International Association for Human and Animals Health Improvement; 6. [Received June 30, 2015; Accepted August 20, 2015]

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1 International Journal of Probiotics and Prebiotics Vol. 10, No. 2/3, pp , 2015 ISSN print, Copyright 2015 by New Century Health Publishers, LLC All rights of reproduction in any form reserved IMPACT OF LACTOBACILLUS ACIDOPHILUS INMIA 9602 Er-2 AND ESCHERICHIA COLI M-17 ON SOME CLINICAL BLOOD CHARACTERISTICS OF FAMILIAL MEDITERRANEAN FEVER DISEASE PATIENTS FROM THE ARMENIAN COHORT 1,5 Marine Balayan, 1,5 Anahit Manvelyan, 2,5 Seda Marutyan, 3 Marianna Isajanyan, 4,5 Vardan Tsaturyan, 1,5 Astghik Pepoyan, 6 Francesco Marotta and 7 Tamas Torok 1 Armenian National Agrarian University, Yerevan, Armenia; 2 Yerevan State University, Yerevan, Armenia; 3 Armenian State Pedagogical University, Yerevan, Armenia; 4 Yerevan State Medical University, Yerevan, Armenia; 5 International Association for Human and Animals Health Improvement; 6 Unit of Healthy Aging by Genomics & Biotech, Montenapoleone Medical Center, Milano, Italy; and 7 Ecology Department, Lawrence Berkeley National Laboratory, Berkeley, USA. [Received June 30, 2015; Accepted August 20, 2015] [Communicated by Prof. Francesco Marotta] ABSTRACT: Forty-nine volunteer patients with familial Mediterranean fever (FMF) disease in remission were involved in a double blind, partly randomized, placebocontrolled clinical trial in order to investigate the impact of the commercial probiotics Narine, containing the probiotic strain Lactobacillus acidophillus INMIA 9602 Er-2 strain 317/402, and Colibakteron, containing Escherichia coli M-17, on blood serum glucose level, erythrocyte sedimentation rate (ESR) and level of C-reactive protein (CRP). The insignificant increasing drift in the blood glucose level has been described after application of probiotics. At the same time, the CRP level in the blood of patients who were reported to have high CRP level in remission decreased after probiotic therapy. Thus, the effects of commercially available probiotics Narine and Colibakteron have been shown on some blood characteristics. KEY WORDS: Blood glucose, colchicine, C-reactive protein, erythrocyte sedimentation rate, Familial Mediterranean fever, probiotic. Corresponding Author: Prof. A. Pepoyan, Armenian National Agrarian University, Yerevan, Armenia; Tel/Fax: ; apepoyan@gmail.com INTRODUCTION In the classification of the European Society of Immunodeficiency Autoinflammatory Diseases (human autoinflammatory diseases - HAIDS) are primary immunodeficiency conditions caused by genetic infringement of interoperability of regulators of inflammation and arised in absence of pathogens (Rameev et al., 2012; Toplak et al., 2012). For some of these diseases amyloidosis progression is possible (Ben-Zvi et al., 2012) and autoimmune aggressions (autoantibodies, autoreactive T-lymphocytes) are not defined. The familial Mediterranean fever (FMF), known also as periodic disease, belongs to the group of autoinflammatory diseases. It is an ancient and widespread autoinflammatory syndrome affecting more than 100 thousand people all over the world (Yamazaki et al.; 2009). FMF affects mainly nationalities originating from the Mediterranean Sea region: Jews, Armenians, Turks, northern Africans, and Arabs (Brik et al., 1999). FMF is a hereditary disease manifested by episodic fever as well as painful abdominal and pleural inflammations. The gene MEFV, which is responsible for FMF manifestation, is localized on a short arm of the 16-th chromosome (16p13.3, MIM no ) (Pras et al., 1992; Pras et al., 1995). The frequency of heterozygous carriers of the mutation of MEFV gene is high among Armenians (1 in 5 healthy people) (Sarkisian et al., 2007). The most prevalent mutation in the Armenian population is M694V, which cause severe cases of FMF (Sarkisian et al., 2007; Touitou et al., 2007). According to the literature, common FMF genotypes among Armenians are M694V/M694V (20.9%), M694V/M726A (18%), M694V/M680I (12.7%), M680I/M726A (9.8%), M680I/ M680I (3.4%), M726A/M726A (2.8%) and 694V/R761H (2.8%) (Sarkisian et al., 2007). As a result of human migration and interethnic marriages, FMF has spread in many countries (Greece, Russia, Bulgaria, Italy, Japan, India, China, etc.) (Ben-Chetrit & Touitou,

2 92 Impact of probiotics on clinical blood characteristics of FMF patients 2009). FMF patients often suffer from arthritis, pericarditis, nephritis, orchitis, vasculitis, meningitis, episcleritis, and erysipeloides (Harutyunyan et al., 2000). Accumulation of amyloid in various organs is a common consequence of regular inflammatory periodic attacks. The most severe complication is considered to be kidney amyloidosis, which can lead to renal failure (Onen, 2006; Hama et al., 2012). During disease attacks in many patients, laboratory-measured values of inflammatory activities increase (ESR, CRP, fibrinogen, leukocytosis, etc.) and return to normal levels during clinical remission, but in other patients, these levels remain elevated (Lachmann et al., 2006). The most effective FMF treatment is the administration of colchicine, which also is effective in the prevention of amyloidosis (Kalinich et al., 2007). Colchicine is considered to be a strong antimitotic drug. It down regulates the inflammation by bonding with tubulin, blocking formation of microtubules and spindles required for fission at the metaphase stage in neutrophil cells (Nidorf et al., 2013). However, the application of colchicine can cause side effects: i diarrhea or constipation accompanied by disorders in gut microbiota (dysbiosis); ii allergic reactions; iii diminishing of quality and quantity of spermatozoa, potentially leading to male sterility; iv impairment of functions of blood-making organs (Lidar et al., 2003). Commercially available probiotics Narine and Colibacteron are often recommended to Armenian FMF patients by physicians. The aim of current investigation was to evaluate effects of probiotic supplementation on blood glucose level, ESR and CRP in Armenian FMF patients. MATERIALS & METHODS Forty-nine FMF patients (age range 17 to 50 years) in remission have been investigated. All patients have taken colchicine in various doses, depending on the form of disease. Patients have been screened for gene mutations using FMF StripAssay (ViennaLab Diagnostics GmbH, Austria) according to manufacturer instructions. Patients that had particularly low quantities of lactobacilli in fecal samples were assigned to take Narine (16 FMF patients), whereas patients with low amount of commensal E. coli were assigned Colibacteron (16 FMF patients). The patients with normal amount of lactobacilli and commensal E. coli were included in the placebo group (16 FMF patients). The patients were involved in a double blind, partly randomized, placebo-controlled clinical trial. ESR measurements were performed by the Panchenkov s method (Blindar VN, 2013). RF, CRP and the level of serum glucose were measured according to standard procedures using Stat Fax 3300 (Awareness technologies). The reference ranges were used according to the recommendations of the Ministry of Health of the Republic of Armenia and norms of the diagnostic laboratory where the tests were performed. Statistical analyses were performed by Student s t test. RESULTS AND DISCUSSION Description of Investigated FMF Patients Among 49 registered patients (7 female and 42 male), the following symptoms were observed: chest attacks (symptoms of pleuritis) in 5.8 %, abdominal attacks (symptoms of peritonitis) in 11.5%, and mixed attacks (pleuritis and peritonitis) in 82.7 %. It should be noted that the frequency of occurrence of periodic disease among men is almost twice that of women (Samuels et al, 1998). Sixty-seven percent of enrolled patients reported experiencing their first attacks before 10 years of age and 15.4% after age 20. Common MEFV mutation patterns were M694V/V726A: 86.2 % patients carried the M694V mutation (4.5 %- with R761H; 4.5%-with M680I(G/C); 4.5%-with P369S; 9 %- with E148Q and 55%- with V726A). Amyloidosis has been reported in 11.5 % patients, affecting males and females equally(50% vs. 50%). The most patients from the present study had a mixed (chest and abdominal) form of FMF. This finding is in accordance with the literature data (Korkmaz, 2012; Obici and Merlini, 2012; Pras, 1982). For these patients. various mutations were revealed: one or two in heterozygous or a compound- heterozygous condition, but all patients carried M694V. Impact of Probiotics on Blood Glucose Level The insignificant increasing drift in the blood glucose level was described after application of probiotics (Table 1). TABLE 1. The Alteration in Blood Characteristics of FMF Patients after Consumption of Probiotics. The data are presented as mean ± Standard Error. * p value of Student s t-test, differences between the parameters of before and after probiotics adoption; ** Values of CRP of FMF patients with its initially high levels Treatment Amount of glucose; mmol/l C-reactive Protein**; mg/l Before probiotics 4.21 ± ± 4.8 After probiotics 4.93 ± 0.10* P < ± 1.47* P < 0.05 Before placebo 4.6 ± ± 8.48 After placebo ± ± 4.71 As it has been mentioned previously (Pepoyan et al, 2015), investigated patients had normal levels of blood glucose during remission, and while the literature reports collateral influence of colchicine on lactose tolerance (Fradkin et al, 1995), no lactase suppression was observed for the investigated Armenian FMF patients (Pepoyan et al, 2015). Lactase is an enzyme from the β-galactosidase family, which hydrolyzes

3 Impact of probiotics on clinical blood characteristics of FMF patients 93 glycoside links in a molecule of lactose and forms galactose and glucose. In humans, lactase is produced by the mature small intestine enterocytes, whose activity changes during ontogenesis (Lomer et al., 2008; Matthews et al., 2005; He et al., 2008). If lactase activity is insufficient for digestion of all ingested lactose, un-hydrolyzed lactose moves to the large intestine where it becomes a nutritious substratum for bifidobacteria, lactobacilli, and lactose-positive E. coli. Lactose utilizing microorganisms ferment lactose into short-chain fatty acids, lactic acid, carbon dioxide, methane, hydrogen, and water, creating an acidic ph (Rienzo et al., 2013). If colchicine interferes with the gut microbiota in some FMF patients, such interference could lead to reduced lactase function. The demonstrated normal lactase activity of Armenian FMF populations was explained by the normal lactase activity of intestine enterocytes or the bacteria utilizing lactose in the large intestine (Pepoyan et al, 2015). Taking into account the non-genomic upregulation of SGLT1 by bacterial metabolites (Arun et al, 2010), the slight increase of blood glucose level after probiotic consumption described during current investigation was probably influenced by lactase activity of probiotics, or probiotic metabolites could have assisted in the absorption of glucose by regulation of SGLT1 (sodium-glucose linked transporter) transporters. Impact of Probiotics on Blood ESR and C - reactive Protein Level In our earlier research it has been shown that abnormalities of ESR as well as CRP in the same group of FMF patients have been associated with patients genders. The serum RF levels were statistically normal for all patients (Pepoyan et al, 2015). ESR was above normal in 37 % of patients. Percentages of women with high ESR were twice that of men (56 % vs 27 %, respectively) (P < 0.05). Usually, elevated ESR values are maintained between FMF attacks, whereas other parameters can be normal at this time. ESR measurement is considered as a screening test because it has no specificity for any certain disease. C-reactive protein, however, is an indicator of an acute phase of the inflammatory process, which is the most sensitive and fastest indicator of tissue damage. CRP is often compared with ESR. Both values increase sharply at the beginning of the inflammatory process, but CRP appears and disappears more quickly than ESR. An advantage of CRP measurement, unlike ESR measurement, is that its value does not depend on the level of leukocytes in blood (Black, 2004). It is known that ESR increases with age and is higher in women than in men (Böttiger, 1967). Although normal ranges that are traditionally used reflect the effects of age and sex, in different countries different upper normal limits for ESR have been established in accordance with the International Committee for Standardization in Hematology (ICSH) recommendation on reference values. To determine ESR values, the method of Westergren is recommended (ICSH, 1993). In Armenia, Panchenkov s method is used most often. The results obtained by using the method of Westergren in the normal range coincide with the results obtained by Panchenkov s method. However, Westergren method is more sensitive to an increase in ESR, and results in the higher range of values obtained by Westergren are increased compared to the results obtained by Panchenkov s method (Blindar VN, 2013). Despite the fact that no significant changes in blood CRP have been shown, the values of CRP appeared above normal in 27% of the investigated patients. In comparison with ESR, the reverse picture is observed among men and women. The number of women with abnormal values of CRP was 2.5 times less than the number of men (P< 0.05): accordingly, 13% and 33% (Pepoyan et al, 2015). Mostly, elevated ESR and CRP were observed in patients with amyloidosis. By a number of scientific works, it has been demonstrated that the increase of CRP (even in a concentration less than 10 mg/l among seemingly healthy people) indicates an elevated risk of atherosclerosis, as well as the myocardial infarction and thromboembolisms (Kolz et al., 2008; Lange et al., 2006). Blood analyses have been re-done after probiotic application for those patients who initially had high levels of inflammatory activity. It was shown that the level of CRP decreased after probiotic therapy (P<0.05) in the blood of all patients with high CRP levels in remission (Table 1). CONCLUSION The effects of commercially available probiotics Narine, containing the probiotic strain Lactobacillus acidophillus INMIA 9602 Er-2 strain 317/402, and Colibakteron, containing Escherichia coli M-17, on blood glucose levels and CRP levels were shown during the current investigations. ACKNOWLEDGEMENTS This work was supported by US Department of Energy, Global Initiative for Proliferation Prevention (GIPP) program through the International Science and Technology Center (ISTC) (Project A-1980). The authors would like to thank Yvette Piceno for a careful review of the manuscript and helpful suggestions. REFERENCES Arun, K., Yasuhiro, K. and Randal, K. (2010). Metabolites produced by probiotic Lactobacilli rapidly increase glucose uptake by Caco-2 cells. BMC Microbiology 10:16. Ben-C. and Touitou, I. (2009). Familial Mediterranean fever in the world. Arthritis & Rheumatism 61: Ben-Zvi, I., Danilesko, I., Yahalom, G., Kukuy O., Rahamimov, R., Livneh, A. and Kivity, S. (2012). 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6 96 Impact of probiotics on clinical blood characteristics of FMF patients