Nasal administration of a non-viable Lactobacillus casei to infant mice modulates lung damage induced by Poly I:C and hyperreactivity in airways.

Viral respiratory infections caused by RNA viruses are one of the most important diseases around the world. The aim of this work was to study whether the nasal administration of non-viable Lactobacillus casei (LcM) was able to enhance respiratory antiviral defenses in young mice challenged with Poly I:C. Three-week-old BALB/c mice were nasally challenged with Poly I:C, used to mimic the pro-inflammatory state of lung infections caused by RNA viruses. LcM was nasally administered 2 days before Poly I:C challenge. Lactate dehydrogenase (LDH) activity, albumin concentration in broncho-alveolar lavages (BAL), wet-to-dry lung weight ratio, and total and differential leukocytes counts in blood were evaluated. Also, α, λ, γ interferons, IL-10, TNF-α, IL-4 in BAL and nasal lavages and total IgE in BAL and serum, were evaluated by ELISA. Poly I:C induced pulmonary injuries while alteration of bronchoalveolar-capillary barrier was reduced by nasal administration of LcM. Moreover, alterations in leukocyte counts induced by Poly I:C were regulated. LcM favorably modulated the cytokines responses triggered by Poly I:C challenge in nasal and lung mucosal compartments. Also, LcM decreased IgE levels in BAL and plasma compared with the Poly I:C group. LcM nasally administered reduced the lung damage induced by Poly I:C and prevented airway hyperreactivity.

Genetic Regulation of Redox Balance in ß-Thalassemia Trait.

ß-Thalassemia (ß-thal) trait is a heterogeneous group of genetic defects leading to decreased ß-globin production, ineffective erythropoiesis, and oxidative stress. The aim is to evaluate the cytoprotective response, at transcriptional and systemic levels, of the variations of global redox balance in ß-thal trait patients. Sixty-six subjects (40 healthy and 26 with ß-thal trait) were analyzed at the Universidad Nacional de Tucumán, Tucumán, Argentina, between 2016 and 2017. The following parameters were evaluated: complete blood count, iron status, hemoglobin (Hb) electrophoresis, Hb A2, thiobarbituric acid reactive species (TBARS), serum catalase (CAT), and superoxide dismutase (SOD) activity, FOXO3a, NRF2, SOD, PRDX2, CAT, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) gene expression. The ß-thal trait group showed a decrease in Hb levels, MCV, and MCH with higher TBARS levels. The SOD activity was significantly increased by 32.0% in ß-thal trait patients respect to the control group. Relative expression of NRF2 was 4.7-fold higher in ß-thal trait than in the control group, while FOXO3a expression was similar in both groups. The SOD, PRDX2, and proinflammatory cytokines transcriptional expression was significantly upregulated in ß-thal trait patients. This is the first study on the genetic regulation of redox balance in ß-thal trait patients in which interesting modifications were observed in the transcript levels of some redox regulators that could be associated with changes in the erythrocyte proteome in this disorder. A better understanding of the pathophysiological mechanisms present in these heterozygous patients would allow adequate therapy in situations such as growth, pregnancy, or high performance sports, favoring a personalized treatment.

Lactobacillus casei beneficially modulates immuno-coagulative response in an endotoxemia model.

: The current study aims at evaluating the effect of the oral administration of Lactobacillus casei CERELA (CRL) 431 on parameters implicated in inflammation-coagulation interaction using a model of acute inflammation induced by lipopolysaccharide (LPS) in mice. Six-week-old Balb/c mice were treated with L. casei for 5 consecutive days. Then treated and untreated mice received an LPS injection (L. casei + LPS and LPS groups, respectively). Liver and kidney were removed, blood samples were obtained, and hemostatic and inflammatory parameters were evaluated at different times post LPS injection. Preventive L. casei administration induced a significant decrease in proinflammatory TNF-α and IL-6 cytokines by decreasing tissue factor expression in liver and kidney. Moreover, the lower expression of tissue factor in the L. casei + LPS group led to a lower activation of the coagulation system, which was observed by the fast systemic restoration of factors VII and V coagulation factors and antithrombin levels. This study highlights the capacity of L. casei to modulate the hemostatic unbalance in an acute endotoxemia model. Our findings showed the ability of L. casei CRL 431 to regulate the immuno-coagulative response. This fact could be helpful to propose new adjunctive strategies addressed to the restoration of physiological anticoagulant mechanisms in sepsis patients.

Cryohemolysis, erythrocyte osmotic fragility, and supplementary hematimetric indices in the diagnosis of hereditary spherocytosis

BACKGROUND: Hereditary spherocytosis (HS) is a chronic hemolytic anemia characterized by microspherocytes in the peripheral blood and increased erythrocyte osmotic fragility (EOF). This study evaluated the cryohemolysis test (CHT); initial hemolysis (IH); immediate and incubated hemolysis percentage in 5.5 g/L NaCl (H5.5); mean corpuscular hemoglobin concentration (MCHC); red blood cell distribution width (RDW); and Hb/MCHC, Hb/RDW, and MCHC/RDW ratios for the diagnosis of HS. METHODS: Data from 13 patients with HS were evaluated at the Instituto de Bioquímica Aplicada and compared with data from 14 unaffected individuals and 11 patients with anemia due to another etiology. Total blood and reticulocyte counts, CHT, and immediate and incubated EOF were performed in all subjects; sensitivity, specificity, efficiency, and Youden index (YI) were calculated. RESULTS: Eight patients with HS had MCHC ≥345 g/L, 10 had RDW ≥14.5%, 12 had IH >5.0 g/L, 11 had immediate H5.5 ≥5%, and 13 had incubated H5.5 ≥50% (the cut-off value to consider HS). The efficiency and YI were: immediate H5.5 (0.94–0.85), incubated H5.5 (0.89–0.82), IH (0.89–0.78), MCHC (0.87–0.62), CHT (0.84–0.54), and Hb/MCHC (0.71–0.56), respectively. The calculated ratios could distinguish subjects with HS from unaffected individuals (P 0.05). CONCLUSION: Although the CHT and supplementary hematimetric indexes were useful to differentiate individuals with SH from healthy controls, they cannot distinguish from anemias of other etiology. CHT and MCHC, in addition to EOF, are recommended for diagnosing HS patients because of their low cost and efficiency.

Safety of a nasal vaccine against Streptococcus pneumoniae using heat-killed Lactobacillus casei as adjuvant.

Streptococcus pneumoniae is a highly important respiratory pathogen that causes infections in children, elderly people and immunocompromised people around the world. Pneumococcal vaccines licensed did not reach to eradicate the pneumococcal infection. In a previous study was demonstrated the effectiveness of a nasal experimental vaccine that consisted in a pneumococcal protective protein A (PppA) co-administrated with heat-killed-Lactobacillus casei (LcM), in mice model of respiratory pneumococcal challenge. In the present work the safety of the experimental vaccine LcM+PppA and its components were evaluated through hematological, biochemical and immune parameters in a model infection with S. pneumoniae. Thus, alanine transaminase activity, creatinine levels, lactate dehydrogenase activity, C reactive protein levels, corticosterone levels in serum, total and differential leukocyte counts in blood and bronchoalveolar lavages (BAL) and IgE in BAL, were evaluated. Experimental vaccine: LcM+PppA nasally administered does not induce harmful effects in our vaccination-infection model. Studied parameters showed LcM+PppA's safety in liver, kidney, pulmonary and systemic levels. Although studies in experimental animals do not guarantee security for the application of the vaccine on humans, they are important evidence for the planning and subsequent clinical trials in humans.

Modulation of the immuno-coagulative response in a pneumococcal infection in malnourished mice nasally treated with Lactobacillus casei.

We studied the systemic effects of the intranasal administration of Lactobacillus casei on the immuno-coagulative response in pneumoccocal infection in immunocompromised mice. Weaned mice consumed a protein-free diet (PFD) for 21 days and were therefore malnourished. Malnourished mice were fed a balanced conventional diet (BCD) for 7 days (BCD group) or a BCD for 7 days with nasal administration of viable L. casei on days 6 and 7 (BCD+LcN group). The malnourished control mice (MNC) received a PFD, whereas the well-nourished control mice (WNC) continually consumed a BCD. At the end of the treatment period, the mice were infected with Streptococcus pneumoniae. At different times after infection, we analysed the following parameters: global coagulation system, activation of coagulation, coagulation inhibitors, platelet count, leukocyte count and myeloperoxidase (MPO) activity, total proteins, albumin and acute phase proteins (APPs). The MNC group showed greater impairment in the coagulation tests and an increase in the positive APPs. These parameters were normalized by the L. casei treatment. However, the number of leukocytes, decreased by malnutrition, was improved only by the administration of L. casei. After infection, the BCD+LcN group showed similar results to those of the WNC group for most of the haemostatic parameters. The BCD+LcN group did not show significant variations in the prothrombin time or in the level of anticoagulant protein C, but showed higher levels of fibrinogen, platelets, albumin, leukocytes and MPO activity compared with the different experimental groups. The intranasal administration of L. casei was effective in modulating the pro-inflammatory aspects of coagulation without affecting coagulation itself.

Inflammation-hemostasis relationship in infected malnourished mice: modulatory effect of Lactobacillus casei CRL 431.

OBJECTIVE AND DESIGN: The coagulation system is considered part of the defense machinery, but its excessive activation can lead to additional damage. We studied the effects of oral administration of Lactobacillus casei CRL 431--a probiotic bacterium--on the activation of coagulation and the relationship with inflammatory parameters during a respiratory infection in malnourished mice. MATERIALS AND METHODS: Malnourished Swiss albino mice were nourished with a balanced commercial diet (BCD) for 7 days or BCD with L. casei for the last 2 days (BCD + Lc). BCD, BCD + Lc, malnourished (MNC) and well-nourished controls (WNC) were infected with Streptococcus pneumoniae. Blood and bronchoalveolar lavage samples were obtained at different times post-infection. RESULTS AND CONCLUSIONS: Malnutrition altered most of the evaluated parameters before and after infection. The repletion diet with supplemental L. casei was the most effective in limiting coagulation activation and normalizing coagulation inhibition mechanisms. These findings will help develop further strategies to reduce the damaging effects of clotting and enhance its beneficial contribution to immune reactions.

Enterocin CRL35 inhibits Listeria monocytogenes in a murine model.

Listeria monocytogenes is a foodborne pathogen causative of opportunistic infections. Listeriosis is associated with severe infections in pregnant women causing abortion or neonatal listeriosis. An alternative to antibiotics are safe novel bacteriocins peptides such as enterocin CRL35 with strong antilisterial activity produced by Enterococcus mundtii CRL35. In the present paper, our goal is to study the effectiveness of this peptide and the producer strain in a murine model of pregnancy-associated listeriosis. A single dose of 5×10(9) colony-forming unit of L. monocytogenes FBUNT (Faculty of Biochemistry-University of Tucumán) resulted in translocation of pathogen to liver and spleen of BALB/c pregnant mice. The maximum level of Listeria was observed on day 3 postinfection. Interestingly, the intragastric administration of enterocin CRL35 significantly reduced the translocation of the pathogen to vital organs. On the other hand, the preadministration of E. mundtii CRL35 slightly inhibited this translocation. Listeria infection caused a significant increase in polymorphonuclear leukocytes at day 3 postinfection compared to the noninfected group. This value was reduced after the administration of enterocin CRL35. No significant changes were observed in either white blood cells or lymphocytes counts. Based on the data presented in the present work enterocin CRL35 would be a promising alternative for the prevention of Listeria infections.

Coagulation activation in an experimental pneumonia model in malnourished mice.

Malnutrition induces a decrease in immunity that affects the ability of the organism to deal with an infectious challenge. The clotting system is considered a branch of immunity and its activation is important in the pathogenesis of an infectious disease. This work was conducted to determine coagulation modifications in malnourished hosts before and during infection. Weaned mice were malnourished via a protein-free diet. Well-nourished control mice (WNC) consumed a balanced conventional diet. Malnourished mice (MN) and WNC were challenged intranasally with Streptococcus pneumoniae. Blood, bronchoalveolar lavages (BAL), and lung samples were taken at different times post infection. The results were that MN showed altered hemostatic tests and fibrin(ogen) deposits in the lung. Thus, an increase in thrombin-antithrombin complexes (TATc) in plasma and BAL was observed. In the MN group, infection induced a rise in TATc in plasma and BAL and increased plasma fibrinogen and fibrin(ogen) deposits in the lung. A decrease in activated protein C and antithrombin in BAL and an early decrease followed by an increase in plasma Factor VIII were also observed. Thus, malnourishment induced a procoagulant state increased by infection. This is the first work that presents results of an exhaustive study of coagulation in malnourished hosts before and during an infection.

Lactobacillus casei modulates the inflammation-coagulation interaction in a pneumococcal pneumonia experimental model.

BACKGROUND: We have previously demonstrated that Lactobacillus casei CRL 431 administration improved the resistance to pneumococcal infection in a mouse model. METHODS: This study examined the effects of the oral administration of Lactobacillus casei CRL 431 (L. casei) on the activation of coagulation and fibrinolytic systems as well as their inhibitors during a Streptococcus pneumoniae infection in mice. RESULTS: The alveolo-capillary membrane was damaged and the coagulation system was also activated by the infection. As a consequence, we could see fibrin(ogen) deposits in lung histological slices, increased levels of thrombin-antithrombin complex (TATc) in bronchoalveolar lavage (BAL) and plasma, decrease in prothrombin activity (PT) and prolonged activated partial thromboplastin time test (APTT) values. Factor VII (FVII) and factor X (FX) were decreased in plasma, whereas fibrinogen (F) and factor VIII (FVIII) were increased. The low levels of protein C (PC) in BAL and plasma proved damage on inhibitory activity. The infected animals showed reduced fibrinolytic activity, evidenced by an increase in plasminogen activation inhibitor-1 (PAI-1) in BAL and plasma. The pathogen induced an increase of TNF-alpha, IL-1beta and IL-6 in BAL and serum a few hours after challenge followed by a significant decrease until the end of the assayed period. IL-4 and IL-10 in BAL and serum were also augmented, especially at the end of the experiment. The animals treated with L. casei showed an improvement of alveolo-capillary membrane, lower fibrin(ogen) deposits in lung and decrease in TATc. APTT test and PT, FVII and FX activity were normalized. L. casei group showed lower F levels than control during whole experiment. In the present study no effect of L. casei on the recovery of the inhibitory activity was detected. However, L. casei was effective in reducing PAI-1 levels in BAL and in increasing anti-inflammatory ILs concentration. CONCLUSION: L. casei proved effective to regulate coagulation activation and fibrinolysis inhibition during infection, leading to a decrease in fibrin deposits in lung. This protective effect of L. casei would be mediated by the induction of higher levels of IL-4 and IL-10 which could regulate the anti-inflammatory, procoagulant and antifibrinolytic effects of TNF-alpha, IL-1beta and IL-6.

Lactobacillus casei: influence on the innate immune response and haemostatic alterations in a liver-injury model.

UNLABELLED: Lactobacilllus casei CRL 431 has the ability to modulate the local and systemic immune responses, which are significantly involved in liver injury caused by hepatotoxins. This work was conducted to determine whether L. casei has a preventive effect on the hepatic damage undergone during an acute liver injury (ALI). METHODS: ALI was induced by an intraperitoneal injection of d-galactosamine (D-Gal). Different groups of mice received 1x 109 L. casei cells/day/mouse for 2 days before D-Gal injection. Blood and liver samples were obtained 0, 6, 12, and 24 h after D-Gal administration. RESULTS: D-Gal induced increases in serum aminotransferases, reduced the number of blood leukocytes, enhanced neutrophil myeloperoxidase activity, increased dead cells, and altered prothrombin time and plasma fibrinogen levels. The preventive treatment with L. casei for 2 days modulated the innate immune response. This effect was shown by the earlier normalization of white blood cell counts, myeloperoxidase activity and aminotransferases levels. However, the haemostatic parameters were only partially recovered. The favourable effects obtained could be due to the capacity of L. casei to moderate the inflammatory response at the site of the injury with less damage to liver tissue.

Yogurt: effect on leukocytes and blood coagulation in an acute liver injury model.

This study determined whether cow or goat yogurt administration has a preventive effect on the hepatic damage undergone during an acute liver injury. Acute liver injury was induced by an intraperitoneal injection of d-galactosamine. Groups of mice were fed with cow or goat yogurt for 2 days or 7 days before the d-galactosamine injection. Blood and liver samples were obtained 12 hours after d-galactosamine inoculation. d-Galactosamine induced an increase in serum amino-transaminases, a reduction in the number of blood leukocytes, an enhancement in neutrophil myeloperoxidase activity, a recruitment of leukocytes toward the liver, an increase in cell death, and an alteration in prothrombin time, activated partial thromboplastin time, and fibrinogen levels. Treatment with cow or goat yogurt was effective at increasing leukocyte number and decrease myeloperoxidase activity. We also observed a decrease in leukocyte accumulation in the liver and a reduction in cell death. Activated partial thromboplastin time and fibrinogen were normalized, but prothrombin time only showed an improvement without reaching normal values. Cow or goat yogurts were effective at protecting against an experimental acute liver injury, especially when administered for 7 days.

Beneficial immunomodulatory activity of Lactobacillus casei in malnourished mice pneumonia: effect on inflammation and coagulation.

OBJECTIVE: The effect of Lactobacillus casei CRL 431 immunomodulatory activity on inflammation and coagulation during pneumococcal pneumonia was investigated in malnourished mice. METHODS: Weaned mice were malnourished after they consumed a protein-free diet for 21 d. Malnourished mice were treated for 7 d with a balanced conventional diet (BCD) with L. casei supplementation (BCD+Lc) or without it. The malnourished control group received only a protein-free diet whereas well-nourished control (WNC) mice consumed BCD ad libitum. Mice were challenged by the intranasal route with pneumococci at the end of each dietary treatment. Lung injury, leukocyte recruitment, cytokine production, coagulation tests, and fibrin(ogen) deposition in lungs were evaluated. RESULTS: Malnourished control mice showed impaired leukocyte recruitment and cytokine production, and more severe lung injuries when compared with WNC mice. Coagulation tests were significantly impaired in malnourished control group versus WNC group. Repletion with BCD or BCD+Lc improved these parameters, but only BCD+Lc mice achieved the values of WNC mice. In addition, the interleukin-10 level was higher in the BCD+Lc group than in the WNC group. CONCLUSION: Repletion with supplemental L. casei accelerated recovery of the defense mechanisms against pneumococci by inducing different cytokine profiles. These cytokines would be involved in the improvement of the immune response and in the induction of a more efficient regulation of the inflammatory process, limiting the injury caused by infection.