA in these malignancies may, in part, originate from the ovarian tumor itself.

The data show that the quantity of local intracellular expression of SAA correlates directly with the grade of malignancy of the ovarian epithelial neoplasias and runs in parallel with the serum value of SAA, CRP and CA-125. Therefore, SAA may have a role in ovarian tumorigenesis [27].

### **5.11. SAA in the female reproductive system**

(see **Figure 5**) did not lead to an efficient paratope subsite of mc29 for the method applied. Finally, all controls were in line with the conclusion that SAA was binding to platelets via the laminin-like and fibronectin-like motives. Since the related binding motives are not chemically identical with laminin or fibronectin, they could have a lower affinity, which may be exerted differently at lower concentrations as compared with higher concentrations, i.e., during the APR. Thus, SAA may play a role in inhibiting and modulating platelet adhesion at vascular injury sites by sharing platelet receptors with other platelet-adhesive proteins. In addition, depending on the kind of disease, the window between bleeding and thrombosis may sometimes be very narrow; how can it be widened? Finally, systemic and local thrombosis are not rare, which are life-threatening sequels of many conditions. These are related to arteriosclerosis, heart conditions, nutrition-related ailments, deranged lipid metabolism, smoking and other drugs, cancer, injuries, bacterial infections and sepsis, mostly in a more advanced age as well as in cases of vascular injury. The role of platelets is central in these and many other diseases, and the concentration-dependent role of SAA and its antidotes (humanized monoclonals and

Intracellular SAA of colon tissue with cancer of progressing stages of anaplasia was examined on formalin-fixed paraffin sections from 26 patients with colon cancer (after SAA plasma levels were shown by others to be elevated in carcinomas, assuming that the elevated SAA is of hepatic origin) [26]. SAA was detected immunohistochemically by using the monoclonal antibodies mc1 and mc29 (specificity, see **Figure 5** and **Table 1**). On normal cells, no reaction or only traces were detected. However, stronger reactions were found in carcinoma cells. The staining intensity increased gradually from dysplasia to the stage of malignant neoplasia. The metastases also showed the presence of SAA, but weaker. In addition, cells, other than colon cells in these sections, also showed the presence of SAA as lymphoid cells of the intestinal wall, inflammatory cells, ganglion cells and endothelial cells. The presence of SAA has been confirmed by in situ hybridization and reverse transcriptase polymerase chain reaction (RT-PCR). The genes of SAA1 and SAA4 in the colon carcinomas were activated. Although the role of SAA in colon carcinoma is unknown, the close association of the increasing grade of malignancy with the increased SAA synthesis may indicate a role of SAA in tumorigenesis. SAA can serve as an adhesive ligand for tumor-cell homing; it induces inflammation, which may be neoplastic. It also induces migration and can be involved in metastasis, or it can be inhibitory

**5.9. Protein SAA enhances plasminogen activation and may contribute to tumor** 

The colon carcinoma cell line HT-29 showed plasminogen activity (PA) enhanced by SAA measured with a chromogenic substrate. This activity could be inhibited using monoclonal antibodies against SAA (mc1 + mc29). The cell line also produced endogenous SAA1 by itself, which could be augmented by exogenous SAA and also by cytokines IL-1b and IL-6. This activity was also inhibited in part by the monoclonal antibodies against SAA [36]. The concomitant overexpression and co-localization of SAA and PA in colon cancer cells raises the possibility of

others) in vivo needs to be explored and then further developed.

**5.8. Presence of SAA in human cancer and other cells**

to attachment [26].

84 Infectious Process and Sepsis

**spread**

Ovarian reproduction includes a kind of inflammatory process [28]. Therefore, the cellular expression and localization of SAA in all stages of follicular development was examined in in vitro fertilization (IVF) patients applying nonradioactive in situ hybridization and immunochemistry with the monoclonal anti-AA (mc1 and mc29) antibodies. In parallel, SAA of follicular fluids and SAA in serum were examined using micro-ELISA. Expression of SAA mRNA was found in all follicular cells (granulosa, thecal and luteal) of all stages of development, from primordial, primary and secondary follicles to corpora lutea and even in oocytes.

The concentration of SAA in serum and in the matched follicular fluid was very closely associated (R2 = 0.80), although both values could vary considerably by a factor of ca. 30× for blood SAA and by 100× for the follicular fluid. In addition, elevated follicular SAA values have a strong correlation with the patients' body mass index. Values over 30 are associated with a reduced pregnancy rate. Taken together, SAA is locally produced by all follicular cells and is a constituent of the follicular fluid. Therefore, it has a role in ovarian development and in the rate of pregnancy, which is reduced when SAA values are too high in overweight female patients with a BMI of over 30.

Finally, since human ovarian epithelial tissues reproduce SAA during reproduction (see above), the neoplastic degenerated cells in ovarian carcinoma continue their SAA synthesis [26].

### **5.12. A role of SAA in the APR of murine septic inflammations**

### *5.12.1. On the role of the APR and APPs in septic mice*

In order to analyze the different steps necessary to overcome an infection by the hepatic APR, an experimental mouse model was applied and shown as an "anti-sepsis circle" (see **Figure 6**) [32]. Using polymicrobial sepsis induced by cecal ligation and puncture (CLP), the various actions begin with mice exposed to a bacterial overload that leads to the IL-6 induction, which is the dominant interleukin and major inductor of the APR. IL-6-deficient mice can still mount an APR, since IL-6 represents one member of a larger group of interleukins with redundant actions. The action of IL-6 is to initiate the intracellular signaling via the hepatic IL-6 receptor gp130 and further induction of STAT3, which is inevitable for developing the full hepatic APR in hepatic cells, including the synthesis of the dominant APP SAA. However, when mice with a deletion of gp130 or STAT3 are treated with CLP, the hepatic synthesis of SAA is not induced and these mice cannot mount an APR anymore and are thus defenseless, and mortality is greatly increased. The missing APR and the missing defense can be reversed by adding myeloid-derived suppressor cells (MDSCs), which are induced by a hepatic APR including SAA. SAA induces and activates the proliferation of bone marrow cells, which include MDSCs. These cells are accepted to be able to also act against the microbial infection. MDSCs are anti-inflammatory in cancer, cancer spread and metastases [27]. They home-in on different organs. In septic mice, they have been examined from spleen and increase their numbers when pg130 and STAT deficiency are overcome by an injection of SAA, cxc1 (KC) or SAA/KC. MDSC can be regarded as a second anti-inflammatory wave induced by SAA and the other components of the APPs when the first wave of anti-inflam-

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When very important functions of SAA (which need to be more clarified) are being blocked by the monoclonal antibodies mc4 and mc29 in CLP mice, the essential MDSCs cannot be produced to the necessary amount and function to cope with the bacterial load so that the mice become defenseless and display a significantly accelerated death rate. This unfavorable situation can be reverted by the injection of SAA, thereby resulting in the former defense with the proliferation of MDSCs, so that the mice survived like CLP-treated mice in this sepsis model [29, 30, 48]. The APP KC has a similar, but not identical, effect. When KC was added to SAA, the recovery from the CLP fate of mice with a murine SAA inactivated by antibodies may even be slightly improved, thus indicating that SAA, although the major and dominant

This demonstrates a cooperative defense of SAA and KC [29]. Cooperation can also be expected from other APPs and constituents in the APP network, including from the greater SAA family. The AA antibodies mc4 and mc29 bind to invariant and therefore very important peptides of SAA as described in detail in Section 4.2, in **Table 1** and **Figure 5**. With these antibodies, life-saving biological functions have been detected and their functions localized to invariant peptides of SAA. This approach could be extended to analyze all the invariant peptides of the SAA family. This can be regarded as a starting point for a possible therapy of a long list of such maladies as severe chronic inflammations and severe chronic infections including sepsis with (induced in vivo or recombinant) SAA isotypes (and their inhibitors as humanized generic SAA antibodies), and with other APPs and constituents of the network of

matory phagocytes is beginning to wane while becoming exhausted [1, 2, 27].

the SAA family, which are able to fortify the "anti-sepsis circle" (**Figure 6**).

**6.1. The septic loop became an "anti-sepsis circle" as a basis for further work**

epitopes of the AA/SAA antibodies (**Table 1**) in order fortify it.

Some essential elements of the cooperative defense against the experimental multi-microbial infections became apparent as shown in **Figure 6**. The pathway from infection procedures passes, through IL-6, gp130 and STAT3, to the APR with the dominant SAA family and its network. This loop has been closed to a circle through the action of at least the SAA1 that assisted in inducing the growth of the MDSCs in the bone marrow. These cells are also shown to be essential in fighting bacterial infection. However, when gp130 or SAA was not available in this model and the "circle" was interrupted, with fatal consequences, the addition of the missing agents restored the circle with its function [29]. It should be an important goal to examine the SAA isotypes in different inflammatory states and diseases in relation to febrile temperatures (**Figures 3** and **4**) and to analyze the functions of all 20 invariant peptides (**Figure 5**) and the

It is also important to define the febrile temperatures by which the individual SAAs separate from HDL (proven in vitro, **Figures 3** and **4**) and get activated to execute their function. A

APP, can be assisted by KC against the bacterial load.

**6. Some remarks**

**Figure 6.** Closing the sepsis loop to the "anti-sepsis circle" schematically.

injection of SAA, cxc1 (KC) or SAA/KC. MDSC can be regarded as a second anti-inflammatory wave induced by SAA and the other components of the APPs when the first wave of anti-inflammatory phagocytes is beginning to wane while becoming exhausted [1, 2, 27].

When very important functions of SAA (which need to be more clarified) are being blocked by the monoclonal antibodies mc4 and mc29 in CLP mice, the essential MDSCs cannot be produced to the necessary amount and function to cope with the bacterial load so that the mice become defenseless and display a significantly accelerated death rate. This unfavorable situation can be reverted by the injection of SAA, thereby resulting in the former defense with the proliferation of MDSCs, so that the mice survived like CLP-treated mice in this sepsis model [29, 30, 48]. The APP KC has a similar, but not identical, effect. When KC was added to SAA, the recovery from the CLP fate of mice with a murine SAA inactivated by antibodies may even be slightly improved, thus indicating that SAA, although the major and dominant APP, can be assisted by KC against the bacterial load.

This demonstrates a cooperative defense of SAA and KC [29]. Cooperation can also be expected from other APPs and constituents in the APP network, including from the greater SAA family. The AA antibodies mc4 and mc29 bind to invariant and therefore very important peptides of SAA as described in detail in Section 4.2, in **Table 1** and **Figure 5**. With these antibodies, life-saving biological functions have been detected and their functions localized to invariant peptides of SAA. This approach could be extended to analyze all the invariant peptides of the SAA family. This can be regarded as a starting point for a possible therapy of a long list of such maladies as severe chronic inflammations and severe chronic infections including sepsis with (induced in vivo or recombinant) SAA isotypes (and their inhibitors as humanized generic SAA antibodies), and with other APPs and constituents of the network of the SAA family, which are able to fortify the "anti-sepsis circle" (**Figure 6**).
