Consumption of an Extremely Biodiverse Probiotic and a Supplement based on Microbial Chondroitin Sulfate is Associated with Very Low Serum Alpha-N- acetylgalactosaminidase (Nagalase) Activity and Decrease of C-reactive Protein Values

Corresponding Author: Marco Ruggiero Silver Spring Sagl, Via Raimondo Rossi 24, Mendrisio 6850, Switzerland Email: info@bravo-europe.com Abstract: We describe the case of a 57-years-old male healthy professional who added to his nutritional plan a novel, extremely biodiverse, probiotic and a supplement based on microbial chondroitin sulfate, vitamin D3 and ultrapure phosphatidylcholine to improve his healthy life expectancy. After three months of such a nutritional regimen, serum alpha-Nacetylgalactosaminidase (nagalase) activity was 0.40 that is a value below the minimum level established for adults (normal values: 0.50-0.95 nMol/mL/min). C-reactive Protein (CRP) was less than 1 (normal values: 0.00-5.00 mg/L), a value significantly lower than that recorded before this experience. At the end of the observation period, serum albumin level was 45 g/L, a value very close to the maximum reference value (normal values: 32.00-46.00 g/L). The Prognostic Inflammatory Nutritional Index (PINI) score was 0.05, a value that is close to the minimum calculated value (normal values: 0.00-1.00) and suggests a very low risk of death for all causes. Observation in silico and experiments in vitro demonstrated that both the probiotic and the supplement formed complexes with human nagalase with an efficiency 100 fold higher than that of purified Gc proteinderived Macrophage Activating Factor (GcMAF). The collection of these observations suggests that the combination of this probiotic and this supplement may lead to overall beneficial effects on health as evidenced by positive changes of indicators of immune system function and healthy aging such as nagalase, CRP, albumin and PINI score.


Introduction
Serum alpha-N-acetylgalactosaminidase (nagalase) and C-reactive Protein (CRP) are two markers of chronic conditions that have been used to monitor a variety of diseases as well as to predict successful aging and healthy life expectancy. Nagalase, an enzyme, was first proposed as a marker for cancer and viral infections and its increased serum activity in these conditions was associated with immune system deficiency since nagalase prevents the formation of Gc protein-derived Macrophage Activating Factor (GcMAF), an immune stimulant cytokine (Yamamoto and Naraparaju, 1997;Yamamoto and Urade, 2005;Yamamoto, 2006). This occurs because nagalase breaks the bond between alpha-Nacetylgalactosamine and the amino acid threonine situated in the third domain of Gc protein (the precursor of GcMAF), thus effectively removing alpha-Nacetylgalactosamine that is considered the active site of GcMAF. Elevated serum nagalase activity has been reported in a variety of tumors ranging from experimental squamous cell carcinomas to human malignant melanomas (Korbelik et al., 1998;Greco et al., 2009); consistent with these observation, successful immunotherapy of cancer was associated with a decrease of serum nagalase activity (Thyer et al., 2013a;Schwalb et al., 2016). The significance of nagalase in oncology is well exemplified by the title of a recent review that reads "Is α-N-acetylgalactosaminidase the key to curing cancer?" (Saburi et al., 2017). However, elevated serum nagalase activity is associated also with non-neoplastic conditions ranging from bacterial infection (Caines et al., 2008) to autism (Bradstreet et al., 2012). Interestingly, in autism, just like in cancer, successful immunotherapy was associated with significant decrease of serum nagalase activity, thus lending credibility to the hypothesis that elevated nagalase activity has to be considered more a pathogenetic factor for those conditions rather than a simple marker. If the literature on nagalase is relatively new and limited to a small, albeit significant, number of peerreviewed papers, the literature on CRP, an acute-phase protein that increases in the presence of infectious and inflammatory processes, is older and ampler and refers to a large number of conditions ranging from cancer (Allin and Nordestgaard, 2011) to autism (Brown et al., 2014), depression (McFarland et al., 2019, cardiovascular diseases (Cozlea et al., 2013), multiple sclerosis (Dowling and Cook, 1976) and anemia (Breiterman-White, 2006) to name a few. In a study on incurable cancer patients published in 2012, we demonstrated that CRP, integrated into the context of the Prognostic Inflammatory Nutritional Index (PINI) score, was a powerful marker for predicting overall survival (Fabris et al., 2012). Similar to nagalase, CRP may bear more pathogenic significance than simply being a marker; according to a recent paper "CRP is pathogenic in a number of diseases including hypertensive cardiovascular and kidney complications, diabetic nephropathy and acute and chronic kidney diseases" (Tang et al., 2017). Because of this, low levels of CRP have been proposed also as determinants for successful aging (Puzianowska-Kuźnicka et al., 2016). As far as aging is specifically concerned, Ferrucci et al. (2005) demonstrated that CRP increases 3-5 fold during aging in women and men, respectively. Oluleye et al. (2013) demonstrated that risk of death for all causes was significantly reduced when CRP was below 3 mg/L when compared with values above 3 mg/L in more than 11,000 subjects whose average age was 63. Therefore, it can be deduced that strategies aimed at lowering serum CRP levels are to be associated with reduced risk of allcause mortality, increased longevity and successful aging, as proposed by Arima et al. (2008).
To increase his potential for healthy longevity, one of the authors of this article, Dr. MC, a Medical Doctor, decided to implement a nutritional strategy aimed at lowering serum nagalase activity and CRP levels and improving other markers of successful aging. This strategy comprised a novel supplement containing lowmolecular-weight microbial chondroitin sulfate, ultrapure phosphatidylcholine and vitamin D 3 , (imuno ® , imuno Corporation, Vanuatu) and an extremely diverse probiotic formula (Freeze-Dried Bravo Colostrum and Probiotic Complex; Silver Spring Sagl, Mendrisio, Switzerland, here abbreviated in PMF -Product of Microbial Fermentation). Composition of PMF in terms of probiotic microbes, phages and plasmids has been recently described in full detail . The supplement used by Dr. MC (imuno ® ) represents the latest evolution of the concept of GcMAF and its mechanism of action and indications have been thoroughly described in peer-reviewed publications (Ruggiero and Pacini, 2018a;2018b;Antonucci et al., 2018;.
Here, we describe the results observed by Dr. MC after three months of assumption of imuno ® and the PMF and we present a collection of in silico and in vitro observations highlighting the molecular mechanisms of action responsible for these results.

Materials and Methods
Activity of nagalase in serum was determined at R.E.D. Laboratories (Zellik, Belgium) through a proprietary method consisting of a two-step immunecapture assay. Nagalase activity in serum is measured kinetically through conversion of a fluorogenic substrate in function of time. The test is standardized against a large serum pool of carefully selected healthy subjects with normal white blood cell count, no laboratory signs of inflammation, CRP less than 1mg/L and no clinical history of immune disease of diabetes. Nagalase activity measurement in healthy subjects has allowed to establish a normal range of substrate conversion between 0.50 and 0.95 nMol/mL/min for adults.
Study of in vitro interaction between GcMAF, imuno ® , or PMF and human nagalase was also performed by R.E.D. Laboratories (Zellik, Belgium) where GcMAF was purified. The experiments were performed using microtiter plates coated with a specific antibody able to capture human nagalase. Samples were incubated with a standardized dilution of a pool of 300 human sera from healthy subjects identified as described above and, after 1 h incubation and exhaustive washing, complexes formed by nagalase and purified GcMAF, used as positive control, or nagalase and imuno ® , or nagalase and PMF, were detected with a horse radish peroxidase conjugate of a rabbit antibody. In order to establish the kinetics of interaction between nagalase and GcMAF, nagalase and imuno ® , or nagalase and PMF, the serum pool was mixed either with 200 ng of purified GcMAF, or with two dilutions (1:50 and 1:100) of imuno ® , or with two dilutions (1:10 and 1:100) of PMF in phosphate buffered saline (PBS). The mixture was then incubated at room temperature for 4, 24, 48, 72 and 120 h.
Values for nagalase complex forming activity in the absence of GcMAF, imuno ® or PMF, that is with only PBS in the reaction mixture, were taken as 1.00. The experiment was repeated twice and the results reported in Figs. 3-7 are the means of the two experiments.
As far as other markers of health are concerned, determination of nagalase activity in serum was complemented by evaluation of another serum enzyme that is useful as cancer and inflammation marker, dipeptidyl peptidase-4 (DPP4) also known as CD26 (Pro and Dang, 2004;Ghorpade et al., 2018). This is an enzyme expressed on the surface of most cell types, including macrophages and T-lymphocytes and is associated with immune regulation, signal transduction and apoptosis (Havre et al., 2008;Hiromura et al., 2018). The PINI score was calculated as described by Fabris et al. (2012).
Since this is a single case report that does not produce generalizable knowledge, nor is it an investigation of an FDA regulated product, an Institutional Review Board (IRB) review is not required for this activity. Likewise, since this case report describes the experience of one of the Authors, Dr. MC, the concept of "informed consent" is implicit in the authorship of the study.

Case Presentation
Dr. MC, Medical Doctor, is an active and energetic professional born in 1962, who works as Corporate Medical Director of Real Health Clinics in Atlanta, GA, as Chief Medical Officer for Health Revival Partners and as Chief Medical Advisor for Tissue Regeneration Technologies, Woodstock, GA, USA; he added imuno ® (0.1 mL twice a week) and PMF (1 capsule per day) to his nutritional regimen without any other change to his nutrition or lifestyle. His experience lasted for three months and ended in July 2019. Blood analyses were performed immediately after the three-month experience.
After three months of consumption of imuno ® and PMF, nagalase activity, expressed as nMol/mL/min, was 0.40 that is a value below the minimum level established for adults (normal values: 0.50-0.95). Consistent with this result, DPP4 activity was 24 nMol/mL/min (normal values: 18.80-33.79), further indicating optimal functioning of the immune system. CRP, expressed as mg/L, was less than 1 (normal values: 0.00-5.00). It is worth considering that a previous blood analysis, performed in November 2018 at Lab Corp (Virtual Clinics, Terre Haute, IN, USA) evidenced a CRP value of 0.29 mg/L that, although within the normal value reference, was significantly higher. At the end of the observation period, serum albumin level was 45 g/L, a value very close to the maximum reference value (normal values: 32.00-46.00 g/L). It is worth noticing that serum albumin levels physiologically decrease with aging and lower levels are associated with increased risk of all-cause mortality (Weaving et al., 2016). Consistent with the high albumin/low CRP values, the PINI score was 0.05 a value that is close to the minimum calculated value (normal values: 0.00-1.00) and suggests a very low risk of death for all causes.

In silico Observation and Experimental Results in vitro
Having observed a significant decrease of serum nagalase activity following assumption of imuno ® and PMF, we performed studies in silico and in vitro to establish the molecular mechanisms of action responsible for the observation of Dr. MC. First, we sought to determine the potential sites of interaction between nagalase and GcMAF, nagalase and imuno ® , or nagalase and PMF using the "Align" tool of Uniprot, a tool designed to "align two or more protein sequences with the Clustal Omega program to view their characteristics alongside each other" (www.uniprot.org). We chose the sequence P02774 VTDB_HUMAN for human GcMAF and the sequence P17050 NAGAB_HUMAN for human nagalase. Figure 1 shows the binding sites of human nagalase as indicated by green arrows. The active sites are indicated by red arrows; the aspartic acid in the sequence DDIQ is indicated by a green and a red arrow since it is both a binding site and the second active site of the enzyme. The binding and active sites were identified by the Align tool of Uniprot. Figure 1 also shows the active site of GcMAF that is encased in a square with black outline and indicated by a black arrow. The sequence TPTELAK is the sequence where alpha-N-acetylgalactosamine is attached to the amino acid threonine. In the inactive form of GcMAF, that is in its precursor, sialic acid and D-galactose are attached to alpha-N-acetylgalactosamine -that is the active site of GcMAF -thus masking it and preventing interaction with its receptor. When sialic acid and D-galactose are removed, alpha-N-acetylgalactosamine can interact with its receptor and GcMAF is active. Nagalase breaks the bond between alpha-N-acetylgalactosamine and the amino acid threonine in the sequence TPTELAK only when GcMAF is in its inactive form, that is when sialic acid and D-galactose are attached to alpha-N-acetylgalactosamine. It is worth noticing that the binding and active sites of nagalase are flanked by stretches of hydrophobic amino acids that are highlighted in indigo in Fig. 1. The active site of GcMAF comprises hydrophobic amino acids and it is also flanked by hydrophobic amino acids. Based on the molecular model we first proposed in 2013 (Thyer et al., 2013b) these hydrophobic interactions are deemed responsible for formation of complexes between nagalase and GcMAF. In PMF, such an interaction is strengthened by the presence of fatty acids that are natural components of milk and colostrum; enhancement of hydrophobic interactions may be one of the factors responsible for the 100 fold higher nagalase complex forming activity observed in PMF as compared to purified GcMAF (Figs. 5-7). It may be worth noticing that the stretches of hydrophobic amino acids flanking the binding and active sites of nagalase may also bind the ultrapure phosphatidylcholine that forms ordered structures of phospholipid bilayers in cell-like membranes comprising the tri-dimensional molecular structure of imuno ® (Ruggiero and Pacini 2018a;2018b). In analogy with PMF, these enhanced hydrophobic interactions may be responsible for the 100 fold higher nagalase complex forming activity observed in imuno ® as compared to purified GcMAF (Figs. 3, 4 and 7). Figure 2 shows the distribution of positively (highlighted in green) and negatively (highlighted in red) charged residues in GcMAF and nagalase with particular reference to the binding and active sites of both proteins that are indicated by squares outlined in black and black arrows. Both active sites of nagalase are constituted by negatively charged residues (aspartic acid). Figure 2 also shows how the active sites of nagalase present alternation of positively and negatively charged residues that may represent the binding sites for the highly sulfated low-molecular-weight microbial chondroitin sulfate of imuno ® that is constituted by moieties alternating negative and positive charges. More specifically, electrostatic interaction between the aspartic acid residues of the active sites of nagalase and the alpha-N-acetylgalactosamine that is a constituent of chondroitin sulfate, complementing the hydrophobic interactions described above, may explain the 100 fold higher nagalase complex forming activity observed in imuno ® as compared to purified GcMAF (Figs. 3, 4 and  7). In other words, here we demonstrate that the alpha-Nacetylgalactosamine of chondroitin sulfate works at the molecular level like the active site of GcMAF with the notable difference that there is only one alpha-Nacetylgalactosamine moiety per GcMAF molecule whereas there are 50 to 100 alpha-N-acetylgalactosamine moieties per chondroitin sulfate molecule. In addition, the peculiar conformation of imuno ® with negatively charged surfaces constituted by carboxyl and sulfonic groups encased in a phospholipid bilayer, may be involved in the binding and electrostatic neutralization of protein inserts characterized by high density of positively charges residues. In particular, it may be involved in binding the sequences TNGTKR, HKNNKS, RSYLTPGDSSSG and QTNSPRRA that are characterized by a high surface concentration of positive charges interspersed with hydrophobic residues (Zunaid et al., 2020). It is worth noticing that neutralization of positively charged residues may represent a strategy that exploits electrostatic interactions between charges on the surface of bioactive molecules. Fig. 1: Sequence alignment between Gc protein-derived macrophage activating factor, GcMAF and nagalase. The sequences of GcMAF (P02774 VTDB_HUMAN) and nagalase (P17050 NAGAB_HUMAN) were obtained and aligned using the Align tool of Uniprot (uniprot.org). The conventional consensus symbols are: "*" indicating that the residues are identical in all sequences in the alignment. ":" indicating that conserved substitutions have been observed. "." indicating that semi-conserved substitutions are observed, that is, amino acids having similar features. Hydrophobic amino acids are highlighted in indigo. The binding sites of nagalase are indicated by green arrows. The active sites are indicated by red arrows; the aspartic acid in the sequence DDIQ is indicated by a green and a red arrow since it is both a binding site and the second active site of the enzyme. The active site of GcMAF is encased in a square with black outline and indicated by a black arrow. The sequence TPTELAK is the sequence where alpha-N-acetylgalactosamine -that is the substrate of nagalase -is attached to the amino acid threonine Fig. 2: Sequence alignment between Gc protein-derived macrophage activating factor, GcMAF and nagalase. Negatively and positively charged amino acids. Positively (highlighted in green) and negatively (highlighted in red) charged residues in GcMAF and nagalase are depicted, with particular reference to the binding and active sites of both proteins that are encased in squares outlined in black and indicated by black arrows Fig. 3: Kinetics of complex formation between purified GcMAF or imuno ® and human nagalase. Values for nagalase complex forming activity in the absence of GcMAF or imuno ® , with only PBS in the reaction mixture, were taken as 1.00. imuno ® , diluted 100 fold, had an initial value (at time 0) higher than that observed with PBS alone, thus demonstrating immediate, intrinsic GcMAF activity. At every time point, imuno ® showed significantly higher activity in comparison with purified GcMAF and, at 120 h, the activity of imuno ® was still well above baseline, whereas GcMAF did not show any residual activity   Figure 3 shows the kinetics of in vitro interaction between human nagalase and imuno ® with resulting formation of nagalase/imuno ® complexes detected as described in the Materials and Methods. Since it is well assessed that nagalase specifically binds GcMAF, formation of complexes between nagalase and imuno ® is direct demonstration of the GcMAF activity of imuno ® . As shown in Fig. 3, purified GcMAF, used as positive control, formed complexes with human nagalase only after 4 h incubation, reached a peak at 48 h and returned below baseline values at 120 h. These kinetics for purified GcMAF were taken as benchmark. imuno ® , diluted 100 fold, had an initial value (at time 0) higher than that observed with PBS alone, thus demonstrating immediate, intrinsic GcMAF activity. At every time point, imuno ® showed significantly higher activity in comparison with purified GcMAF and, at 120 h, the activity of imuno ® was still well above baseline, whereas GcMAF did not show any residual activity. Therefore, it may be argued that the GcMAF activity of imuno ® is more than 100 fold higher than that of purified GcMAF and more sustained in time. These results are consistent with the in silico observation described above as well as with the fact that chondroitin sulfate, one of the constituents of imuno ® , has many more alpha-N-acetylgalactosamine moieties per molecule than GcMAF. Figure 4 shows the same results represented as 3D areas in order to highlight the significantly higher GcMAF activity of imuno ® as compared to purified GcMAF.  and human nagalase after 120 h incubation. Values for nagalase complex formation activity in the absence of GcMAF, imuno ® , or PMF, with only PBS in the reaction mixture, were taken as 1.00. imuno ® , diluted 1:50 showed higher GcMAF activity than imuno ® diluted 1:100 just like PMF, diluted 1:10 was more active than PMF diluted 1:100. imuno ® and PMF at the highest dilution (1:100) were significantly more active than control (PBS alone) or purified GcMAF Experimental data obtained in vitro and represented in Figs. 5 and 6 demonstrate the GcMAF activity that is present in PMF. Since it is well assessed that nagalase specifically binds GcMAF, formation of complexes between nagalase and PMF is direct demonstration of the presence of active GcMAF in PMF as we described since 2011 (Pacini et al., 2011). Figure 5 and 6 shows the kinetics of in vitro interaction between human nagalase and PMF with resulting formation of nagalase/PMF complexes. In analogy with what observed with imuno ® , PMF at 1:100 dilution had an initial value (at time 0) higher than that observed with PBS alone, thus demonstrating the presence of immediate, intrinsic GcMAF activity. At every time point, PMF showed significantly higher activity in comparison with purified GcMAF and, at 120 h, the GcMAF activity of PMF was still well above baseline, whereas purified GcMAF did not show any residual activity (Fig. 7). Therefore, it may be argued that the GcMAF activity in PMF is more than 100 fold higher than that of purified GcMAF and more sustained in time. The 100 fold higher activity of PMF in comparison with purified GcMAF can be explained taking into consideration two elements.
1. Naturally formed GcMAF in PMF is associated with vitamin D 3 , fatty acids and glycosaminoglycanssuch as chondroitin sulfate -that are normal constituents of milk and colostrum. Since 2013, we proposed a molecular model describing how non-covalent association of these constituents in a multi-molecular complex significantly enhanced the biological activity of GcMAF (Thyer et al., 2013b). This model was independently confirmed by a recent study demonstrating that association with vitamin D 3 significantly increased the immune stimulating activity of GcMAF (Greilberger and Herwig, 2020).
2. Phages in PMF synthesize proteins with activities superimposable to that of GcMAF that complement the effects of the naturally formed GcMAF that is present in PMF. For example, the protein RAD52 that is the human homolog of the protein Sak encoded by Lactococcus phage ul36, shows significant similarity with the active site of GcMAF. When the sequences of human RAD52 (P43351) and GcMAF (P02774) were obtained and aligned using the Align tool of Uniprot, alignment showed a striking similarity between the active site of GcMAF that is the sequence TPTELAK and the sequence DPAQTSD of RAD52. Since it is well known that phages stimulate the immune system and it is also known that such a stimulation is associated with macrophage activation , it is may be hypothesized that GcMAF-like proteins encoded by phages contribute to the overall GcMAF activity of PMF. Figure 7, shows the formation of nagalase/GcMAF, nagalase/imuno ® , or nagalase/PMF complexes after 120h incubation. It is worth noticing that imuno ® diluted 1:50 showed higher GcMAF activity than imuno ® diluted 1:100 just like PMF diluted 1:10 was more active than PMF diluted 1:100. These results demonstrate a classic dose-response relationship for both imuno ® and PMF as far as their respective GcMAF activity is concerned. In addition, both imuno ® and PMF at the highest dilution (1:100) were significantly more active than the control (PBS alone) or purified GcMAF. It is worth considering that these results were obtained in vitro, that is in the absence of any variable or confounding factor that may affect interpretation of results previously observed in clinical settings (Thyer et al., 2013a; present case report).

Discussion
The changes observed during the three-month experience here described, support the hypothesis that such a nutritional approach provides health benefits ranging from immune system support to reduction of age-related low-level inflammation. A decrease of serum nagalase activity can be interpreted as the result of modulatory activities of imuno ® and PMF on the immune system with particular reference to GcMAF activity and macrophage activation (Ruggiero and Pacini 2018a). As demonstrated here, both imuno ® and PMF have intrinsic, direct GcMAF activity according to the results of the experiments in vitro performed by an independent laboratory and described in Figs. 3-7. It is therefore conceivable that the regimen adopted by Dr. MC (1 capsule of the probiotic per day and 0.1 mL of the supplement twice a week) represents the ideal combination to obtain the most benefit from the two complementary approaches. As far as the decrease of CRP is concerned, it is worth noticing that imuno ® was designed to optimize mitochondrial function as we described in a recent paper (Pacini and Ruggiero, 2019c) and it is well assessed that aging and chronic conditions are associated with, or possibly caused by, mitochondrial dysfunction that leads to chronic energy imbalance with resulting oxidative stress, systemic inflammation and elevation of CRP values (Gajewski et al., 2017). Therefore, it is plausible that optimization of mitochondrial function by imuno ® , together with the antiinflammatory properties of PMF thoroughly described in two recent articles  was responsible for the observed decrease of CRP that was in turn responsible for the ultra-low PINI score recorded at the end of the three-month experience.
The Authors wish to underline the concept that the results here described are from one individual and not part of a wider study. However, the Authors believe the observations like this may benefit those addressing the impact of nutrition on health and successful aging. We consider that this approach is consistent with that proposed by Aronson (2005) who encourages reports of single cases in medicine. Since the results observed in this report cannot be generalized at this stage, further studies are warranted to evaluate whether the nutritional approach here described will yield similar results in other subjects either in the context of healthy aging or in the presence of pathologic conditions. These words of caution notwithstanding, it is worth mentioning that the combination of the probiotic and the supplement here described proved very effective in the nutritional approach to a case of multiple myeloma (Antonucci et al., 2019b) thus lending credibility to the hypothesis that such a combination may bring substantial benefits in a number of diverse conditions.

Conclusion
The experience of Dr. MC suggests that the combination of a novel supplement based on lowmolecular-weight chondroitin sulfate, ultrapure phosphatidylcholine and vitamin D 3 (imuno ® ) and an extremely diverse probiotic featuring strains with known healthy properties, may lead to overall beneficial effects on health as evidenced by positive changes of indicators of immune system function and healthy aging such as nagalase, CRP and albumin.