**5. Chromogranin A, a new marker of severity**

196 Antimicrobial Agents

infections of central nervous system, bone infections and sometimes for the pulmonary infections which require a more concentrations to get treated (Dehority, 2010). *S. aureus* have also developed resistance to the Vancomycin due to the use at low level concentrations and recently, *S. aureus* was isolated that had got the *VanA* gene from the *Enterococcus spp* (

In our group, we have examined the synergically effects of three CGA-derived peptides (CAT, CTL and CHR) with Minocyclin, Amoxicillin and Linezolide. To demonstrate that antimicrobial peptides are able to reduce the doses of antibiotics used and to potentiate the activity of antibiotics, antimicrobial tests were carried out by combining the antibiotic peptides at doses below the MIC. The comparison was made with the antibiotic or peptide

Minocyclin has a MIC of 2 µg/ml alone against the *S. aureus* ATCC 49775, but when it was combined with CTL at a concentration corresponding to 75% of the MIC, the, concentration of Minocycline was lowered to 0.5 µg/ml. Similar data were obtained by the use of the two others peptides (Figure 6). Thus we demonstrate that amidated bCTL acts synergistically with Minocycline against *S. aureus*. In addition CTL acts synergistically with Voriconazole

Fig. 6. Fractional inhibitory concentration (FIC) of the chromogranin derived peptides combined with the antimicrobials (Minocycline against the *Staphylococcus aureus* and Voriconazole against *Candida albicans* and *Candida tropicalis*). FIC in range of ≤0.5 gives a synergistic effect, ≤0.5 - <2 is an additive effect but if more than 2 have an antagonistic effect.

Some of the strains of *Shigella* got resistance to antibiotics. A 9-year study of shigellosis in Malaysia, show that 58.4% of the studied strains were resistant to tetracyclin and 53.8% to trimetropin-sulfamethoxasol (Banga Singh et al., 2011). In China, another study establish for *Shigella* the resistance to aztrenam (30,8%), ampicillin (92,3%), piperacilline (61,5%), ceftazidime (30,8%), cefotaxime (30,8%), gentamicine (53,8%) (Zhang et al., 2011). Furthermore, *Vibrio cholera* was also described to develop several resistances against

Sievert et al., 2008) which leads to drug resistance.

against *Candida albicans* and *Candida tropicalis.*

separately at the same doses.

**4.2 From** *Shigella* 

antibiotics (Lamrani et al., 2010).

In clinical practice, CGA has been used as a marker of pheochromocytomas (O'Connor et al., 1984), carcinoid tumors (O'Connor & Deftos, 1986; Syversen et al., 1993), neuroblastomas (Hsiao et al., 1990), neuroendocrine tumors (Berruti et al., 2005), and neurodegenerative diseases (Rangon et al., 2003). Recent data have shown CGA to be a useful prognostic indicator in patients with chronic heart failure (Omland et al., 2003, suggesting that CGA may have some association with cardiovascular diseases. Furthermore, a pilot study has shown CGA to be a predictor of mortality in patients with acute myocardial infarction (Estensen et al., 2006). Characterization of the severity of organ failures and prediction of patient outcome are of major importance for physicians who care for critically ill patients. Multiple organ failure (MOF) remains the main problem in intensive care because of its impact on morbidity, mortality, and resources (Baue et al., 1998). MOF can develop as a consequence of multiple causes, such as infection, systemic inflammatory response syndrome (SIRS), myocardial infarction, septic shock, leading to the activation of various endogenous cascades, cellular dysfunction and death (Baue et al., 1998).

In a recent study we have evaluated whether unselected critically ill patients at ICU (Hautepierre Hospital, Strasbourg, France) admission demonstrate increased plasma CGA concentrations and whether CGA can be of any interest in the care of patients at high risk of death. Patients older than 18 years were recruited consecutively over 3 months during 2007. Exclusion criteria included: duration of stay >24 h and conditions known to increase CGA concentrations independently of acute stress [i.e., a history of documented neuroendocrine tumors (O'Connor & Deftos, 1986) or chronic treatment with proton pump inhibitors before admission (Giusti et al., 2004)*.* Patients who required surgical interventions were also excluded. Of the 120 participants included in the study, 70 patients had a primary diagnosis severe infection, and 50 had a SIRS. Serum CGA concentrations were measured with a commercial sandwich RIA kit (a gift of Cisbio Bioassays, Marcoule, France. In the central 95% of the healthy population, serum CGA concentrations range from 19µg/L to 98µg/L. In neuroendocrine system tumors, the CGA serum concentration varies from the typical range up to 1200 µg/L, depending on the biological and structural characteristics of the tumor, as well as on the extent of tumor spread (Degorce et al., 1999). As a control Procalcitonin (PCT) concentrations were measured on the Kryptor system (Brahms Diagnostic) with the timeresolved amplified cryptate emission methodology in accordance with the manufacturer's recommendations. The Simplified Acute Physiological Score II (SAPS II) and the Logistic Organ Dysfunction System (LODS) score were calculated at admission according to published standards (Levy et al., 2003; Le Gall et al., 1993)*.* Our data show that CGA concentration was positively but weakly correlated with age, PCT concentration, creatinine concentration, SAPS II, and LODS score (*P* <0.001 for all variables) and was correlated with CRP concentration (Zhang et al., 2008). Thirty-three deaths occurred during the median follow- up time of 23 days. The death rates for CGA and PCT are shown by quartiles in Figure 7. Statistical analysis revealed a significant difference in death rates between CGA quartile 4 and CGA quartiles 1, 2, and 3 (*P* < 0.001, log-rank test). The death rate for CGA quartile 3 was also significantly different from that of CGA quartile 1 (*P*= 0.033).

The Natural Antimicrobial Chromogranins/Secretogranins-Derived

candidiasis rat model (Etienne et al., 2005).

**7. Conclusions** 

**8. Acknowledgement** 

**9. References** 

202X.

Peptides – Production, Lytic Activity and Processing by Bacterial Proteases 199

antifungal activity by interacting with the fungal membrane and penetrating into the cell. *In vitro* studies demonstrated that such an antifungal coating is able to inhibit the growth of yeast *Candida albicans* by 65% and completely stop the proliferation of filamentous fungus *N.crassa*. The cytotoxicity of such a coating was also assessed by growing human gingival fibroblasts at its surface. Finally, the antifungal coating of poly (methylmethacrylate), a widely used material for biomedical devices, is successfully tested in an *in vivo* oral

CGs family emerges as prohormones able to modulate homeostatic processes in response to excessive stimulations such as microbial infections. The studies concerning the expression of CGs and their antimicrobial peptides in patients with inflammatory diseases and the correlation with the proteolytic processes occurring in these pathologies vs. controls are crucial to understand the involvement of these prohormones and their derived peptides in innate and adaptive immunities. Calcium is a universal secondary messenger involved in many cellular signal transduction pathways, regulating crucial functions such as secretion, cell motility, proliferation and cell death. The calcium-dependent immunomodulatory properties of CHR and CAT are important for the understanding of their involvement in inflammatory mechanisms. In sum, these linear peptides may represent prototypic lead molecules useful for the development of new therapeutic agents and also biomaterials.

This study was funded by INSERM, the University of Strasbourg (UDS) and INRA (JFC). We are grateful to financial assistance from the Odontology Faculty of the University of Strasbourg (MA, RA). We thank Charlotte Bach for the excellent technical assistance, Bernard Guérold for the preparation of the synthetic peptides, Thomas Lavaux for fruitful discussions, Cisbio Bioassays Research and Development for providing the ELISA kit required for CGA dosages, EA44-38 for the isolation of bacteria and the preparation of

Aardal, S., Helle, K.B., Elsayed, S., Reed, R.K. & Serck-Hanssen, G. (1993). Vasostatins,

Abtin, A., Eckhart, L., Mildner, M., Ghannadan, M., Harder, J., Schroder, J.M. & Tschachler,

Aerts A.M., François, I.E., Cammue, B.P. & Thevissen, K. (2008). The mode of antifungal

65, No. 12, (July 2008), pp. 2069-2079, ISSN: 1420-682X.

comprising the N-terminal domain of chromogranin A, suppress tension in isolated human blood vessel segments*. Journal of Neuroendocrinology,* Vol. 5, No. 4, (August

E. (2009). Degradation by stratum corneum proteases prevents endogenous RNase inhibitor from blocking antimicrobial activities of RNase 5 and RNase 7*. Journal of Investigative Dermatology*, Vol. 129, No. 9, (March 2009), pp. 2193-2201, ISSN: 0022-

action of plant, insect and human defensins*. Cellular and Molecular Life Science,* Vol.

bacterial supernatants and Martine Rivet for the preparation of the manuscript.

1993), pp. 405-412, ISSN: 0953-8194.

Fig. 7. Kaplan–Meier analysis: cumulative incidence of death by CGA and PCT quartiles. (A), Median (interquartile range) for CGA concentration data: quartile 1, 35 \_g/L (30–53 \_g/L); quartile 2, 84 \_g/L (77–94 \_g/L); quartile 3, 174 \_g/L (151–197 \_g/L); quartile 4, 563 \_g/L (355–974 \_g/L). Each quartile includes 30 patients. (B), ROC curve to test the ability of CGA (black line), SAPS II (black dashed line), and PCT (gray dashed line) to predict outcome.

ROC curves for CGA, PCT, and SAPS II are shown in Figure 7. To assess the best positive likelihood ratio, we chose the cutoff value that was associated with the best specificity. For CGA, we chose a cutoff value of 255µg/L, which produced a sensitivity of 0.63 and a specificity of 0.89 (positive likelihood ratio, 5.73; negative likelihood ratio, 0.42; AUC, 0.82). A cutoff value of 65 for SAPS II produced a sensitivity of 0.61 and a specificity of 0.85 (positive likelihood ratio, 4.07; negative likelihood ratio, 0.46; AUC, 0.87). For a PCT cutoff value of 4.82 µg/L, sensitivity and specificity were 0.60 and 0.71, respectively (positive likelihood ratio, 2.07; negative likelihood ratio, 0.56; AUC, 0.73). To conclude, in this clinical study of critically ill nonsurgical patients, we demonstrate that plasma CGA is a strong and independent prognostic in consecutive critically ill nonsurgical patients. The over expression of complete CGA suggests that for these patients the processing machinery to procuce antimicrobial peptides in not correct.

#### **6. Insertion of synthetic antimicrobial chromogranins-derived peptides in biomaterials**

The surface of medical devices is a common site of bacterial and fungal adhesion, first step to the constitution of a resistant biofilm leading frequently to chronic infections. In order to prevent such complications, several physical and chemical modifications of the device surface have been proposed. In a previous study, we experimented a new type of topical antifungal coating using the layer-by-layer technique. The nanometric multilayer film obtained by this technique is functionalized by the insertion of a CgA-derived antifungal peptide (CGA 47-66, Chromofungin). We show that the embedded peptide keeps its antifungal activity by interacting with the fungal membrane and penetrating into the cell. *In vitro* studies demonstrated that such an antifungal coating is able to inhibit the growth of yeast *Candida albicans* by 65% and completely stop the proliferation of filamentous fungus *N.crassa*. The cytotoxicity of such a coating was also assessed by growing human gingival fibroblasts at its surface. Finally, the antifungal coating of poly (methylmethacrylate), a widely used material for biomedical devices, is successfully tested in an *in vivo* oral candidiasis rat model (Etienne et al., 2005).
