Archive for the ‘brain’ tag
Adhesion of Human Probiotic Lactobacillus rhamnosus to Cervical and Vaginal Cells and Interaction with Vaginosis-Associated Pathogens
1. Introduction
Bacterial vaginosis (BV) is the most frequent vaginal infectious disorder in women of childbearing age with prevalences ranging from 10% to 50%. In addition to the physiological burden that induces BV, it can cause serious sequelae such as preterm birth and facilitate the acquisition of sexually transmitted diseases. The cause of BV remains poorly understood, and no specific infectious agents have been identified. However, the disorder is characterized by modifications of the genital tract microflora, including a reduction in or absence of lactobacillus colonization and overgrowth of several anaerobic bacteria. The vaginal ecosystem in healthy premenopausal women harbors a microbiota dominated by Lactobacilli , that is, being increasingly recognized as protecting it from invading pathogens, including those that cause urinary tract infections and sexually transmitted diseases. Different mechanisms are potentially involved in the activity of Lactobacilli against pathogens, including the competitive exclusion of genitourinary pathogens from receptors present on the surface of the epithelial cells. Under healthy conditions, cervicovaginal cells are constantly exposed to the normal vaginal microbiota.
The recommended treatment regimens for vaginal infections are oral or intravaginal antibiotics, but these conventional treatments are associated with frequent recurrences. Alternative therapeutic agents need to be sought, and it has been suggested that the administration of Lactobacilli can restore ecological balance in the vagina by controlling the infectivity of pathogenic microbes, but the treatment is still a subject of debate. Several clinical trials have been performed to investigate the effects of specific strains, mainly with L. acidophilusand L. rhamnosus species , but no definitive conclusions as to whether these probiotics represent an effective and safe method for treating women with BV can be drawn. The behavior of the probiotics in the vaginal tract is likely to be strain specific and therefore, it is important to determine the characteristics of the strain to be used as a therapeutic agent. The most relevant properties in this context are likely to be adhesion to cervicovaginal cells and adequate pathogen growth inhibition. In vitro studies assessing these properties might not be able to fully simulate the in vivo behavior, but they could be reliable indicators when selecting the probiotic strain. The purpose of this study was to determine the in vitro adherence of a well characterized L. rhamnosusprobiotic strain, Lcr35, and its ability to inhibit growth of three vaginosis-associated pathogens. We used immortalized morphologically and functionally distinct epithelial cell lines from normal endocervix, ectocervix, and vagina to characterize Lcr35 epithelial interactions pertinent to the lower female genital tract and determined its antimicrobial activity against Prevotella bivia, Gardnerella vaginalis, and Candida albicans in coculture experiments.
2. Materials and Methods
2.1. Adhesion Assay
Adhesion assays were performed with epithelial cells from normal human vagina (VK2/E6E7 ATCC-CRL-2616), ectocervix (Ect1/E6E7 ATCC-CRL-2614), and endocervix (End1/E6E7 ATCC-CRL-2615), immortalized by expression of the E6 and E7 genes of human papillomavirus type 16. The morphological and immunocytochemical characteristics of the immortalized lines closely resembled those of their tissues of origin and primary cultures and are likely to represent the different compartments of the vaginal tract.
The cell lines were maintained in keratinocyte serum-free medium (Gifco BRL 17005-042) supplemented with human recombinant EGF (0.1 ng/mL), bovine pituitary extract (0.05 mg/mL), and calcium chloride (0.4 mM) at with a 5% in air atmosphere.
Adhesion of the Lcr35 was assayed by seeding cell lines in 24-well tissue culture plates at epithelial cells/well and allowing them to grow to complete confluence (cells/well). After gentle washing of the cell monolayer, the adhesion capacity of Lcr35 was determined by adding multiplicity of infection (MOI,1), (MOI, 10), and (MOI, 100) bacteria from an overnight culture in de Man, Rogosa, Sharpe (MRS) agar medium. Bacterial cells were previously washed in phosphate buffered saline and resuspended in the cell culture medium. Adhesion was monitored after 1 and 3 hours of incubation carried out at under 5%. The monolayers were washed three times with 1 mL of Dulbecco’s phosphate buffered saline, detached by addition of 0.1% TritonX-100 solution and the number of viable bacteria determined by plating serial dilutions of the suspensions onto MRS agar plates. For qualitative analysis, the cell monolayers and the bacteria were methanol fixed and stained by addition of a 10% Giemsa solution.
2.2. Growth Inhibition of Vaginosis-Associated Pathogens
The effect of Lcr35 on the growth of three pathogens was investigated using the following strains: Candida albicans ATCC10231, Prevotella bivia ATCC29303, and Gardnerella vaginalis ATCC14018. Coculture assays were performed in either Sabouraud broth (Candida) or brain heart infusion supplemented with yeast extract (1%), maltose (0.1%), glucose (0.1%), and horse serum (10%) (Prevotella and Gardnerella). Each pathogen (UFC) was incubated alone (control) and with the Lcr35 (UFC) at under anaerobic conditions for the two vaginosis-associated bacteria (AnaeroGen, Oxoïd). Aliquots were removed after 4, 8, and 24 hours of incubation, serially diluted and plated on appropriate media (Sabouraud, Gardnerella, or MRS) to determine the bacterial colony counts of both the pathogens and Lcr35. Statistical analyses of the data were performed using the Mann-Whitney test.
3. Results
3.1. Adhesion of Lcr35 to Cervical and Vaginal Cells
The ability of Lcr35 to adhere to vaginal and cervical cells . Whatever the MOI and the cell line, the probiotic strain was able to adhere to the cell surface monolayer. The highest number of adherent bacteria was observed with the vaginal cell line, with an average of 4.75 10e5 CFU per after 1 hour of incubation. No major difference was observed between the levels of adhesion obtained after 1 hour and 3 hours of incubation (data not shown), suggesting that adhesion occurs rapidly after the initial contact between the cells and the bacteria. Microscopical observations of Giemsa-stained preparation showed typical chains of Lcr35 randomly dispersed on the cell surface
3.2. Growth Inhibition of Vaginosis-Associated Pathogens
The antagonist effect of Lcr35 against three main pathogens, P. bivia, G. vaginalis, and C. albicans, was assessed in coculture assays and compared with the growth ability of each pathogen in the same culture medium. A decrease in the cell division rate of the three microorganisms tested was observed from 4 hours of coincubation. When the viable bacteria in the mixed suspension were counted over a longer period of time, bactericidal activity was detected between 8 and 24 hours of incubation for all pathogens with the Prevotellastrain being the most susceptible (4-log10 units decrease in the number of viable cells). In no case there was a bactericidal effect against Lactobacilli; the number of viable Lcr35 cells was either constant over the incubation period (coculture with C. albicans) or increased (coculture with G. vaginalis and P. bivia) (data not shown).
4. Discussion
Lactobacillus species in the female urogenital system act as a barrier to infection and contribute to the control of the vaginal microbiota by competing with other microorganisms for adherence to epithelial cells, displacing pathogen biofilm, and/or inhibiting the growth of potential pathogens. Hence the use of probiotic strains of Lactobacilli is potentially interesting both as preventive and curative agents.
Unlike the use of vaginal epithelial cells collected from healthy premenopausal women, assays performed with immortalized epithelial cell lines, which closely resemble the epithelial differentiation patterns of normal human tissues, are more accurate for standardizing tested bacterial adherence and allow comparison of different research approaches. The three epithelial cell lines tested in this study were developed from normal human vagina, ectocervix, and endocervix tissue, and their characteristics closely resembled those of their tissues of origin and primary cultures . We can thus speculate that adhesion assays performed with this material reproduce more faithfully the in vivo situation than experiments performed with any cell line derived from human carcinoma of the lower genital tract mucosa. This is particularly important when comparing bacterial strains belonging to the complex Lactobacillus genus that includes bacterial strains with highly specific characteristics. Using these cell lines, we observed specific adhesion of an L. rhamnosus strain, Lcr35, previously selected for its probiotic features . Adhesion occurred even at a low MOI (1:1) and within less than 1 hour of contact, which corresponds to a highly dynamic process.
Adhesion of Lcr35 to vaginal epithelial cells would allow colonization of the vaginal mucosa and therefore could limit the overgrowth of pathogens, but the second main property of a potential probiotic used as a therapeutic agent against pathogenic microorganisms is direct impairment of their growth. In this study, we demonstrated that Lcr35 showed bactericidal activity against both P. bivia and G. vaginalis in the range of killing stipulated for the bactericidal activity of antimicrobial activity (
2 log-unit). In a previous study, Atassi et al. demonstrated that the bactericidal activity of Lactobacilli toward these two vaginal bacterial pathogens was strain dependent and occurred within the first hours of coculture . In our experiments, a longer incubation time was required to observe bactericidal activity, probably because of the different experimental parameters used in the two assays. We previously showed that the Lcr35 probiotic strain was also able to kill several pathogens. The mechanism(s) underlying this activity has not been elucidated but is likely to be multifaceted and probably includes the production of hydrogen peroxide, lactic acid, and antibacterial compounds. It has been recently shown that G. vaginalis organized in biofilms is more resistant to 
and lactic acid than planktonic cultures . G. vaginalis is the predominant species observed within biofilms present on the vaginal epithelium in bacterial vaginosis , and Saunders et al. recently showed that strains of Lactobacilli were able to disrupt G. vaginalis preformed biofilms . It would therefore be interesting to test the biofilm activity of Lcr35 against G. vaginalis and to determine if the sessile form of Lcr35 also exhibits antibacterial activity against G. vaginalis in mixed biofilm assays.
The antagonist activity of Lcr35 was not limited to bacterial pathogens since the strain was also able to reduce the viability of C. albicans. Several strains of Lactobacilli have shown inhibitory effects against C. albicans , which is the species most often associated with candidiasis. By interfering with Candida overgrowth in the patients’ intestinal or vaginal tract, Lactobacilli could provide colonization resistance and maintain low numbers of yeasts, especially when administered together with antibiotics.
Relevant clinical trials have suggested that intravaginal or oral administration of Lactobacillus strains is able to increase the numbers of vaginal Lactobacilli and restore the vaginal microbiota to normal. Lcr35 was isolated from a human intestinal and not vaginal microbiota and does not belong to the four main species of Lactobacilliconsidered to be predominantly linked to the vaginal microflora, L. crispatus, L. jensenii, L. gasseri, and L. iners , but it has been shown to survive within the human gastrointestinal tract . Furthermore, Petricevic and Witt recently showed in a clinical study that topical administration of Lcr35 enhances the restoration of the vaginal flora after antibiotic treatment of BV. Thus, it might be an excellent candidate for use as a prophylactic agent, taken orally or applied topically. In vivo studies to evaluate its feasibility as such are in progress.
5. Conclusion
Maintenance or reconstruction of the normal composition of the vaginal microflora by applying properly selectedLactobacilli may be of prophylactic value in preventing or curing genitourinary system infections in women. In the light of our experiments, it seems that the probiotic strain L. rhamnosus Lcr35 would be a good candidate as a protective agent against both bacterial vaginosis and Candida vaginitis since it was able to adhere to vaginal and cervical cells and to antagonist the growth of vaginosis-associated pathogens. Clinical studies are now required to assess the in vivo efficacy of such a therapy.
Stem cell therapy for neurodegenerative diseases: Progress and prospects
Although neurodegenerative diseases have different causes, the dysfunction and loss of specific groups of neurons is common to all these disorders and may allow the development of similar therapeutic approaches to the treatment of diseases like Alzheimer’s disease (AD) and Parkinson’s disease (PD). The efforts to treat the neurodegenerative diseases by existing methods of cellular therapy are insufficiently effective. The modern methods do not provide correct restoration of cytoarchitecture and pattern of connections (the rewiring of specifically organized long-distance connections), which are essential to achieve a significant functional recovery. This article discusses existing methods of neural stem cell therapy and provides example of new approach to the treatment of various neurodegenerative diseases.
Neurodegenerative diseases are an assortment of central nervous system disorders characterized by neuronal loss and intraneuronal accumulation of fibrillary materials. Abnormal protein-protein interactions may allow the precipitation of these proteins, forming extracellular and intracellular aggregates. These abnormal interactions could play a role in the dysfunction and neuronal death that characterizes several common neurodegenerative diseases, such as Alzheimer’s Disease (AD) and Parkinson’s Disease (PD).
AD is the most common cause of dementia, with aging a major contributor to its onset. Currently, it is estimated that 40% of people over age 80 are afflicted with AD. Autopsy examination of a patient’s brain reveals gross cerebral atrophy, signifying loss of neurons and the presence of large numbers of extracellular neuritic plaques and intracellular neurofibrillary tangles. Plaques and tangles are found predominantly in the frontal and temporal lobes, including the hippocampus. In more advance cases, the pathology extends to other regions of the cortex. Similar plaques and tangles do occur in normal ageing brains.
PD is more common in people 60 years old and older. In the US, PD affects 1.5 million people. The degeneration and loss of dopaminergic neurons in PD causes akinesia, rigidity and tremor. Cell transplantation for the treatment of PD is the promising approach that has received most attention.
Cell therapy for PD
The potential of cell therapy for neurodegenerative diseases was demonstrated on implantation of different types of stem cells in the animals with PD (Kim J-H et al 2002, Parati EA et al 2003). Transplantation of stem cells into rat brain resulted in reinnervation of the striatal neurons and partial recovery of motor deficit associated with dopamine deficiency (Kim J-H et al 2002). The same results were obtained after transplantation of fetal dopaminergic neurons in clinical trials (Piccini P et al 2000, Freed CR et al 2001). It is possible to use different types of stem cells to generate dopaminergic neurons. Today the process of dopaminergic neurons differentiation from embryonic stem cells (ESC) in vitro is most effective and understandable (Kim J-H et al 2002, Isacson O, Ann Neurol 2003, Isacson O, Lancet Neurol 2003, Barberi T et al 2003). Recent progress in human therapeutic cloning (Woo Suk Hwang et al 2004) makes this way to generate neurons more and more attractive. Differentiation of ESC in vitro and transplantation of dopaminergic neurons in the animal models of PD resulted in functional integration of implanted cells into recipient’s brain and partial recovery of motor functions (Kim J-H et al 2002, Barberi T et al 2003).
Although transplantation of neurons into striatum in PD model has a higher effectiveness in comparison with transplantation of neurons in other neurodegenerative disorders, it is too early to speak about full restoration of motor deficit associated with parkinsonism. In case of PD significant functional recovery requires cell replacement with, at least partial repair of original connections with neurons in the striatum. If such connections do not exist the full regress of motor deficit is impossible because dopamine release is under feedback control. This fact emphasizes the importance to develop effective methods to
The method to enhance accuracy of regeneration (Potential therapeutic strategy)
After transplantation stem cells make decisions regarding fate and patterning in response to external signals from extracellular environment and neighboring cells. The effectiveness of neural stem cell therapy may be facilitated by the ability to manipulate these signals in a temporal and spatially appropriate fashion (Liu CY et al 2003). The future methods of therapy could include in vitro processing of stem cells before implantation, supporting and guiding the cells after implantation with the help of nanorobots, as well as the in vivo creation of molecular scaffold (The Samuel I. Stupp Laboratory – sistagirl.ms.northwestern.edu, Silva GA et al 2004) for stimulating their growth in the correct direction.
During experiments on neonatal rats (Englund U et al 2002) the potential ability of neural stem cells to establish appropriate long-distance axonal projection after region-specific differentiation were shown. Unfortunately, adult brain, as compared to neonatal, has unfavorable conditions for axon growth in the correct direction. It is for this reason the stimulation of new neurons growth, for example along the surface of neurons in the zone of progressive degeneration, is necessary. The reconstruction of dysfunctional neural circuits may be facilitated in the following way . The proposed strategies are designed to increase accuracy of dysfunctional neurons regeneration.
Management of Chronic Pain
Comprehensive treatment for chronic pain must address both physical and psychological aspects.
Pain is a leading reason that people seek medical care. Annually, pain is estimated to burden the U.S. economy with $100 billion in direct costs and $61 billion in productivity losses. These losses, which amount to a mean of 4.6 hours weekly, are largely due to diminished performance at work.1 Perhaps more significant, but more difficult to quantify, is the emotional distress and diminished quality of life that pain inflicts on individuals and their loved ones.
WHAT IS PAIN?
Pain, as defined by the International Association for the Study of Pain is “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage.”2 Thus, pain is an experience that comprises a physical sensory component and an affective cognitive component. The physical element involves a nociceptive, neuropathic, or inflammatory stimulus that is transmitted via the spinal cord to the thalamus and then to areas of the cerebral cortex, where it is perceived. Acute pain serves a biological purpose as an alert to potential, immediate, or ongoing injury.
Chronic pain is pathologic, serves no biological purpose, and can result from peripheral sensitization, in which processes (such as changes in gene expression in the dorsal root ganglia) augment the synthesis of various peripheral nociceptors. In turn, inputs to the dorsal horn of the spinal cord are amplified, leading to activation of N-methyl-D-aspartate (NMDA) receptors and subsequent spinal cord hyperexcitability. Destruction of inhibitory interneurons and sprouting of nerve fibers at the level of the dorsal horn also can contribute to central sensitization; in addition, disruption of descending inhibitory neural pathways can trigger central hypersensitivity and chronic pain.3
Chronic pain can persist after an initial injury has healed, or it can result from ongoing pathologic processes. Although specific periods (typically 6 months) often are used to define chronic pain, a more appropriate definition of the condition is simply pain that persists longer than it should. Chronic pain often involves neural activity in specific brain areas that are distinct from those that mediate acute pain; for example, unrelenting back pain is processed by areas that also process negative emotions.4 In addition, chronic pain can have widespread effects on overall brain functioning, thereby causing depression, anxiety, and decision-making difficulties in ways that acute pain does not.
WOMEN AND PAIN
Conditions such as headache, pelvic pain, rheumatoid arthritis, and facial pain are more common in women than in men; accordingly, women are two to three times more likely than men to suffer from chronic pain. Compared with men, women also tend to perceive pain as more severe and to report lower pain thresholds and higher pain ratings in research studies. Such sex differences could be secondary to the influence of hormones, family history, traditional sex roles, cognitive factors, and how the central nervous system processes pain.5
INTERDISCIPLINARY TREATMENT OF CHRONIC PAIN
Comprehensive treatment of chronic pain must address both physical and psychological aspects; thus, interdisciplinary approaches to pain management involve medical management, physical therapy, occupational therapy, biofeedback, vocational and recreational therapy, and psychological counseling.
Rational polypharmacy (evidence-based use of multiple medications) is a cornerstone of medical management of pain syndromes, which can encompass sleep and mood disorders. Treatment options include nonsteroidal anti-inflammatory agents, antiepileptic drugs such as gabapentin (for neuropathic pain), antidepressants, and opioid drugs. Use of chronic opioid therapy has risen substantially, along with concerns about drug abuse and addiction; accordingly, treatment recommendations have been developed by the American Pain Society and the American Academy of Pain Medicine to provide guidance about patient selection, risk factors for abuse, and opioid management plans.6 Chronic opioid treatment is appropriate if analgesia is achieved, adverse side effects do not occur, activities of daily living improve, and aberrant behavior or signs of abuse are not present (sometimes referred to as the four A’s). In addition to documentation of the four A’s, clinicians’ use of opioid contracts with patients, as well as random urine screens, all help to justify prescribing these agents as part of therapy for patients with chronic pain. Other medical strategies involve steroid injections (epidural or delivered directly to affected joints) to treat pain flares, and, when indicated, surgical approaches.
Physical therapy serves to allay the patient’s fear of movement; to teach the difference between “hurt” and “harm” sensations; to increase endurance, range of motion, and tolerance for sitting, standing, and walking; and to improve posture and strength. Occupational therapy addresses body mechanics, pacing, functional mobility and activities of daily living, and ergonomic modifications in the workplace. Biofeedback helps the patient to master diaphragmatic breathing and progressive muscle relaxation with the goals of alleviating muscle tension, reducing physical and emotional distress, and encouraging mindfulness of pain in an effort to divert attention away from it.
Managing pain largely entails attending to its psychological features and their impingement on overall emotional status. Sleep disturbances, anxiety, and depression can be treated with psychotherapy and, when required, pharmacologic agents. Along with medical management, cognitive-behavioral therapy (CBT) is often a mainstay of treatment.7 CBT is based on the theory that thoughts affect feelings and behaviors, which, in turn, influence how one experiences pain. CBT teaches individuals how to identify negative dysfunctional pain-related thoughts and how to replace them with more-adaptive thoughts. Ideally, one learns coping skills that can be applied to daily situations, including pain flares.
A key goal of CBT — indeed, of all the therapies for chronic pain — is to foster the belief that patients have the power to manage their pain by improving their physical abilities and by learning how to control their thoughts, behaviors, and reactions. Having a healthy sense of command of oneself includes being both physically and socially active and integrated. Unremitting pain leads to lower physical activity levels, resulting in diminished function and greater likelihood of depression; maintaining higher activity levels leads to less pain, thereby breaking the cycle.
Maintaining a sense of control is undeniably challenging for women who are in continual pain. Isolation and depression commonly accompany chronic pain conditions, which makes treatment difficult. Women must learn to maintain direction over their own lives and take time to care for themselves while also meeting the challenges of employment, caring for their families, and remaining socially integrated. Being able to do all of this, in addition to handling a debilitating condition, can be demoralizing and exhausting — physically, emotionally, and financially. Likewise, managing chronic pain conditions can be challenging and potentially draining for clinical pain-care teams. The treating clinician has a responsibility not only to educate the patient that her chronic pain is a disease but also to stop ongoing and unnecessary laboratory and radiologic testing after the diagnosis has been made definitively.
CONCLUSION
Collaboration among therapists, psychologists, and other supportive resources is crucial to delivering effective pain treatments. Helping women with chronic pain to take ownership of their conditions and to actively curb physical and emotional aspects will boost their functional mobility, productivity, and quality of life while also shrinking the associated financial burden.