**3. SAA-HDL and febrile temperatures**

### **3.1. Temperature-induced structural changes of SAA in serum**

When examining a patient's acute-phase serum (APS) with elevated SAA in immunodiffusion (ID) at different temperatures and different times using a polyclonal AA antiserum in comparison with isolated control AA, this resulted in the three different precipitation patterns presented in **Figure 2**. In (a), one recognizes a line of identity of AA with all four patients' sera as if the SAA (probably SAA1 and SAA2) reaction were done with pure SAA. At 4°C in (b), however, there is no reaction with SAA-HDL in serum. This is due to the hiding of the AA-reactive parts of SAA through HDL. However, when the temperature was switched to room temperature after the reaction in (b) at 4°C the SAA containing serum resulted in a strong line after releasing the SAA from HDL in (c) as seen in (a). However, different from the pattern in (a), the precipitation line of AA-anti-AA is somewhat independent of the SAAanti-AA line, thus indicating that the homologous AA-anti-AA line seems to be more stable than the SAA-anti-AA line. These results show that SAA-HDL is stable in full APS at 4°C where the AA-reactive sites are covered by HDL. When at room temperature (in ID buffer), where SAA is released from HDL and is now accessible to antibodies for precipitation, it is reactive. Therefore, the separation of SAA from HDL is temperature dependent [15]. These results became only fully explainable through Section 3.2, where the separation of SAA from HDL became clear [14].

In addition, we prepared recombinant SAA2 and, when added to normal human serum, it was possible to repeat exactly that behavior reported in **Figure 2**. This shows that SAA alone can reproduce this phenomenon [16].

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a monoclonal immunoglobulin κ-light chain and was named ALκ. The first sequence identifying the chemical nature of inflammation-induced amyloid in monkey and human amyloid was published by Benditt et al. [11], which was named amyloid A (AA). The first anti-AA antibodies were prepared in rabbits where a serum protein in patients suffering from inflam-

and was in serum approximately 180 KDa by calibrated gel filtration [12] and thus ready to monitor the isolation of the soluble with anti-AA reactive precursor. This isolation of serum protein began in summer 1972 and was monitored with another rabbit anti-AA antibody. Its chromatographic separation from serum yielded a native 200 ± 20 kDa AA reactive protein, which was further chromatographically isolated in 5 M guanidine-HCl. The AA reactive

protein had the same N-terminal amino acid sequence as AA, it was named serum amyloid A (SAA) [13]. Since SAA was larger than AA, a limited proteolytic cleavage had to be presumed in order for the former to generate AA. During the isolation of SAA and its purification to one size by gel filtration, by isoelectric focusing, however, eight SAA bands of different isoelectric point named A-H were identified with anti-AA antibodies (with AAE as the major SAA species for the planned radioimmunoassay), thus indicating the first signs of a polymorphism of

When examining a patient's acute-phase serum (APS) with elevated SAA in immunodiffusion (ID) at different temperatures and different times using a polyclonal AA antiserum in comparison with isolated control AA, this resulted in the three different precipitation patterns presented in **Figure 2**. In (a), one recognizes a line of identity of AA with all four patients' sera as if the SAA (probably SAA1 and SAA2) reaction were done with pure SAA. At 4°C in (b), however, there is no reaction with SAA-HDL in serum. This is due to the hiding of the AA-reactive parts of SAA through HDL. However, when the temperature was switched to room temperature after the reaction in (b) at 4°C the SAA containing serum resulted in a strong line after releasing the SAA from HDL in (c) as seen in (a). However, different from the pattern in (a), the precipitation line of AA-anti-AA is somewhat independent of the SAAanti-AA line, thus indicating that the homologous AA-anti-AA line seems to be more stable than the SAA-anti-AA line. These results show that SAA-HDL is stable in full APS at 4°C where the AA-reactive sites are covered by HDL. When at room temperature (in ID buffer), where SAA is released from HDL and is now accessible to antibodies for precipitation, it is reactive. Therefore, the separation of SAA from HDL is temperature dependent [15]. These results became only fully explainable through Section 3.2, where the separation of SAA from

In addition, we prepared recombinant SAA2 and, when added to normal human serum, it was possible to repeat exactly that behavior reported in **Figure 2**. This shows that SAA alone



mations was detected immunochemically. This protein had an α<sup>1</sup>

SAA [13]. In addition, in plasma, SAA is bound to HDL [14].

**3.1. Temperature-induced structural changes of SAA in serum**

**3. SAA-HDL and febrile temperatures**

protein had an α<sup>2</sup>

72 Infectious Process and Sepsis

HDL became clear [14].

can reproduce this phenomenon [16].

**Figure 2.** Immunochemical comparison of SAA-HDL, SAA and AA. Immunodiffusion (ID) at different temperatures [14, 15]. The ID was performed in 1.5% Seakem agarose in 0.03 M barbital buffer, pH 8.6 with the same reagents in each of the three plates à 6 wells ((a)-(c)). Top and bottom well contained AA (0.1 mg/ml), the middle well contained polyclonal rabbit anti-AA antibodies undiluted and the 4 side wells contained elevated SAA-HDL containing APS from 4 patients at 1/10 diluted. Plate (a) after diffusion over night at room temperature, plate (b) at 4°C over night and plate (c) first at 4°C over night as plate B at 4°C followed by room temperature for 6 h similar to plate (a).

### **3.2. The molecular size of the SAA and SAA-HDL at different febrile temperatures**

Temperature-dependent molecular weight determination of AA-antigenic proteins of acutephase serum (APS) has been performed using an ACA-34 gel filtration column in PBS with the enzyme inhibitor phenylmethylsulfonylfloride (PMSF) under various temperatures as shown in **Figure 3**. The size grading was done by the serum proteins IgM, IgG, albumin and, in addition, cytochrome C and the salt marker N-ε-DNP-lysine. The proteins were identified by way of the size position in the column by immunodiffusion as SAA-HDL at a size of ca. 180–200 kDa or SAA at 12.5 kDa. The different temperatures were kept with a temperature-controlled glass jacket, that is at 37°C in column run A, at 38°C in B, at 40°C in C and at 42°C in D. E was run as D, but without enzyme protection by PMSF, thus showing some degradation of SAA [18].

At a normal body temperature of 37°C, AA-containing proteins are at a single position as that of the SAA-HDL stable complex in A (fractions 34–37). However, already at 38°C, the stable complex SAA-HDL begins to dissociate as shown in **Figure 3**, run B. AA antigenic proteins appear at three positions, that is first of all at the void volume at fractions 19–20 (which has not been further analyzed, but could be related to aggregated SAA and/or its derivatives), secondly at the position of the stable SAA-HDL complex at fractions 34–36 and thirdly at the position of the HDL-free SAA at 53–56, as determined by the antigenic differentiation as seen in **Figure 2**. This size differentiation may also indicate functional heterogeneity, as the different affinities of SAA to HDL. This dissociation begins at 38°C and progresses with diminution of the SAA-HDL complex until run C. SAA-HDL disappeared at a "threshold of life" in run D at 42°C and above where the SAA species was maximized and the broadest was seen at fraction 53–56. This shows a temperature-induced gradual dissociation of SAA from HDL at the different febrile temperatures, which was shown here in vitro. This may also occur under systemic and local, acute-phase conditions, with the release of different SAA isotypes at different temperatures, for functions to be discovered. Finally, the SAA monomers released at different temperatures differ

the SAA-HDL complex at low temperatures. The hiding of the antigenic determinants disappeared gradually from 38°C (the 5th sample) until it is completely above 42°C with the

very strong exposure of formerly concealed antigenic AA determinants detected after the strongest exposure appears at

**Figure 4.** Electrophoresis of SAA-HDL across a continuous temperature gradient in agarose [18]. An APS was applied (see horizontal arrow) in 13 identical samples containing SAA-HDL across a temperature gradient. The small crosshatched bar on the cm ruler denotes 37°C at the left margin and 42°C at the right one. Electrophoresis was done in 1.5% agarose (Seakem ME) in 25 mm barbital buffer, pH 8.6 followed by a standard Western blot developed with a mixture of the monoclonal anti-AA antibodies [19] mc1 + mc4 + mc29 at 1 + 1 + 1, 1/100 (see **Table 1**). One recognizes SAA-HDL

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


gradually increasing the temperature, the SAA release increases gradually while the SAA-HDL fades away up to the extreme exposure at 42°C and beyond, in agreement with the stepwise separation of SAA from HDL shown in **Figure 3**. Also important during this gradual temperature-induced separation from HDL seems to be that the morphology of the dots is different. They changed by their shape in the longitudinal direction, which is consistent with the fact that SAA is not uniform and consists of a group of homologous, but chemically different, SAAs demonstrating different isoelectric points (13 reviewed by [1, 2]). Another observation concerns the AA-antigenic species below 37°C in the first four samples. These slow, arc-like uniform samples could represent an SAA species, which is always active as a monomer regardless of temperature. This species seems to be less acidic. It could be a type of SAA species for the general protection under normal condition. In sample 5, this "arc SAA" is overlaid by a more acidic SAA released from the acute-phase proteins (APPs) SAA1 or SAA2, thus changing the spots to a more longitudinal pattern. With increasing temperature, the SAA spots become thicker and increase more in the longitudinal direction. This "arc SAA" needs to be analyzed since it does not seem to be part of the intact SAA-HDL complex (we did not check for SAA4). Finally, far above a temperature of 42°C, the SAA species seems to be stable. Parallel to the gradual release of SAA, at the same time, the SAA-HDL complex while losing SAA is gaining more negative charges with increased temperatures. Moreover, the trailing of SAA in samples 5–8 possibly indicates the gradual separation of differently



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75

appearance of large amounts of SAA (intensive staining of the band with α<sup>2</sup>

febrile temperatures between 38°C and 42°C (see cross-hatched bar). See vertical arrow at 42°C- 43°C.

while the SAA freed from HDL (as in **Figure 3**) starts to appear at 38°C at the α<sup>2</sup>

charged SAA species.

at the left-side site marking the α<sup>1</sup>

**Figure 3.** Size separation of SAA-HDL at febrile temperatures. SAA-HDL in a patient's acute-phase serum with a common cold was separated in A at 37°C, B at 38°C, C at 40°C and D at 42°C by gel filtration. All individual fractions (20–70) were examined and semi-quantified by ID using polyclonal rabbit anti-AA antibodies [15, 17].

in size. SAA in B is somewhat smaller than SAA in C. In addition, both appear at 42°C in D together as a broad combination of the two SAAs in B and D. In conclusion, SAA separated from HDL at 38°C in B has a lower affinity to HDL and is smaller, and SAA with a higher affinity for HDL is larger. Different isotypes and sizes of SAA are known [1, 2, 13]. The acute-phase SAAs, aSAA1 and aSAA2, are each 12.5 kDa with 104 amino acids and the constitutive SAA (cSAA), which is 14 kDa and has 112 amino acids. Since SAA1 has the lowest affinity for HDL and is the most amyloidogenic SAA, it could have separated from HDL in run at 38°C in B already, while SAA4, which is somewhat larger than the aSAAs, could be a component in the C. These indications can be solidified using isoelectric focusing or SAA-isotype-specific antibodies [1, 2].

### **3.3. Gradual dissociation of SAA-HDL during a continuous temperature gradient**

While these experiments above were done stepwise, one by one, a more precise dissociation of the SAA-HDL separation was performed by electrophoresis in 1.5% agarose across a continuous temperature gradient in a single flat gel, as shown in **Figure 4**. The two sides between the agarose gel were kept at a constant temperature of 15°C in T1 and of 65°C in T2 [17].

The results in **Figure 4** show two horizontal bands of samples of one patient in the form of dots across the temperature gradient. The SAA-HDL band of α<sup>1</sup> -electrophoretic mobility is marginally stained due to the concealing of the AA-antigenic determinants of SAA within

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**Figure 4.** Electrophoresis of SAA-HDL across a continuous temperature gradient in agarose [18]. An APS was applied (see horizontal arrow) in 13 identical samples containing SAA-HDL across a temperature gradient. The small crosshatched bar on the cm ruler denotes 37°C at the left margin and 42°C at the right one. Electrophoresis was done in 1.5% agarose (Seakem ME) in 25 mm barbital buffer, pH 8.6 followed by a standard Western blot developed with a mixture of the monoclonal anti-AA antibodies [19] mc1 + mc4 + mc29 at 1 + 1 + 1, 1/100 (see **Table 1**). One recognizes SAA-HDL at the left-side site marking the α<sup>1</sup> -electrophoretic mobility (upper band). This band fades beginning from 38°C to 42°C, while the SAA freed from HDL (as in **Figure 3**) starts to appear at 38°C at the α<sup>2</sup> -electrophoretic mobility with the very strong exposure of formerly concealed antigenic AA determinants detected after the strongest exposure appears at febrile temperatures between 38°C and 42°C (see cross-hatched bar). See vertical arrow at 42°C- 43°C.

the SAA-HDL complex at low temperatures. The hiding of the antigenic determinants disappeared gradually from 38°C (the 5th sample) until it is completely above 42°C with the appearance of large amounts of SAA (intensive staining of the band with α<sup>2</sup> -mobility). By gradually increasing the temperature, the SAA release increases gradually while the SAA-HDL fades away up to the extreme exposure at 42°C and beyond, in agreement with the stepwise separation of SAA from HDL shown in **Figure 3**. Also important during this gradual temperature-induced separation from HDL seems to be that the morphology of the dots is different. They changed by their shape in the longitudinal direction, which is consistent with the fact that SAA is not uniform and consists of a group of homologous, but chemically different, SAAs demonstrating different isoelectric points (13 reviewed by [1, 2]). Another observation concerns the AA-antigenic species below 37°C in the first four samples. These slow, arc-like uniform samples could represent an SAA species, which is always active as a monomer regardless of temperature. This species seems to be less acidic. It could be a type of SAA species for the general protection under normal condition. In sample 5, this "arc SAA" is overlaid by a more acidic SAA released from the acute-phase proteins (APPs) SAA1 or SAA2, thus changing the spots to a more longitudinal pattern. With increasing temperature, the SAA spots become thicker and increase more in the longitudinal direction. This "arc SAA" needs to be analyzed since it does not seem to be part of the intact SAA-HDL complex (we did not check for SAA4). Finally, far above a temperature of 42°C, the SAA species seems to be stable. Parallel to the gradual release of SAA, at the same time, the SAA-HDL complex while losing SAA is gaining more negative charges with increased temperatures. Moreover, the trailing of SAA in samples 5–8 possibly indicates the gradual separation of differently charged SAA species.

in size. SAA in B is somewhat smaller than SAA in C. In addition, both appear at 42°C in D together as a broad combination of the two SAAs in B and D. In conclusion, SAA separated from HDL at 38°C in B has a lower affinity to HDL and is smaller, and SAA with a higher affinity for HDL is larger. Different isotypes and sizes of SAA are known [1, 2, 13]. The acute-phase SAAs, aSAA1 and aSAA2, are each 12.5 kDa with 104 amino acids and the constitutive SAA (cSAA), which is 14 kDa and has 112 amino acids. Since SAA1 has the lowest affinity for HDL and is the most amyloidogenic SAA, it could have separated from HDL in run at 38°C in B already, while SAA4, which is somewhat larger than the aSAAs, could be a component in the C. These indications can be solidified using isoelectric focusing or SAA-isotype-specific antibodies [1, 2].

**Figure 3.** Size separation of SAA-HDL at febrile temperatures. SAA-HDL in a patient's acute-phase serum with a common cold was separated in A at 37°C, B at 38°C, C at 40°C and D at 42°C by gel filtration. All individual fractions

(20–70) were examined and semi-quantified by ID using polyclonal rabbit anti-AA antibodies [15, 17].

74 Infectious Process and Sepsis

**3.3. Gradual dissociation of SAA-HDL during a continuous temperature gradient**

dots across the temperature gradient. The SAA-HDL band of α<sup>1</sup>

While these experiments above were done stepwise, one by one, a more precise dissociation of the SAA-HDL separation was performed by electrophoresis in 1.5% agarose across a continuous temperature gradient in a single flat gel, as shown in **Figure 4**. The two sides between the agarose gel were kept at a constant temperature of 15°C in T1 and of 65°C in T2 [17].

The results in **Figure 4** show two horizontal bands of samples of one patient in the form of

marginally stained due to the concealing of the AA-antigenic determinants of SAA within


### **3.4. Activation of the SAA by separation from HDL under febrile temperatures and consequences**

by the organism as a response to various stimuli, exemplified by bacterial invasion. It could represent some sort of a "gear shift" for providing a graded response in order to release special SAAs to provide adequate amounts, which are necessary "tools" for survival. This could occur in concert with other agents including other APPs and cytokines of the APR network. The possible therapeutical manipulation of the body's temperature ("the gear shift") in vivo

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In a collaborative study, each of the eight species-specific polyclonal AA antibodies against eight species (including humans) was immunohistochemically tested against the AA amyloids of eleven different species, including those of humans. The results showed a strong reactivity only with the homologous species and with only some cross-reaction with a related species. The reactivity was in general species specific, but a universal generic AA antibody

The next step was to produce murine monoclonal antibodies against AA and SAA [20]. Their value and merit have been documented by the inventors Köhler and Milstein [38]. Monoclonal antibodies are represented by one amino acid sequence and have the value of a chemical reagent. We selected 20 stable clones (see **Table 1**), which were epitope mapped [31] and immunohistochemically tested on AA amyloids in 10 different mammals, many humans and 9 different birds. Some cross-reactivity with some monoclonals was detected. Most of the 19 AA amyloids tested could be identified with the two monoclonals mc4 and mc29, showing that most of these AA amyloids have some peptides in common and these antibodies recognize the same or very similar epitopes of AA in different species. In addition, antibodies of all clones were tested for binding with 15 synthetic SAA peptides in only 4 clones the epitope could be identified. These included the known clones mc4 and mc29 (see above), and the two new ones, mc1 and mc20 (see **Table 1**). In APS, two different charge variants of SAA have

The cause of the failing reactivity of most of the synthetic peptides with most of the monoclonals may be due to the presence of more discontinuous epitopes. This could also be deduced from the fact that SAA shows multiple short peptides that alternate between the invariable

Moreover, since mc21 was negative with the linear peptides, but reacted very strongly with AA amyloid in tissues, it was epitope mapped differently. It was mapped with endoproteinase Asp-N-generated peptides from a pure and partially amino acid-sequenced human AA (KIR) protein of 8.4 kDa. Of the 11 distinct peptides separated by RP-HPLC, mc21 reacted only with a single peptide, which was aa 33–42 of SAA [32]. This peptide is almost identical with the largest invariant peptide of SAA (see **Figure 5**). Two other monoclonals mc9 and mc13 did not show any reaction with these 11 distinct HPLC peaks [32] although they were reactive with AA in tissue sections. Here again, in linear SAA peptides, the discontinuous epitopes of SAA may not be preserved.

(red) and the variable (white) peptides, as shown in **Figure 5** (see also below).

needs the precise analysis of this phenomenon in vivo first.

could not be obtained in these eight polyclonal antibodies [37].

**4.1. Polyclonal and monoclonal antibodies prepared against AA and SAA**

**4. Application of antibodies**

been detected with these monoclonals [22].

Taken together [15, 17, 18, 21], it is clear that the mechanism of separation of SAA from HDL in vitro is also strictly regulated in vivo by body temperatures above 37°C. Therefore, this is a key mechanism that can be induced and activated basically by two different manifestations. The most common is the orthologic APR activation [33] of SAA. This occurs with a maximal SAA concentration of up to 1000 times within a day as a systemic "biochemical thunderstorm" with a myriad of activating and inhibiting events simultaneously, which are not understood in detail today [1, 2]. During these events, the cause of the APR will be eradicated and the APR becomes curative. With this beneficial outcome, the normal immune homeostasis returns in a foreseeable future. However, when the APR cannot overcome its initial cause, it will become a pathologic APR [33] with a "persistent biochemical thunderstorm" and lacking a self-driven cure. The consequences can be summarized in an exhaustion of the resources of the organism and decline of the metabolic activity through a multitude of clinically challenging conditions exemplified by severe viral and bacterial chronic inflammations, systemic inflammatory response syndrome (SIRS) or uncontrolled chronic infections, sepsis and septic shock [1, 2]. Moreover, when the infection remains limited, a local APR will take care of it.

The functions of the four human isotypes, SAA1, SAA2, (SAA3 in humans is only transcribed in some cells) and SAA4 have not been fully analyzed. They have arisen through gene duplications, thus indicating important individual functions either alone or in combination. As described before, the human acute-phase A-SAA has two very similar isotypes, A-SAA1 and A-SAA2, in the APR mostly synthesized in the liver and expressed in most body cells (see below) and the constitutive C-SAA4 and some allotypes in SAA1 and SAA2. For a review of the SAA heterogeneity and its known functions, see the reviews [1, 2].

Another discovery was the discontinuous separation of SAA from HDL described above at different temperatures, meaning that not all SAA molecules are being separated from HDL at a single temperature except for the temperature of 42°C (**Figures 3** and **4**). In fact, these figures show that the separation of SAA is spreading out over the whole febrile temperature range starting from 38°C to 42°C and above. In addition, based on these observations in **Figure 4**, it is possible that SAA isotypes and allotypes are separated from HDL at different febrile temperatures and thereafter fulfill their different functions locally or systematically as individual SAA species as is also to be derived from **Figure 3**. Another indication for the differential release of the SAA species can be detected in **Figure 4** in the different shapes of the protein blots of the SAAs devoid of HDL, thus indicating possible SAAs with distinct isoelectric points (IP). In **Figure 4**, there are free dots before 37°C named (for convenience) "arc SAA," the least acidic SAA. The SAA species released from HDL after 37°C ("38 SAA") are probably the more acidic ones. In this sense, the dot changes also occur later on 39°C-, 40°C-released SAA, etc. Analyzing the spots for the identity of the various SAA species could show whether these indications did discover a mechanism by which the different SAA species can be released from HDL and thereby are being activated at specific temperatures alone or with other SAAs for special purposes, which need to be analyzed. These points may also be of therapeutical interest. This proposed temperature selection of SAA isotypes could specify the needed APR function for a specific purpose. The increase of the organism's temperature is being induced by the organism as a response to various stimuli, exemplified by bacterial invasion. It could represent some sort of a "gear shift" for providing a graded response in order to release special SAAs to provide adequate amounts, which are necessary "tools" for survival. This could occur in concert with other agents including other APPs and cytokines of the APR network. The possible therapeutical manipulation of the body's temperature ("the gear shift") in vivo needs the precise analysis of this phenomenon in vivo first.
