The vaginal microbial flora in non-specific vaginitis

The facultative and strictly anaerobic vaginal microbial flora was investigated in 40 women with non-specific vaginitis and in 40 control women seen in private gynaecological practice.Gardnerella vaginalis, anaerobic gram-negative bacilli, anaerobic gram-negative and gram-positive cocci were all associated with non-specific vaginitis (p<0.001), whereas lactobacilli occurred less frequently in non-specific vaginitis than in controls (p<0.01). The most common anaerobes wereVeillonella parvula, Bacteroides bivius, Bacteroides assaccharolyticus, Bacteroides capillosus andPeptococcus asaccharolyticus. Anaerobic gram-negative curved rods were found in 11% of cases of non-specific vaginitis. A characteristic pattern of short chain organic acids was found on gas liquid chromatographic analysis of vaginal secretions in non-specific vaginitis. A succinate/lactate peak ratio of 0.3 or more was found in 75% of women with non-specific vaginitis (p<0.001). Clue cells, a Positive amine test, a pH higher than 5.0, and the absence of lactobacilli on a Gram stained vaginal smear strongly correlated with non-specific vaginitis (p<0.001).     

Effects of lactobacilli on parameters of non-specific resistance of mice

The viability ofLactobacillus plantarum in vivo and the effects of viable and heat-killed lactobacilli on parameters of non-specific resistance were studied. After intravenous administration of 10⁸ viable lactobacilli, which is a dose with optimal adjuvant activity, viable lactobacilli could be isolated from spleens for more than 1 week and from livers and lungs for more than 3 weeks. Both viable and heat-killed lactobacilli stimulated the clearance of colloidal carbon, viable bacteria stimulated initially to a higher extent. Doses of 10⁸ viable and heat-killed lactobacilli, but not less, stimulated non-specific resistance toListeria monocytogenes and caused splenomegaly. Doses as small as 10⁵ viable and heat-killed lactobacilli induced substantial natural killer (NK) cell activity in the peritoneal exudate 4 days after i.p. administration. Higher doses generally caused a dose-dependent increase of NK cell activity. Viable lactobacilli injected in the paw and to a lesser extent heat-killed bacteria caused a proliferative response in the draining popliteal lymph node, which peaked at day 5. Results are discussed in relation to adjuvanticity and comparisons are made with bacterial agents already used in immunotherapy.     

Effect of perorally administered lactobacilli on macrophage activation in mice.

The effect of perorally (p.o) administered Lactobacillus casei and L. bulgaricus on macrophage activation in mice was studied. L. casei and L. bulgaricus were administered p.o. to mice for 8 days. The macrophage activation was measured on days 2, 3, 5, and 8 of lactobacillus administration by using biochemical and functional criteria. We measured the release of lysosomal hydrolases, the level of a nonlysosomal enzyme, and in vitro phagocytic activity of mouse peritoneal macrophages. All the assays were performed comparatively with mice inoculated with L. casei and L. bulgaricus (viable and nonviable cells) intraperitoneally (i.p.) at the same dose as for p.o. administration. The phagocytic activity was significantly higher in mice treated i.p. than in control mice. For p.o. administration, there was an increase only when L. casei was used. L. bulgaricus had little effect. No differences were found between viable and nonviable cells. The phagocytic function of the reticuloendothelial system was tested by the carbon clearance test, which showed that L. casei and L. bulgaricus accelerate the phagocytic function in mice treated p.o and i.p., from day 2 onward. These observations show that L. casei and L. bulgaricus given by p.o. administration are able to activate macrophages in mice and suggest that these bacteria, when passing through the intestinal tract, may be responsible for the enhanced host immune response. This fact is very significant because the diet includes fermented and manufactured products containing lactobacilli.     

Human phagocytic cells as oxygen metabolite scavengers

Human neutrophils or monocytes decreased hydrogen peroxide (H₂O₂) concentrationsin vitro. Neutrophils or monocytes decreased H₂O₂ concentrations as well as human erythrocytes. Treatment with aminotriazole or azide decreased both phagocyte and erythrocyte catalase activity and the ability of each cell to decrease H₂O₂ concentrationsin vitro. Prestimulation of phagocytic cells with phorbol myristate acetate (PMA) or opsonized zymosan decreased neither their catalase activity nor their ability to decrease H₂O₂ concentrations. The results suggest that unstimulated or stimulated phagocytic cells can scavenge H₂O₂ and may potentially decrease H₂O₂-mediated tissue injury. The H₂O₂ scavenging potential of phagocytic cells is due at least partially to their catalase activity.This work was supported in part by grants from the National Institutes of Health (P50 HL40784), Johnson and Johnson, Council for Tobacco Research Inc., Procter and Gamble, and Tambrands, Inc.     

Single administration of enterococcal preparation (FK-23) augments non-specific immune responses in healthy dogs

Influence of a single administration of heat-killed Enterococcus faecalis FK-23 preparation (FK-23) on non-specific immune responses was evaluated in healthy dogs. The dogs received a single administration of FK-23 at a dose of 100 mg/kg perorally and thereafter a complete blood count (CBC) , leukocyte differential count, phagocytic activity of peripheral blood neutrophils and a lymphocyte blast transformation (LBT) test were assessed. Although FK-23 had no effect on the CBC or leukocyte differential count, treatment of the drug caused a 1.4-fold increase in neutrophil phagocytosis compared to FK-23 non-treated healthy dogs. LBT activities of phytohemagglutinin (PHA) and pokeweed mitogen (PWM) were also activated to twice the control levels and that of concanavalin A (Con A) was increased to one and a half times by the FK-23 treatment. Thus, a single administration of FK-23 appears to augment host resistance through stimulation of the non-specific immune responses in vivo.     

Effects of thiolic antioxidants on in vitro mouse peritoneal macrophage functions

Since leukocytes have the potential to produce oxygen free radicals that damage biomolecules, we have evaluated the influence of thiolic antioxidants (glutathione, N-acetylcysteine and thioproline) on selected hematoimmunological characteristics. We used peritoneal leukocytes incubated at 0.5, 1.0 and 5.0 mM concentration of antioxidants. Following thiol treatment, we found a stimulation of phagocytic activity, and markedly decreased incidence of programmed cell death of peritoneal leukocytes, both basal and induced by H₂O₂. Low concentrations of thiolic compounds also increased the activity of catalase, glutathione peroxidase and glutathione reductase. On the contrary, only the highest concentration of N-acetylcysteine and thioproline reduced superoxide dismutase activity. Tested antioxidants can lead to a decrease of the oxidative stress, and could provide a nutritional benefit for evaluated immunological characteristics.     

Influence of lactic acid bacteria and their spent culture supernatant on hydrogen peroxide-induced interleukin-8 synthesis and necrosis of Caco-2 cells

Intestinal epithelial cells are confronted with many noxious stimuli which play an important role in the mucosal immune response. In the present study, Caco-2 cells treated with hydrogen peroxide were used as a model system for studying inflammatory responses and induction of cell death. Live lactobacilli and their concentrated spent culture supernatant (SCS) were applied for studying possible short- and long-term protective effects against hydrogen peroxide-mediated oxidative stress in Caco-2 cells. The secretion of pro-inflammatory cytokine interleukin-8 (IL-8) was investigated in non-filter grown Caco-2 cells, while transepithelial electrical resistance and cell death was studied in filter grown Caco-2 cells. Pre-incubation of Caco-2 cells with Lactobacillus plantarum 2142 did not decrease IL-8 levels induced by 1 mM hydrogen peroxide, nor did lactobacilli suppress IL-8 levels induced by hydrogen peroxide when Caco-2 cells were treated simultaneously with 1 mM hydrogen peroxide and L. plantarum 2142. Thus, lactobacilli did not exert a long-term protective effect against hydrogen peroxide in non-filter grown Caco-2 cells. However, the concentrated SCS of lactobacilli was able to reduce IL-8 levels by more than 6-fold, as determined 24 h after treatment. A short-term effect of lactobacilli was observed in filter grown Caco-2 cells, as they inhibited cell death induced by hydrogen peroxide in the concentration range 10-40 mM. Pre-incubation of epithelial cells with L. plantarum 2142 or simultaneous exposure to hydrogen peroxide and lactobacilli protected Caco-2 cells against cell death. In spite of the presence of lactobacilli, the permeability of membrane increased (transepithelial electrical resistance decreased), and exhibited a similar characteristic pattern as under treatment with hydrogen peroxide alone.     

Effectiveness of vaginal tablets containing lactobacilli versus pH tablets on vaginal health and inflammatory cytokines: a randomized, double-blind study

The purpose of this study was to evaluate the effectiveness of lactobacilli on vaginal health and proinflammatory cytokines. Sixty-seven patients with bacterial vaginosis (BV), 50 with intermediate flora and 42 with normal vaginal flora were enrolled in this double-blind study. The subjects were randomized to receive probiotic lactobacilli vaginal tablets (L. brevis CD2, L. salivarius subsp. salicinius, L. plantarum) or the vaginal pH tablet (active comparator). Cervico-vaginal lavage was collected to measure the concentrations of IL-1β, TNFα and IL-6 by ELISA. Neutral sphingomyelinase activity was also quantified in both arms before and after treatment. The probiotic vaginal tablet was well tolerated and no side effects were reported. The study demonstrated a cure rate of nearly 80?%; i.e., 32?% of the women could restore normal vaginal flora and 47?% had improved Nugent score, whereas 20?% of the subjects did not clear BV in the first follow-up (after 8?days treatment). The pH tablet containing pH lowering compounds induced resolution of BV and restoration of normal vaginal flora in 74 % and 26?%, respectively. The lactobacilli tablet was found to be better than the pH tablet in preventing BV in healthy subjects. A significant reduction in IL-1β and IL-6 vaginal cytokines was observed after treatment with lactobacilli, while the active comparator did not have any effect on local proinflammatory cytokines. Vaginal neutral sphingomyelinase activity was not modified in either group. Vaginal tablets containing lactobacilli can cure BV and reduce vaginal inflammatory response.     

Evidence for interplay among antibacterial-induced gut microbiota disturbance,neuro-inflammation,and anxiety in mice

The aim of the present study was to determine whether there is the mechanistic connection between antibacterial-dependent gut microbiota disturbance and anxiety. First, exposure of mice to ampicillin caused anxiety and colitis and increased the population of Proteobacteria, particularly Klebsiella oxytoca, in gut microbiota and fecal and blood lipopolysaccharide levels, while decreasing lactobacilli population including Lactobacillus reuteri. Next, treatments with fecal microbiota of ampicillin-treated mouse (FAP), K. oxytoca, or lipopolysaccharide isolated from K. oxytoca (KL) induced anxiety and colitis in mice and increased blood corticosterone, IL-6, and lipopolysaccharide levels. Moreover, these treatments also increased the recruitment of microglia (Iba1⁺), monocytes (CD11b⁺/CD45⁺), and dendritic cells (CD11b⁺/CD11c⁺) to the hippocampus, as well as the population of apoptotic neuron cells (caspase-3⁺/NeuN⁺) in the brain. Furthermore, ampicillin, K. oxytoca, and KL induced NF-κB activation and IL-1β and TNF-α expression in the colon and brain as well as increased gut membrane permeability. Finally, oral administration of L. reuteri alleviated ampicillin-induced anxiety and colitis. These results suggest that ampicillin exposure can cause anxiety through neuro-inflammation which can be induced by monocyte/macrophage-activated gastrointestinal inflammation and elevated Proteobacteria population including K. oxytoca, while treatment with lactobacilli suppresses it.     

Co-cultures of Human Coronary Smooth Muscle Cells and Dimethyl Sulfoxide-differentiated HL60 Cells Upregulate ProMMP9 Activity and Promote Mobility—Modulation By Reactive Oxygen Species

Vascular cells and leukocytes, involved in the development of atherosclerosis, produce cytokines and/or reactive oxygen species (ROS) and matrix metalloproteinases (MMPs) implicated in cell mobility. We investigated by co-culture experiments the effects of human coronary smooth muscle cells (HCSMC) on MMPs characteristics and mobility of neutrophil-like dimethyl sulfoxide-differentiated HL60 cells (≠HL60). The effects of superoxide dismutase (SOD) and catalase were also analyzed. All the studied MMP2 characteristics remained unchanged. HCSMC stimulated MMP9 protein level, activity and mobility of ≠HL60 cells and expressed and secreted a variety of cytokines implicated in atherosclerosis. SOD and catalase increased MMP9 expression, protein level and activity of ≠HL60, but migration of ≠HL60 cells was only decreased by catalase, demonstrating that ROS are more efficient in modulating MMP9 activity of ≠HL60 than their mobility. Finally, HCSMC being able to stimulate ≠HL60, their co-cultures may represent an in vitro approach to study cellular interactions occurring in vivo during atherosclerosis.     

Effect of carnosine on immunocompetent cells from alcoholic patients

We studied the effect of natural dipeptide carnosine on phagocytosis, respiratory burst in neutrophils, and subpopulation composition of lymphocytes from healthy donors and alcoholic patients. Carnosine in vitro produced different effects on immunocompetent cells from healthy donors and patients with alcoholism. In patients with alcoholism phagocytic activity of leukocytes and generation of reaction oxygen species increased under the influence of carnosine in a concentration of 0.01 mM, but decreased after treatment with this compound in a concentration of 1 mM. Carnosine in both concentrations stimulated the respiratory burst, but had no effect on the count of phagocytic cells in healthy donors. Carnosine in a concentration of 0.01 mM increased the number of lymphocytes carrying apoptosis markers (CD95{su+}) in patients with alcoholism not receiving therapy. Our results indicate that carnosine holds much promise for the therapy of alcoholism.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 9, pp. 289–292, September, 2004     

Chemotactic factor inactivator release from rat leukocytes

Rat neutrophils, alveolar macrophages, and peritoneal macrophages each released chemotactic factor inactivator (CFI) activities under conditions of endocytosis (uptake of opsonized zymosan particles and preformed immune complexes). CFI activities in supernatant fluids from phagocytizing leukocytes were found for the chemotactic factors from the third (C3) and the fifth (C5) components of complement and for the bacterial chemotactic factor (present in culture supernatant fluids fromEscherichia coli.) CFI activity could also be demonstrated in homogenates obtained from disrupted leukocytes. CFI release from intact leukocytes was dependent on the duration of incubation of leukocytes with the phagocytic stimulus. No quantitative relationships were noted between the amount of CFI activity and the amount of-glucuronidase in phagocytic supernatant fluids released from leukocytes. The release of CFI activities from phagocytizing leukocytes may represent a regulatory (turn-off) mechanism for the inflammatory response.Supported in part by NIH grants AI 09651 and HL 23192.     

Hydrogen peroxide enhances phagocytosis of Pseudomonas aeruginosa in hyperoxia

Mechanical ventilation with hyperoxia is a necessary treatment for patients with respiratory distress. However, patients on mechanical ventilation have increased susceptibility to infection. Studies including ours have shown that reactive oxygen species (ROS), generated by exposure to prolonged hyperoxia, can cause a decrease in the phagocytic activity of alveolar macrophages. Hydrogen peroxide (H?O?) is a form of ROS generated under hyperoxic conditions. In this study, we examined whether treatment with H?O? directly affects macrophage phagocytic ability in RAW 264.7 cells that were exposed to either 21% O? (room air) or 95% O? (hyperoxia). Moderate concentrations (ranging from 10 to 250 μM) of H?O? significantly enhanced macrophage phagocytic activity and restored hyperoxia-suppressed phagocytosis through attenuation of hyperoxia-induced disorganization of actin cytoskeleton and actin oxidation. These results indicate that H?O? at low-moderate concentrations can be beneficial to host immune responses by improving macrophage phagocytic activity.     

Peroxide-inducible catalase in Aeromonas salmonicida subsp. salmonicida protects against exogenous hydrogen peroxide and killing by activated rainbow trout, Oncorhynchus mykiss L., macrophages

Aeromonas salmonicida subsp. salmonicida expresses a single cytoplasmically located catalase which was found to be inducible by exposure to 20 microM hydrogen peroxide in mid-exponential phase resulting in a 4 fold increase in activity. Subsequent exposure to 2 mM peroxide in late-exponential/early-stationary phase resulted in further induction of catalase activity which increased to 20 fold higher levels than those found in uninduced cultures. Exponentially induced cultures were protected against subsequent exposure to 10 mM peroxide which was lethal to non-induced cultures. Bacteria subjected to induction in mid-exponential and early-stationary phase were resistant to 100 mM peroxide, although viability was greatly reduced. Growth of the bacterium under iron-restricted conditions had no effect on the peroxide induction of catalase. As current evidence indicates, the latter is an iron-co-factored heme catalase, this result suggests that catalase induction has a high priority in the metabolism of iron. Furthermore, exposure to peroxide also induces expression of periplasmic MnSOD. A. salmonicida MT423 was resistant to normal rainbow trout macrophages, but was susceptible to killing by activated macrophages. However, if catalase was induced by prior exposure to 20 microM peroxide during mid-exponential phase, A. salmonicida was resistant to killing by activated macrophages. The ability of A. salmonicida to upregulate periplasmic MnSOD and cytoplasmic catalase production under iron restricted conditions and low level peroxide (conditions expected to exist during the early stages of an infection) may be vital for its ability to withstand attack by phagocytic cells in vivo.     

Characterization of Opsonins for Bacteroides fragilis in Immune Sera Collected from Experimentally Infected Mice

Serum collected from mice experimentally infected with Bacteroides fragilis 23745 (immune serum) was analyzed for its ability to opsonize the in vitro ingestion of this organism by mouse peritoneal macrophages. B. fragilis was shown to be phagocytized most efficiently in the presence of immune serum although normal mouse serum demonstrated reduced, but significant, opsonic activity. Phagocytosis was greater in the presence of serum collected from animals inoculated twice with the organism than in the presence of serum from once-inoculated animals. The increased opsonic activity of serum from twice-inoculated animals compared to singly inoculated animals was associated with increases in immunoglobulin G1(IgG1), IgG2a, and IgG2b but not IgM. Adsorption analysis of immune serum with homologous or heterologous bacterial antigens indicated that both antibody and complement act synergistically in opsonizing B. fragilis, although either alone may effectively opsonize this organism. Further evaluation of antibody-mediated opsonization revealed that prior treatment of heat-inactivated immune serum with the reducing agent 2-mercaptoethanol caused a slight, but significant, decrease in opsonic activity, thus indicating that IgM is a minor opsonizing antibody for B. fragilis. When ingestion of a B. fragilis stock strain (23745) was compared to a recent clinical isolate (C-1), it was observed that the stock strain was more easily phagocytized in the presence of normal mouse serum, thus suggesting a possible anti-opsonic-phagocytic property of the clinical strain. In addition, the clinical isolate was phagocytized to a significantly greater degree in an aerobic than an anaerobic environment. Subsequent analysis of in vitro killing of B. fragilis 23745 by peritoneal macrophages reflected the previous results in that optimal killing occurred in the presence of immune serum, although normal serum promoted phagocytic killing to an intermediate degree. Thus, these studies implicate both antibody and complement, either alone or in combination, in the opsonization of B. fragilis. Moreover, the virulence of clinical B. fragilis strains may relate to their refractoriness to opsonization and phagocytosis.     

Effects of microbial metabolites on catalase activity and growth of Staphylococcus aureus 6538 P

The effects of cell extracts and supernatants ofLactobacillus spp. andCorynebacterium spp. on catalase activity and growth ofStaphylococcus aureus 6538 P were studied. Intra- and extracellular metabolites of lactobacilli and corynebacteria inhibited catalase activity ofS. aureus 6538 P. The growth ofS. aureus 6538 P decreased after incubation with lactobacillus metabolites. The inhibitory effect of intra- and extracellular metabolites of lactobacilli and corynebacteria on catalase activity ofS. aureus is a possible pathway of microbial interrelations responsible for the formation and/or development of microbial biocenoses. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 7, pp. 80–82, July, 2000     

Effects of microbial metabolites on catalase activity and growth ofStaphylococcus aureus 6538 P

The effects of cell extracts and supernatants ofLactobacillus spp. andCorynebacterium spp. on catalase activity and growth ofStaphylococcus aureus 6538 P were studied. Intra- and extracellular metabolites of lactobacilli and corynebacteria inhibited catalase activity ofS. aureus 6538 P. The growth ofS. aureus 6538 P decreased after incubation with lactobacillus metabolites. The inhibitory effect of intra- and extracellular metabolites of lactobacilli and corynebacteria on catalase activity ofS. aureus is a possible pathway of microbial interrelations responsible for the formation and/or development of microbial biocenoses. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 7, pp. 80–82, July, 2000     

Coconut oil protects against light-induced retina degeneration in male Wistar rats

The retinoprotective effect of Cocos nucifera oil (CNO) was investigated. Twenty male Wistar rats weighing 140?g and 180?g were randomly divided into four groups comprising of five animals each. The control group received distilled water. Retinal degeneration was induced in the remaining three groups by exposing the animals to 5,000?lux of bright white light for two hours. Prior to the light exposure, the light model group (LMG) received distilled water for 14 days, low Cocos nucifera oil (LCNO) group received 5?ml/kg of CNO for 14 days, and the high Cocos nucifera oil (HCNO) group received 10?ml/kg of CNO for 14 days. The treatments continued for 7 days after exposure to light. On the eight day, the animals were euthanised and their retinas isolated. The right retinas and occipital cortices of the animals were prepared for histological evaluation while the homogenates of the left retinas were used for biochemical assay. The results show that CNO significantly (p?<?0.05) reduced caspase-3 activity from 1.15?±?0.054?ng/ml to 0.434?±?0.095?ng/ml (LMG versus LCNO) and malondialdehyde concentration. There was no significant difference in the total antioxidant capacity in the retinas of the rats. However, LMG showed a significant increase in catalase activity. CNO was able to preserve the retinal morphology while LMG showed a distorted retinal layer and significant reduction (p?<?0.05) in retina thickness. CNO was unable to prevent perineural vacuolations in the occipital cortices of the rats. In conclusion, Cocos nucifera oil produced retino-protective effect via anti-oxidative and anti-apoptotic mechanisms.     

Higher values of hepatic lipase activity in postmenopause: relationship with atherogenic intermediate density and low density lipoproteins

OBJECTIVE: To investigate the enzymatic activity of hepatic lipase (HL) in postmenopausal women (PMW) and reproductive age women (RAW); and to evaluate the relationship between this enzyme and the atherogenic intermediate density lipoproteins (IDL) and low density lipoproteins (LDL), and antiatherogenic high density lipoproteins (HDL) and its subfractions (HDL2 and HDL3). DESIGN: We studied 55 PMW receiving no hormonal treatment in a cross-sectional study in comparison with a control group of 55 RAW, matched by body mass index. Follicle-stimulating hormone was > 40 mUI/ml in PMW and 3-12 mUI/ml in RAW. PMW presented at least 1 year of natural menopause and no more than 10 years of amenorrhea with E2 serum concentration < 15 pg/ml. RESULTS: HL activity was significantly higher in PMW versus RAW (14.0 +/- 1.4 vs. 10.9 +/- 0.4 micromol of fatty acids/ml of postheparin plasma, respectively, mean +/- SEM, p < 0.001). In PMW, IDL cholesterol showed a positive correlation with LDL cholesterol (r = 0.28, p < 0.05), and HDL2 cholesterol was inversely correlated with HL activity (r = 0.31, p < 0.05). HL was positively correlated with plasma concentration of LDL cholesterol in both groups (r = 0.27, p < 0.05). The higher values of HL activity and IDL cholesterol were independent of age. CONCLUSIONS: Higher HL activity is associated with a more atherogenic profile in PMW.     

VIP modulation of immune cell functions

Neuropeptides have recently been shown to modulate the immune response. Vasoactive intestinal peptide (VIP) released from nerve endings and from immune cells modulates the mobility and adherence of lymphocytes and macrophages, phagocytic cell functions (phagocytosis and free radical production), the lymphocyte proliferative response, lymphokine and immunoglobulin production and the natural killer cell activity, with opposite effects in vitro on these immune cell functions. The VIP receptor heterogeneity and the different action mechanisms of VIP-mediated immunoregulation could explain, at least in part, the different VIP effects observed on lymphoid and phagocytic cells. The evidence supports the theory that VIP acts not as an inhibitor, but as a modulator of immune functions, as previously thought, and that this neuropeptide may play a relevant role in vivo.