Tween 80 and respiratory growth affect metabolite production and membrane fatty acids in Lactobacillus casei N87.

AIMS: To evaluate the effect of cultivation (anaerobiosis vs respiration) and Tween 80 supplementation on the production of metabolites and on the composition of membrane fatty acids (FAs) in Lactobacillus casei N87. METHODS AND RESULTS: Anaerobic and respiratory growth, with or without Tween 80 supplementation, was carried out in a chemically defined medium. Production of biomass, organic acids, volatile organic compounds (VOCs), consumption of amino acids and changes in membrane FAs were investigated. Respiration altered the central metabolism rerouting pyruvate away from lactate accumulation, while Tween 80 had a minor effect on metabolic pathways. VOCs were mainly affected by growth conditions and significant amounts of diacetyl were produced by respiratory cultures. Respiration increased desaturation of membrane lipids and Tween 80 improved the production of essential polyunsaturated FAs. Palmitic acid decreased in Tween-supplemented aerated cultures. CONCLUSIONS: Combination of Tween 80 and respiratory growth promoted production of biomass and aroma compounds and affected the composition of membrane FAs in Lact. casei N87. SIGNIFICANCE AND IMPACT OF THE STUDY: Respiration might be exploited in Lact. casei as a natural strategy for the enhanced production of aroma compounds.

Seminal anti-Müllerian hormone levels during recombinant human follicle-stimulating hormone treatment in men with idiopathic infertility undergoing assisted reproduction cycles.

A prospective study was designed to investigate the effects of recombinant human follicle-stimulating hormone (rhFSH) on seminal anti-Müllerian hormone (AMH) levels in men with idiopathic oligoasthenoteratozoospermia (iOAT), researching possible relationships between the seminal AMH behavior and the response to the treatment. Thirty-nine men who were candidates for intracytoplasmic sperm injection (ICSI) because of iOAT were enrolled. Patients were treated on alternately days with 150 IU of rhFSH for at least 3 months before assisted reproduction cycles. Main outcome measures were seminal AMH concentrations before and after rhFSH therapy. After treatment, 16 subjects (responders) showed an improvement in their sperm count compared to baseline (7.6 ± 2.9 vs. 19.3 ± 7.7, p < 0.01) whereas 23 men (non-responders) experienced no sperm modifications. Baseline seminal AMH concentrations were significantly higher in responders than in non-responders (53.0 ± 30.6 vs. 34.6 ± 18.5, p < 0.025). Following therapy, a greater increase in AMH levels was observed in responders compared to non-responders (Δ = 24.8 ± 36.4 vs. Δ = 6.4 ± 11.2, p < 0.028). Seminal AMH levels significantly and positively correlated with sperm count (after rhFSH treatment rho = 0.647, p < 0.001). Our study suggests that rhFSH improves sperm count in a quota of iOAT men, and the subjects who respond to the treatment have higher baseline seminal AMH concentrations than the patients who are not responsive. Seminal AMH could be helpful to select those infertile men who may benefit from rhFSH treatment.

Modified chemically defined medium for enhanced respiratory growth of Lactobacillus casei and Lactobacillus plantarum groups.

AIMS: Members of the Lactobacillus casei and Lactobacillus plantarum groups are capable of aerobic and respiratory growth. However, they grow poorly in aerobiosis in the currently available chemically defined media, suggesting that aerobic and respiratory growth require further supplementation. METHODS AND RESULTS: The effect of Tween 80, L-alanine, L-asparagine, L-aspartate, L-proline and L-serine on anaerobic and respiratory growth of Lact. casei N87 was investigated using a 2(5) factorial design. The effectiveness of modified CDM (mCDM) was validated on 21 strains of Lact. casei and Lact. plantarum groups. Tween 80 supplementation did not affect anaerobic growth, but improved respiratory growth. L-asparagine, L-proline and L-serine were stimulatory for respiring cells, while the presence of L-aspartate, generally, impaired biomass production. mCDM promoted the growth of Lact. casei and Lact. plantarum, with best results for strains showing a respiratory phenotype. CONCLUSIONS: The nutritional requirements of anaerobic and respiratory cultures of members of the Lact. casei and Lact. plantarum groups differ. Tween 80 and selected amino acids derived from pathways related to TCA cycle, pyruvate conversion and NADH recycling are required for respiration. SIGNIFICANCE AND IMPACT OF THE STUDY: The availability of mCDM will facilitate the study of aerobic metabolism of lactobacilli under controlled conditions.

Biochemical analysis of respiratory metabolism in the heterofermentative Lactobacillus spicheri and Lactobacillus reuteri.

AIMS: This study evaluated the aerobic and respiratory metabolism in Lactobacillus reuteri and Lactobacillus spicheri, two heterofermentative species used in sourdough fermentation. METHODS AND RESULTS: In silico genome analysis, production of metabolites and gene expression of pyruvate oxidase, pyruvate dehydrogenase and cytochrome oxidase were assessed in anaerobic and aerobic cultures of Lact. reuteri and Lact. spicheri. Respiring homofermentative Lactobacillus casei N87 and Lact. rhamnosus N132 were used for comparison. Aerobiosis and respiration increased the biomass production of heterofermentative strains compared to anaerobic cultivation. Respiration led to acetoin production by Lact. rhamnosus and Lact. casei, but not in heterofermentative strains, in which lactate and acetate were the major end-products. Lactobacillus spicheri LP38 showed the highest oxygen uptake. Pyruvate oxidase, respiratory cytochromes, NADH oxidase and NADH peroxidase were present in the genome of Lact. spicheri LP38. Both Lact. spicheri LP38 and Lact. rhamnosus N132 overexpressed pox in aerobic cultures, while cydA was up-regulated only when haeme was supplied; pdh was repressed during aerobic growth. CONCLUSIONS: Aerobic and respiratory growth provided physiological and metabolic advantages also in heterofermentative lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: The exploitation of oxygen-tolerant phenotypes of Lact. spicheri may be useful for the development of improved starter cultures.

Environmental pollution due to cadmium: measure of semen quality as a marker of exposure and correlation with reproductive potential.

PURPOSE: Contradictory reports exist in the literature regarding an association of cadmium with parameters of semen quality. The aim of the study was to assess cadmium levels in both blood and seminal plasma and to analyze the relationships between cadmium concentrations and lifestyle and semen parameters. MATERIAL AND METHODS: Fifty healthy male volunteers were recruited to provide semen and blood samples. Each patient completed an extensive questionnaire regarding his occupation, residence, social status, diet, water source, smoking habits, and medical and surgical history. Semen analysis was carried out according to WHO guidelines. Detection of cadmium in both semen and blood samples was carried out by means of atomic absorption spectrophotometer. RESULTS: Mean concentrations of cadmium were 8.18 ± 1.6 ng/ml in blood samples and 2.56 ± 0.9 ng/ml in semen samples. Cadmium blood levels were significantly higher in men from industrialized areas and in current smokers, but were not correlated with semen levels. A significant positive correlation was found between cadmium blood levels, number of immotile spermatozoa, and teratozoospermia index (TZI). Significant inverse relationships between cadmium blood concentration and type-a and type a + b motility were found. CONCLUSIONS: The present data show a significant correlation between blood cadmium concentrations, cigarette smoking, occupational exposure, and parameters of semen quality. Such a reduction in spermiogenetic function could be an early marker of a toxic effect by cadmium pollution.

Selection of mutants tolerant of oxidative stress from respiratory cultures of Lactobacillus plantarum C17.

AIMS: Lactobacillus plantarum is a lactic acid bacterium involved in the production of many fermented foods. Recently, several studies have demonstrated that aerobic or respiratory metabolism in this species leads to improved technological and stress response properties. METHODS AND RESULTS: We investigated respiratory growth, metabolite production and stress resistance of Lact. plantarum C17 during batch, fed-batch and chemostat cultivations under respiratory conditions. Sixty mutants were selected for their ability to tolerate oxidative stress using H2 O2 and menadione as selective agents and further screened for their capability to growth under anaerobic, respiratory and oxidative stress conditions. Dilution rate clearly affected the physiological state of cells and, generally, slow-growing cultures had improved survival to stresses, catalase production and oxygen uptake. Most mutants were more competitive in terms of biomass production and ROS degradation compared with wild-type strain (wt) C17 and two of these (C17-m19 and C17-m58) were selected for further experiments. CONCLUSIONS: This work confirms that, in Lact. plantarum, respiration and low growth rates confer physiological and metabolic advantages compared with anaerobic cultivation. SIGNIFICANCE AND IMPACT OF THE STUDY: Our strategy of natural selection successfully provides a rapid and inexpensive screening for a large number of strains and represents a food-grade approach of practical relevance in the production of starter and probiotic cultures.

Temperature and respiration affect the growth and stress resistance of Lactobacillus plantarum C17.

AIMS: The aim of the study is to gain further insight on the respiratory behaviour of Lactobacillus plantarum and its consequences on stress tolerance. METHODS AND RESULTS: We investigated the effect of temperature and respiration on the growth and stress (heat, oxidative, freezing, freeze-drying) response of Lact. plantarum C17 during batch cultivations. Temperature as well as respiration clearly affected the physiological state of cells, and generally, cultures grown under respiratory conditions exhibited improved tolerance of some stresses (heat, oxidative, freezing) compared to those obtained in anaerobiosis. Our results revealed that the activities in cell-free extracts of the main enzymes related to aerobic metabolism, POX (pyruvate oxidase) and NPR (NADH peroxidase), were significantly affected by temperature. POX was completely inhibited at 37°C, while the activity of NPR slightly increased at 25°C, indicating that in Lact. plantarum, the temperature of growth may be involved in the activation and modulation of aerobic/respiratory metabolism. CONCLUSIONS: We confirmed that respiration confers robustness to Lact. plantarum cells, allowing a greater stress tolerance and advantages in the production of starter and probiotic cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on respiratory metabolism on a strain other than the model strains WCFS1; novel information on the role of temperature in the modulation of aerobic/respiratory metabolism in Lact. plantarum is presented.

Aerobic metabolism and oxidative stress tolerance in the Lactobacillus plantarum group.

Aerobic metabolism and response to oxidative stress and starvation were studied in 11 Lactobacillus plantarum, L. paraplantarum and L. pentosus strains in order to assess the impact of aerobic metabolism on the growth and on the stress response. The strains were grown in aerobiosis without supplementation (AE), with hemin (AEH) or with hemin and menaquinone (AEHM) supplementation and in anaerobiosis (AN) in a complex buffered substrate. Growth rate, biomass yield, glucose and O2 consumption, production of lactic acid and H2O2, catalase activity, oxidative and starvation stress tolerance were evaluated. Aerobic growth increased biomass yield in late stationary phase. Further increase in yield was obtained with both hemin (H) and menaquinone (M) addition. With few exceptions, the increase in biomass correlated with the decrease of lactic acid which, however, decreased in anaerobic conditions as well in some strains. Addition of H or H + M increased growth rate for some strains but reduced the duration of the lag phase. H2O2 production was found only for aerobic growth with no supplementation due to catalase production when hemin was supplemented. To our knowledge this is the first study in which the advantages of aerobic growth with H or H + M in improving tolerance of oxidative stress and long-term survival is demonstrated on several strains of the L. plantarum group. The results may have significant technological consequences for both starter and probiotic production.

Isolated autoimmune response: definition, analysis of the prevalence in an outpatient rheumatology clinic, relationship to pre-clinical autoimmune disease and infections by hepatotropic viruses.

OBJECTIVE: To define the clinical significance of non-organ specific autoantibody positivity in patients in whom routine clinical and laboratory examinations did not detect any disease that might have caused the serological finding. METHODS: Out of 1,120 patients consecutively admitted to an outpatient rheumatology clinic, 28 were referred for the evaluation of an autoantibody positivity unrelated to the clinical status. These patients and 28 sex- and age-matched controls underwent a specific work-up with the aim of detecting any underlying infection or autoimmune disease. RESULTS: Eight of the 28 patients (28.5%) were found to be affected by a previously undetected disease: 3 chronic hepatitis C, 3 Sjögren's syndrome, and 2 autoimmune thyroiditis. The remaining 20 did not show any autoimmune or hepatic disease, although 4 of them showed active infection by HBV (n = 1) or HGV (n = 3) and 15 had had a previous infection by hepatotropic viruses (HBV, CMV or EBV). After a follow-up lasting 6-54 months, none of the last 20 patients developed any autoimmune or chronic hepatic disease. CONCLUSIONS: A diagnostic work-up is necessary in patients presenting with unexpected autoantibody positivity in order to detect an underlying pre-clinical autoimmune disease and/or unexpected hepatic infection. Patients in whom such a work-up fails to point out any condition should be further followed in order to make an early diagnosis of autoimmune disease.

Effects of benzodiazepines on immunodeficiency and resistance in mice.

Our results indicate that benzodiazepine (Bz) treatment time, greater than 2-3 months, induce a decrease of both specific and nonspecific responses. Mice treated for different times with diazepam or chlordemethyldiazepam showed decreased survival to experimental Salmonella typhimurium infections after three months of treatment. Adherence, expressed as the polymorphonuclear cells (PMN) capacity to attach to nylon wool, was impaired after 7 days of treatment. Longer treatments further increase this impairment. PMN from mice treated with Bz for 90 days also demonstrate on impaired chemotaxis and phagocytosis for Saccharomyces cerevisiae. Monocytes from mice treated for 7 days secreted more IL-1 alpha then controls; the antibody titer in mice given to prolonged treatment progressively diminished compared to controls. Con A or LPS stimulated lymphocytes showed an increase of H3-thymidine incorporation from mice treated for a short time and conversely a decreased incorporation when taken from mice that underwent longer treatments. Benzodiazepines were therefore found to affect PMN chemotaxis and phagocitosis, general immunity and survival of mice to infections.

Effect of measles-mumps-rubella vaccination on polymorphonuclear neutrophil functions in children.

Adherence, metabolic burst and chemotaxis of polymorphonuclear neutrophils (PMNs) were examined in 15 children before and seven days after measles-mumps-rubella vaccine administration. In all children, PMN functions were significantly reduced on the seventh day. Adherence, metabolic burst and chemotaxis tested in three subjects one month after vaccination had returned to normal values. Only two children presented transient hyperpyrexia. We conclude that measles-mumps-rubella vaccine administration suppresses PMN functions without clinical consequences. This is probably because attenuated strains of vaccine viruses do not replicate in lymphoid tissues as extensively as do wild-type strains.

The presence in human spermatozoa of sites binding some proteins of the outer membrane of Escherichia coli.

In previous works, we noted that porins, surface components of Gram negative bacteria, are toxic for human spermatozoa. In this work we see that porins of E. coli labeled with I125 (0.1-1.0 microgram/ml) bind to spermatozoa, showing the presence of saturable molecular configurations of 0.5-0.6 micrograms/ml. In the presence of non labeled porins, the binding of labeled porins to the spermatozoa is greatly reduced. At saturating concentrations of 0.6 micrograms/ml labeled porins, the binding reaches a saturation plateau at about 15 min.

Antimicrobial agents induce monocytes to release IL-1 alpha, IL-6, and TNF, and induce lymphocytes to release IL-4 and TNF tau.

Evaluation was carried out on the action of different antibiotics on the release of cytokines. Experiments were done in vitro on monocytes and on human lymphocytes. Results show that the majority of the antibiotics tested are able to induce the release of one or more cytokines from their respective producing cells. Among the beta-lactams the most active were the cephalosporins (cephalexin, cefamandol, ceftazidin, and a sulbactam-ampicillin combination) in inducing the release of TNF, IL-1 alpha, and IL-6 from monocytes, and releasing IL-4 and IFN-tau from lymphocytes. The sulbactam-ampicillin combination and cefamandole were extremely active in the production of IFN-tau. Among the lincosamides, clindamycine notably stimulated the release of TNF and IL-6, while lincomycine induced a notable increment of IL-4 from monocytes. Teicoplanin is a very strong inducer of TNF, IL-1 alpha and IL-6.

Neutrophil function in rapidly progressive and adult periodontitis.

Our study focused on the functional characteristics of neutrophil leukocytes (PMN) in two different forms of periodontal disease:--Adult Periodontitis (AP) and Rapidly Progressive Periodontitis (RPP). Specifically neutrophil leukocytes in gingival fluid (GF) and in peripheral blood (PB) were studied. In our experimental studies we evaluated PMN hydrophobicity and adhering capacity, metabolic burst, chemotaxis response and N-formyl-L-methionyl-leucyl-phenylalanine (FMLP) receptor by using a chemotactic peptide labeled with 3H and by evaluating the binding on PMN. These functional characteristics were found markedly reduced in the RPP group, while in the AP group, they were comparable to those of a healthy control group. No difference between local (GF) and systemic (PB) values was detected.

Protein A and other surface components of Staphylococcus aureus stimulate production of IL-1 alpha, IL-4, IL-6, TNF and IFN-gamma.

Studies were carried out on the ability of protein A (PA) and of muramic acid (MA) from S. aureus to induce the release of cytokines both from monocytes and lymphocytes in vitro. Results show that protein A induces the greatest activity, compared to the activity already known for the theicoic acid (TA) and for muramyl dipeptide (MDP). At concentration of 10 micrograms/ml; PA induces roughly +180% release of TNF with respect to controls, while release of IL-1 alpha is about 500% control values, and is higher than those obtained when cells are treated with TA and MDP; IL-6 release is higher than that stimulated by Con A, used as standard challenge. At PA concentrations of 5 micrograms/ml, IL-4 release is about five times higher than that induced by Con A. Release of IFN-gamma showed similar dose-dependent stimulations. Muramic acid (MA) is particularly active in inducing the release of cytokines from target cells, inducing TNF release of about +75% with respect to the controls. This increase is less than that obtained with PA. Also IL-4 and IFN-gamma are released by PA in quantities higher than those induced by TA and MDP. Our results lead us to believe that during infections by Gram-positive bacteria, their surface components are able to induce a series of chain reactions ranging from the inflammatory to the immunologic responses which are also conditioned by release of cytokines.

Correlation between modification of membrane phospholipids and some biological activity of lymphocytes, neutrophils and macrophages.

Our study considered the possibility of modifying the functional response of human neutrophils, of mouse lymphocytes and macrophages treated with phospholipids having different polar groups, different isomerisms with saturated and unsaturated fatty acids from C12 to C20 carbon atoms. The results are as follows. a) Most of the phospholipids containing fatty acids from C12 to C20 cause inhibition of the blastogenic capacity of the polyclonal activators tested. b) The phospholipids tested cause a decrease in adherence of polymorphonuclear leukocytes with the exception of the phosphatidyl-choline containing saturated and unsaturated fatty acids. c) A decrease in polymorphonuclear leukocytes migrational capacity almost always occurs. d) The cells treated with L-phosphatidyl-ethanolamine having fatty acids from C14 to C17 show an increase in chemiluminescence; those treated with phosphatidyl-choline and L-phosphatidyl-glycerol show a decrease of the chemiluminescence; L-phosphatidic acid and L-phosphatidyl-ethanolamine having Microbial fatty acids (FAs) at C16 cause a decrease in the formation of phagolisosomes in the macrophages tested.

[Neutrophil function and microbial associations in rapidly progressing periodontitis and chronic periodontitis in the adult]. / Funzionalità dei neutrofili e associazioni microbiche nella parodontite rapidamente progressiva e nella parodontite cronica dell'adulto.

Our study was focused on the functional characteristics of neutrophil leukocytes (PMN) and the subgingival microflora in two different forms of periodontal disease: 1) adult periodontitis (AP); 2) rapidly progressive periodontitis (RPP). Our study dealt with the functional characteristics of neutrophil leukocytes in the gingival fluid and in the peripheral blood. These were found markedly reduced in the RPP group, while, in the AP group, they were comparable to those of a healthy control group. No difference between local and systemic values was detected. Moreover, some samples of subgingival plaque were taken from two groups of patients, affected by AP and RPP respectively. The above samples showed a predominance of Gram-negative flora over Gram-positive flora, and of anaerobic flora over the aerobic one, and the predominance of specific pathogens in each of the two forms of periodontal disease. The subgingival plaque samples taken at the end of the periodontal treatment from five out of ten patients affected by RPP showed inverse ratios, as well as the absence of the previously detected pathogens. The findings underline the relevance of tests of leukocytes functionality and that of microbiological analysis to allow correct diagnosis of dubious forms of periodontal disease and the checking of the posttreatment results.

[Role of the PMN in periodontal disease. Study of a clinical case]. / Il ruolo dei PMN nella malattia parodontale. Studio di un caso clinico.

The Authors examine PMN's morphofunctional characteristic and show PMN's role in establishing periodontal disease. Then they show a clinical case of Rapidly Progressive Periodontitis in a patient with neutrophil function alteration.

Decreased adherence of polymorphonuclear neutrophils in children with viral infection.

The adherence of polymorphonuclear neutrophils was examined in 16 children affected by enteritis, pneumonia, hepatitis and infectious mononucleosis. The results were compared with those obtained in 30 healthy adult volunteers and in 15 healthy children of the same age. Adhesiveness was significantly higher in adults than in healthy children, and significantly higher in healthy children than in children with viral infection. In 7 patients tested one month after regression of the disorder, PMN adhesiveness had returned to normal.

Effect of Salmonella typhimurium porins on biological activities of human polymorphonuclear leukocytes.

The effect of Salmonella typhimurium porins on human polymorphonuclear leukocytes (PMNs) was studied. Labeled porins were shown to bind to the PMNs, and could be completely displaced by unlabeled porins. The binding caused modifications of membrane integrity and of the physico-chemical characteristics of the PMN surface, e.g. decreased oxidative burst, decreased hydrophobicity and altered cell morphology. The porins acted as both chemotaxins and chemotaxinogens. When PMNs were preincubated with porins their migration in the presence of commonly used chemoattractants (serum activated by zymosan or N-formyl-L-methionyl-L-leucyl-L-phenylalanine) was inhibited.