Lactobacillus plantarum Lp62 exerts probiotic effects against Gardnerella vaginalis ATCC 49154 in bacterial vaginosis.

The severe side-effects elicited by conventional antibiotic therapy and the recurrence of Bacterial vaginosis-associated bacteria and bacterial resistance have led to the development of novel alternative therapies, among which genital probiotics are widely used. In this study, we aimed to evaluate the antimicrobial activities of Lactobacillus plantarum Lp62 and its supernatant against Gardnerella vaginalis, using both in vitro and in vivo approaches. In vitro assays were used to evaluate the viability of the strain and the antimicrobial activities of the supernatant in different pH ranges. An in vivo assay was performed on female BALB/c mice, wherein the animals were divided into eight groups: four control groups and four treated groups (for curative and preventive therapies). After infecting and treating the mice, the animals were killed to quantify the bacterial load using qPCR, evaluate leucocyte cellular response, determine vaginal cytokine levels and perform cytokine tissue gene expression. Our analyses revealed significant activity of the strain and its supernatant against G. vaginalis. Preliminary in vitro tests showed that the strain grew with equal efficiency in different pH ranges. Meanwhile, the presence of halo and inhibition of pathogen growth established the significant activity of the supernatant against G. vaginalis. We observed that both micro-organisms are resident bacteria of mouse microbiota and that the lactobacilli population growth was affected by G. vaginalis and vice versa. We also observed that the treated groups, with their low bacterial load, absence of leucocyte recruitment, reduced cytokine levels in the vaginal lavage and normalized cytokine gene expression, successfully controlled the infection.

Efficacy of oral administration of lactic acid bacteria isolated from cocoa in a fermented milk preparation: reduction of colitis in an experimental rat model.

We investigated the probiotic potential of lactic acid bacteria (LAB) obtained from cocoa fermentation using an experimental rat model of colitis. Cocoa beans were collected from fermentation boxes every 12 h for 5 days to isolate the microorganisms. Strains were isolated by serial dilution and plating on MRS agar. Gram-positive and catalase-negative rods were subjected to DNA extraction, polymerase chain reaction, and sequencing. Ten strains were randomly pooled and used to prepare a fermented milk drink that was used to treat the experimental colitis. A parallel group was treated with a single strain drink. Serum concentrations of cytokines and IgA, total and differential counts of blood leukocytes, and histological appearance were compared with the untreated control colitis group. Eighty strains of LAB were identified as Lactobacillus fermentum (68) and Lactobacillus plantarum (12). The multi-strain LAB pool significantly reduced the total number of leukocytes. There was a significant reduction in the percentage of neutrophils and monocytes compared with the control colitis group. IFN-γ concentration was downregulated in animals treated with the LAB pool. IL-10 and IgA increased significantly in the group treated with the strains. Histological analysis showed that the LAB pool reduced the inflammatory infiltrate and restored tissue architecture. The group treated with the single strain LAB drink (L. fermentum) showed no signs of inflammation remission. The results confirm the probiotic action of cocoa-derived LAB in the treatment of experimental colitis. Studies using isogenic models and humans will clarify the mechanisms of immune response modulation in inflammatory bowel disease.

Serum profile of cytokines interferon gamma and interleukin-10 in ewes subjected to artificial insemination by cervical retraction.

This study evaluated the influence of artificial insemination (AI) by cervical retraction (CRI) on serum levels of interferon gamma (IFNγ) and interleukin-10 (IL-10) in ewes. Synchronized pluriparous Santa Inês ewes were subjected to natural mating (NM, n = 8) and AI, which was performed for a fixed time (55 ± 1 hour) by CRI (n = 8) or laparoscopy (n = 8). Ewes were classified as pregnant, with return to estrus (RE) or with embryonic loss (EL). Blood samples were collected on Day 0, Day 3, Day 5, Day 12, and Day 17 (Day 0 = AI/NM) for progesterone dosage and cytokines were quantified from Day 0 to Day 12. Progesterone levels were constant, except for a decrease in ewes with RE at Day 17 (P < 0.05). Regardless of the reproductive method used, there was no difference in the IFNγ and IL-10 levels at any time, with averages of 642.1, 713.2, and 741.2 pg/mL for IFNγ and 667.1, 616.8, and 721.1 pg/mL for IL-10 when using CRI, laproscopy, and NM, respectively. Regarding the physiological status, ewes with EL had lower serum levels of IFNγ and IL-10 than pregnant ewes and ewes with RE, regardless of the reproductive method used, with averages of 769.1, 714.9, and 555.7 pg/mL for IFNγ and 713.8, 699.3, and 578.7 pg/mL for IL-10 in pregnant ewes, ewes with RE and EL, respectively (P < 0.01). In conclusion, AI by CRI in Santa Inês ewes does not alter the profile of serum cytokines IFNγ and IL-10 and does not induce an inflammatory reaction that can compromise pregnancy.

Denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA to monitor changes in mouse gut bacterial communities during Salmonella enterica serovar Enteritidis latent infection.

Changes in intestinal microbial flora during a 4-week period of Salmonella enterica serovar Enteritidis colonization in resistant mice (latent carrier animals) were evaluated using a culture independent method involving denaturing gradient gel electrophoresis. The contents of the ileocecal portion of the intestines produced 26 bands. Fifty-seven percent of the bands were expressed in more than 80% of the samples. Forty percent of the bands present in the negative control were common to all samples, and 60% differed from those obtained 12 h and 1, 5, 10, and 28 days post-inoculation (PI). A dendrogram distinguished the negative control as the external group, and 2 clusters were formed with 76% similarity, separating the 12-h PI and 3-day PI time points from the others. These groupings were also revealed through multivariate analysis in a principal component analysis and the Venn diagram. The production of interferon γ 12 h and 3 days PI may explain this brief imbalance in microbiota that was quickly reversed in the subsequent days. These findings demonstrate that S. enterica serovar Enteritidis can colonize the gut and persist in balance with the microbiota of resistant hosts.

Lactic acid bacteria dynamics during spontaneous fermentation of cocoa beans verified by culture-independent denaturing gradient gel electrophoresis.

Cocoa is naturally fermented in the field before the cocoa seeds are removed for processing. We assessed the dynamics of lactic acid bacteria during cocoa fermentation in Bahia, Brazil. During five days of fermentation, temperature and pH were measured and beans were collected for genomic DNA extraction every 12 h. The DNA was used as a template for amplification with Lac1-Lac2 and Lac3-Lac2 for denaturing gradient gel electrophoresis analyses. pH values ranged from 3.34 to 4.98, while the temperature varied from 23° to 50°C. Lac1-Lac2 primers permitted detection of 11 operational taxonomic units. Twenty-eight operational taxonomic units were obtained with the primer pair Lac3-Lac2. It was observed that there were variations between the numbers of operational taxonomic units throughout the process, probably because of changes in pH and temperature. The greatest similarity in amplified samples was obtained with the primers Lac3-Lac2.

Detection of Salmonella Enteritidis in asymptomatic carrier animals: comparison of quantitative real-time PCR and bacteriological culture methods.

Quantification of Salmonella in asymptomatic carrier animals can be used to assess microbial risk and monitor the level of contamination in domestic animals used for food production. We examined the sensitivity, specificity and accuracy of real-time qPCR, without pre-enrichment or selective enrichment stages, for the quantification of S. enterica serovar Enteritidis in resistant mice, as a model of asymptomatic carrier animal. The results were compared with those obtained by traditional bacteriological culture methods, the gold standard test. Two hundred and forty-three samples, including spleen, liver, mesenteric lymph nodes, portions of intestine, intestinal content of the ileocecal portion, and feces, were collected from a group of 27 C57BL/6 mice, that had been intragastrically inoculated with high doses of S. enterica serovar Enteritidis. The real-time qPCR assay presented a consistent linearity of the standard curve (r(2) = 0.999), with very low differences between melting temperatures, and low coefficients of variation in intra- (< 1%) and interassay (< 2%) comparisons. The primers were highly specific; there was no amplification with other Salmonella serovars or with DNA from uninfected tissues and feces from mice. The detection limit of the technique was defined as 32 copies of S. enterica serovar Enteritidis. A sensitivity of 90%, a specificity of 77% and an accuracy of 79% were obtained. The higher sensitivity of PCR was reflected in a kappa coefficient of 0.41, showing moderate agreement between tests. We conclude that real-time qPCR is a good alternative for diagnostic scanning in asymptomatic carrier animals, due to its high sensitivity and rapidity.

Tissue and serum immune response in chronic hepatitis C with mild histological lesions.

The immunopathogenesis of chronic hepatitis C virus (HCV) infection is a matter of great controversy and has been suggested to involve a complex balance between cytokines with pro and anti-inflammatory activity. We investigated the expression of inflammatory cells and cytokines in the liver and serum of 51 chronically HCV infected patients and compared them to data from two sets of normal controls: 51 healthy blood donors and 33 liver biopsies of healthy liver donors. We also assessed the relationship between selected cytokines and cell populations in hepatic compartments and the disease stage. Compared with controls, hepatitis C patients had a greater expression of portal TNF-alpha, TGF-beta and CD4(+) and acinar IFN-gamma, TNF-alpha, IL-1beta and IL-4, as well as a higher serum concentration of IL-2, IL-10 and TGF-beta. Significant positive correlations were found between portal CD4+ and TNF-alpha, portal CD8(+) and TGF-beta, portal CD45(+)RO and TNF-alpha, acinar CD45(+)RO and IFN-gamma and acinar CD57(+) and TGF-beta. In conclusion, we have shown that (i) in this sample of predominantly mild disease, the immune response was associated with a pro-inflammatory response pattern, (ii) CD4(+) T-lymphocytes played a major role in orchestrating the immune response and (iii) these events primarily took place in the portal space.

Loxosceles venom-induced cytokine activation, hemolysis, and acute kidney injury.

Herein, we describe a confirmed case of Loxosceles spider bite that illustrates the critical complications seen in loxoscelism, including skin necrosis, rhabdomyolysis, hemolysis, coagulopathy, acute kidney failure, and electrolyte disorders. Upon initial assessment, laboratory studies revealed the following: the white blood cell count was 29,400 WBCs/mm(3), hemoglobin was 9.2g/dL, and the platelet count was 218,000 cells/mm(3). Coagulation studies revealed the following: international normalized ratio, 1.83; activated partial-thromboplastin time, 62 s; D-dimer, 600 ng/mL (normal range <500 ng/mL); free protein S, 37% (normal range=64-114%); protein C, negative; and antithrombin III, negative. Various serum levels were abnormal: urea, 110 mg/dL; creatinine, 3.1mg/dL; indirect bilirubin, 3.8 mg/dL; creatine kinase, 1631 U/L; lactate dehydrogenase, 6591 U/L; potassium 6.2 mmol/L. Urine tests were positive for hemoglobin and bilirubin. In addition, concentrations of interleukin-6 and tumor necrosis factor-alpha were notably elevated in the serum. In conclusion, physicians must be alert to the possibility of loxoscelism when a patient presents with the clinical and laboratory findings described above, especially if the patient resides in an endemic area. Advances in our understanding of multiple pathways and mediators that orchestrate the response to Loxosceles venom might reveal new possibilities for the management of loxoscelism.

Imbalance of IL-2, IFN-gamma and IL-10 secretion in the immunosuppression associated with human paracoccidioidomycosis.

Patients with paracoccidioidomycosis (PCM) display a certain degree of immunecompromise characterized by lymphocyte hyporesponsiveness to the main Paracoccidioides brasiliensis antigen (gp43). To determine whether cytokines are involved in this state, we evaluated the secretion of IL-2, IL-10 and IFN-gamma by peripheral blood mononuclear cells (PBMC) from patients with the acute (AF) and chronic (CF) forms of PCM and from healthy, P. brasiliensis-sensitized subjects. gp43-stimulated PBMC from healthy subjects produced substantial amounts of IL-2, IFN-gamma and IL-10, whereas PBMC from AF and CF patients produced low levels of IL-2 and IFN-gamma but substantial amounts of IL-10. Phytohaemagglutinin-induced cytokine secretion was comparable among AF and CF patients and healthy subjects, suggesting integrity of non-specific cellular immune mechanisms in PCM. gp43-pulsed adherent cells, but not non-adherent cells, were the main source of IL-10. Moreover, IL-2 and IFN-gamma secretion correlated inversely with the amount of specific antibodies produced by patients and healthy subjects. Our results suggest that the imbalance in cytokine production of patients with PCM plays a role in the gp43-hyporesponsiveness and the marked (non-protective) antibody production of these patients.

Characterization of the cellular immune function of patients with chronic mucocutaneous candidiasis.

Chronic mucocutaneous candidiasis (CMC) is a rare syndrome characterized by persistent and refractory infections of the skin, nails and mucosal tissues by yeasts of the genus Candida. Defects in the cellular limb of the immune system are well documented in CMC patients, but non-specific immune defects, such as myeloperoxidase deficiency or phagocyte chemotaxis disorders, have also been described. Nonetheless, the underlying defect(s) remains poorly understood, and further studies are required. We studied eight CMC patients without endocrinopathies, who showed (i) low normal proliferative response to phytohaemagglutinin (PHA), (ii) partially defective response to pokeweed mitogen (PWM), and (iii) impaired response to Candida and PPD antigens. Furthermore, peripheral blood mononuclear cells (PBMC) from CMC patients produced lower levels of type-1 cytokines (IL-2 and interferon-gamma) in response to Candida antigens, compared with control individuals. Conversely, we did not observe an enhancement of IL-4 and IL-10 in the patients, suggesting that, even though Th1 cytokines are decreased, the Th2 response is not increased in CMC. Nevertheless, the synthesis of these cytokines was normal when induced by PHA. We also observed an increased antigen-induced apoptosis in lymphocytes from the patients compared with controls, and this applied both to Candida and PPD antigens. Lastly, innate immunity defects were investigated. We observed an impairment of natural killer activity against K-562 target cells in half of the studied patients. These findings corroborate the extensive clinical and laboratory variability of CMC, which requires further studies on a larger number of patients to be better understood.

Diagnostic equivalence of conventional and fast spin echo magnetic resonance imaging of the anterior cruciate ligament of the knee.

Many techniques and pulse sequences have been devised for the assessment of the anterior cruclate ligament. The present study compares fast spin echo (FSE) imaging to conventional spin echo imaging at a field strength of 1.5 T in an effort to determine if these sequences are diagnostically equivalent. Where available, arthroscopy was also done. A total of 52 patients were imaged using both FSE and conventional spin echo sequences. Eight volunteers were used as controls. Arthroscopy was performed on 10 patients. The anterior cruciate ligament was assessed in a blinded fashion by three radiologists. The Kappa statistic was then used to determine the percentage agreement between FSE and conventional spin echo imaging. Fast spin echo sequencing demonstrated a sensitivity of 100%, a specificity of 94.8% and an accuracy of 96.3% when compared to arthroscopy. Conventional spin echo imaging and arthroscopy had a sensitivity of 100%, specificity of 84.6% and an accuracy of 88.9%. The remaining 34 patients who did not undergo arthroscopy were followed clinically because clinical and imaging findings were not suggestive of ACL tears. These demonstrated 72% agreement between FSE and conventional spin echo imaging using the Kappa statistic, with regards to calling ACL normal or having only a low-grade partial tear. Fast spin echo imaging produces images of the anterior cruciate ligament that have similar diagnostic accuracy to conventional spin echo images (P < 0.05) within a much shorter scan time. These results however, require further validation in a larger group, preferably with arthroscopic correlation.

Adamantinoma of the tibia: plain-film, computed tomography and magnetic resonance imaging appearance.

The authors describe the computed tomography (CT) and magnetic resonance imaging (MRI) characteristics of adamantinoma of the tibia in a 29-year-old man. Adamantinoma is a rare, aggressive osteolytic lesion occurring primarily in the diaphyseal portion of long bones. Because of its rarity the MRI features have been infrequently reported. In this case MRI provided more accurate information about the extent and invasiveness of the tumour than either plain radiography or CT.

Imaging after arthroplasty.

Arthroplasty is now an important and frequently performed orthopedic procedure. This article focuses on the imaging of abnormalities frequently encountered in patients who have undergone arthroplasty. The emphasis is on plain film analysis, although arthrography, computed tomography and nuclear medicine are also discussed. Among the problems illustrated are loosening of the prosthesis, component fatigue or failure, and infection. The authors recommend techniques to optimize imaging. Although most of the entities illustrated involve hip arthroplasty, the principles are valid for arthroplasty at other sites.

Magnetic resonance imaging of a giant nonfunctioning retroperitoneal paraganglioma.

The authors describe the magnetic resonance imaging (MRI), computed tomographic, ultrasonographic and angiographic appearance of a giant nonfunctioning retroperitoneal paraganglioma. To their knowledge, the MRI appearance of a nonfunctioning paraganglioma has not been previously described. The MRI characteristics of the nonfunctioning tumour were similar to those of a functioning paraganglioma and included hyperintensity in T2-weighted images and intense enhancement after intravenous administration of contrast agent.