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.

Men and women are different indeed. Besides the obvious external anatomical differences, the brains of men and women tend to operate differently. Through recent research, scientists have been able to examine the human brain more than ever. With the use of MRI, fMRI (functional Magnetic Resonance Imaging), and PET (Positron Emission Tomography) researchers are able to see the brain in 2 to 3 dimensions. This enables them to conduct brain research on a functioning human brain. They are able to see what parts of the brain are activated when a specific task is given. With this new technology, scientists conduct more strenuous tests on subjects in order to see what part, or parts, of the brain is responsible for various functions. This research is done in controlled studies where they rule out social and environmental factors. With this in mind, a great deal of differences may exist in the brains of males and female. Male and female differentiated brains are not necessarily in one anatomically correct body. A male can have a female differentiated brain, and a female a male differentiated brain. When we speak in terms of male and female, we are talking about the brain itself, and not the external or physical appearance of the body.

Anatomically, the brains of both male and female are quite different. At birth, a boy’s brain is between 12-20% larger than that of girls. On average, the male brain weighs 11-12% more than a woman. This is due to the larger physical stature of men. Male’s larger muscle mass, and larger body size require more neurons to control them. This does not suggest that due to the larger brain, males are smarter than females. There are several differences between the two hemispheres. The left hemisphere, which is important to communication, is thicker in female oriented brains. This may conclude why women become more proficient with language skills. The corpus callosum is thicker allowing the free flow of communication between both hemispheres of the brain. Allowing more synapses between the two hemispheres. The female differentiated brain is able to use both sides of the brain, during communication. This allows them to become multitasked while conversing. When female subjects were asked to recall vocabulary and definitions, the entire brain lit up like a Christmas tree. The male brain on the other hand, only had highly concentrated neuron activity in the left hemisphere. The ability for the female differentiated brain to operate in this manner is due in part to the larger corpus callosum that allows more transmissions between the two hemispheres. The male differentiated brain has a thicker right hemisphere. This may be the reason males tend to be more spacial, and mathematical. The corpus callosum is thinner than it’s female counter part. This is the reason why when men communicate; they tend to only use one side of their brain. Male oriented brains, hardly express feelings. This is due to the use of the right hemisphere only. While men hold their emotions in until bursting point, women express how they feel. The corpus callosum allows this. Being larger, women use both hemispheres creating more synapses between the two sides of the brain. Male brains separate language, in the left, and emotions in the right, while the female’s emotions are in both hemispheres. This helps explain why the male brain has a hard time expressing its feelings. The male brain has its vocabulary making power seated only in the left hemisphere enabling him to develop a large vocabulary. Hence all the technical terms and terminology he may use. Whereas the female brain becomes more proficient in the vocabulary she already has using her emotions and feelings for others to aid in the production of language.

The hypothalamus is another area of the brain with differences between males and females. The hypothalamus is located at the base of the brain and regulates many of the body’s basic functions. Eating, sleeping, temperature control, and reproduction are some of the basic functions it provides. Many of which are primal in state. The preoptic area is involved in mating behavior. In males this area is about 2 times larger than in females, but also contains 2 times more cells. This enlargement is dependent on the amount of male sex hormones or androgens.

Scientists have discovered a region in the cortex called the inferior-parietal lobule (IPL) in the parietal cortex. This area is significantly larger in men than in women. More specifically, the left side IPL is larger than the right in men. Whereas the right side is larger than the left in the female brain. This is the same area that was shown to be larger in the brain of Albert Einstein, as well as in other physicists and mathematicians (Sabbatini). It seems that the IPL correlates with the mathematical ability. The IPL lets the brain process input from the senses and aid in selective attention and perception. Studies have shown that the right IPL is linked to understanding spatial relationships and the ability to sense relationships between body parts (Sabbatini). The left on the other hand, is linked with perception of time and speed, and the ability to rotate 3-D figures in your brain.

Sex hormones play a significant role in developing a male or female differentiated brain. This lies in the mother’s hormone levels during pregnancy. Studies in lab animals have proved that altering hormone levels during pregnancy can produce brains with male or female traits depending on the type of hormone added to the pregnant female. For instance, if testes of newborn male rats are removed, they tend to develop thicker left hemispheres than rats whose testes are still intact. This is a female trait. If a pregnant female monkey is injected with testosterone, the offspring will show one or more male traits. These traits are known as the five characteristics of the male-differentiated brain: aggression, competition, self-assertion, self-confidence, and self-reliance. These characteristics are related to levels of testosterone whether in males or females. Men who act as if they have female differentiated brains, in fact have lover levels of testosterone, and women who behave with male differentiated brains possess higher levels of testosterone than normal. The male differentiated brain tends to be one that is aggressive, spatial, and math proficient. The female differentiated brain is one in which is nurturing, and verbose. There can be a relationship between body asymmetry and gender behavior. The amount of androgens and estrogens in the body can affect both gender behavior and body asymmetry. These hormones are passed from mother to newborn during the embryonic stage of life. For example: if a female embryo receives an excess of androgen during pregnancy, she is likely to have a male appearance, behavior and male differentiated brain. On the other hand, if a male embryo receives an excess of estrogen. Male appearance, and behavior is prevalent, but with a female differentiated brain. When a female embryo is subjected to a large amount of estrogen, she has an excessive female appearance and behavior. The same is true for males when they receive large amounts of androgen during pregnancy. They tend to be a super male with lots of hair and very aggressive. Various events can cause this to happen during pregnancy. The unborn child can be subjected to various hormones during crucial periods during pregnancy. Mutations in the chromosomal matter may cause one of the events to occur. Major or sustained stress levels will suppress testosterone levels; renal dysfunction will produce too much testosterone. Injections for diabetes will cause an increase in estrogen, barbiturates, and exercise. Spurt exercise will cause an increase in testosterone, while sustained exercise like a long run or jogging will lower the amount. While the brain is immersed in hormones during pregnancy, the true affect does not appear until puberty begins and the brain becomes activated due to the full immersion of the hormones in the body.

Understanding that the differences between the male and female differentiated brain is not limited to just the external anatomical sex of the person. Many factors determine whether or not the brain is male or female. The actual size of the brain corresponds to the size of the individual. Does a larger brain determine the amount of knowledge one can possess? No. Through the research, we know that male differentiated brains tend to be more mathematical, spatial, and aggressive. Where the female differentiated brain tends to be more nurturing and verbose. This is not to say that women are not capable of higher achievement in math, but on average males tend to achieve higher test scores on standardized tests in math. Depending on the amount of hormonal activity during the embryonic stage of development may indicate whether a person’s brain is male or female differentiated.

Studies that have looked at differences in the brains of males and females have focused on:

(1) Total brain size: In adults, the average brain weight in men is about 11-12% MORE than the average brain weight in women. Men’s heads are also about 2% bigger than women’s. . This is due to the larger physical stature of men. Male’s larger muscle mass, and larger body size require more neurons to control them. This does not suggest that due to the larger brain, males are smarter than females.

(2) Cell number: men have 4% more brain cells than women , and about 100 grams more of brain tissue. this may explain why women are more prone to dementia (such as Alzheimer’s disease) than  men, because although both may lose the same number of neurons due to the disease, “in males, the functional reserve may be greater as a larger number of nerve cells are present, which could prevent some of the functional losses.”

(3) Cellular connections: while men have more neurons in the cerebral cortex, women have a more developed neuropil, or the space between cell bodies, which contains synapses, dendrites and axons, and allows for communication among neurons .

(4) Corpus callosum: it is reported that a woman’s brain has a larger corpus collusum, which means women can transfer data between the right and left hemisphere faster than men. Men tend to be more left brained, while women have greater access to both sides.(however other studies have told a different story).

(5) Hypothalamus: LeVay discovered that the volume of a specific nucleus in the hypothalamus (third cell group of the interstitial nuclei of the anterior hypothalamus) is twice as large in heterosexual men than in women and homosexual men, thus prompting a heated debate whether there is a biological basis for homosexuality .

(6) Language: two areas in the frontal and temporal lobes related to language (the areas of Broca and Wernicke) were significantly larger in women, thus providing a biological reason for women’s notorious superiority in language-associated thoughts. For men, language is most often just in the dominant hemisphere (usually the left side), but a larger number of women seem to be able to use both sides for language. This gives them a distinct advantage. If a woman has a stroke in the left front side of the brain, she may still retain some language from the right front side. Men who have the same left sided damage are less likely to recover as fully. Curiously, oriental people which use pictographic (or ideographic) written languages tend also to use both sides of the brain, regardless of gender.

(7) Inferior parietal lobule (IPL): it is a brain region in the cortex, which is significantly larger in men than in women. This area is bilateral and is located just above the level of the ears (parietal cortex). Furthermore, the left side IPL is larger in men than the right side. In women, this asymmetry is reversed, although the difference between left and right sides is not so large as in men. This is the same area which was shown to be larger in the brain of Albert Einstein, as well as in other physicists and mathematicians. So, it seems that IPL’s size correlates highly with mental mathematical abilities. Studies have linked the right IPL with the memory involved in understanding and manipulating spatial relationships and the ability to sense relationships between body parts. It is also related to the perception of our own affects or feelings. The left IPL is involved with perception of time and speed, and the ability of mentally rotate 3-D figures .

(8) Orbitofrontal to amygdale ratio (OAR): In one project, they measured the size of the orbitofrontal cortex, a region involved in regulating emotions, and compared it with the size of the amygdala, implicated more in producing emotional reactions. The investigators found that women possess a significantly larger orbitofrontal-to-amygdala ratio (OAR) than men do. One can speculate from these findings that women might on average prove more capable of controlling their emotional reactions.

(9) Limbic size: females, on average, have a larger deep limbic system than males. This gives females several advantages and disadvantages. Due to the larger deep limbic brain women are more in touch with their feelings, they are generally better able to express their feelings than men. They have an increased ability to bond and be connected to others . Females have a more acute sense of smell, which is likely to have developed from an evolutionary need for the mother to recognize her young. Having a larger deep limbic system leaves a female somewhat more susceptible to depression, especially at times of significant hormonal changes such as the onset of puberty, before menses, after the birth of a child and at menopause. Women attempt suicide three times more than men. Yet, men kill themselves three times more than women, in part, because they use more violent means of killing themselves (women tend to use overdoses with pills while men tend to either shoot or hang themselves) and men are generally less connected to others than are women. Disconnection from others increases the risk of completed suicides.