Lactulose ingestion causes an increase in the abundance of gut-resident bifidobacteria in Japanese women: a randomised, double-blind, placebo-controlled crossover trial.

The genus Bifidobacterium comprises various bacterial species, and the complement of species within the human intestinal tract differs from individual to individual. The balance of these bifidobacterial species remains poorly understood, although it is known that the abundance of bifidobacteria increases following the ingestion of prebiotics. We previously conducted a randomised, placebo-controlled, double-blind, crossover study of 2 g/day lactulose ingestion for 2 weeks in 60 Japanese women. To study the effect of lactulose ingestion on each bifidobacterial species, here, we measured the abundance of each of the principal bifidobacterial species. After lactulose ingestion, the log cell counts of the Bifidobacterium adolescentis group (8.97±0.08 vs 9.39±0.08, P=0.0019), Bifidobacterium catenulatum group (9.45±0.10 vs 9.65±0.10, P=0.0032) and Bifidobacterium longum group (9.01±0.07 vs 9.29±0.07, P=0.0012) were significantly higher than in the placebo ingestion control group. However, the log cell counts were similar for Bifidobacterium breve (8.12±0.12 vs 8.33±0.12, P=0.20), Bifidobacterium bifidum (9.08±0.12 vs 9.42±0.14, P=0.095) and Bifidobacterium animalis subspecies lactis (8.65±0.53 vs 8.46±0.46, P=0.77). Cluster analysis of the log cell count data at the bifidobacterial species level revealed three distinct clusters, but the combinations and ratios of the constituent bifidobacteria were not affected by lactulose ingestion. Furthermore, principal coordinate analysis of the intestinal microbiota in the lactulose and placebo ingestion groups using Illumina MiSeq showed no significant differences in the intestinal microbiota as a whole. These results suggest that 2 g/day lactulose ingestion for 2 weeks significantly increases the abundance of intestinal bifidobacteria, but does not affect the intestinal microbiota as a whole.

Prebiotic effect of two grams of lactulose in healthy Japanese women: a randomised, double-blind, placebo-controlled crossover trial.

Sixty healthy Japanese women with a defaecation frequency of 2-4 times/week participated in this randomised, double-blind crossover trial. Participants received 2 g/day lactulose for 2 weeks and placebo in a random order, separated by a washout period of 3 weeks. Eight participants were excluded who did not satisfy the conditions, and therefore data from 52 were analysed. The primary outcome was defaecation frequency and the secondary outcomes were the number of defaecation days, faecal consistency, faecal volume, and the number and percentage of Bifidobacterium in faeces. The defaecation frequency (times/week) was significantly higher during lactulose (4.28±0.23) than placebo (3.83±0.23) treatment (delta (Δ) 0.45 [95% confidence interval (CI) 0.10-0.80], P=0.013). The defaecation days (days/week) was significantly higher during lactulose (3.77±0.17) than placebo (3.47±0.17) treatment (Δ0.30 [95% CI 0.04-0.56], P=0.024). Faecal consistency using the Bristol Stool Scale (/defaecation) was significantly higher during lactulose (3.84±0.10) than placebo (3.68±0.10) treatment (Δ0.16 [95% CI 0.00-0.31], P=0.044). Faecal volume (/week) was significantly higher during lactulose (21.73±3.07) than placebo (17.65±3.07) treatment (Δ4.08 [95% CI 0.57-7.60], P=0.024). The number of Bifidobacterium in faeces (log colony forming units/g faeces) was significantly higher during lactulose (9.53±0.06) than placebo (9.16±0.06) treatment (Δ0.37 [95% CI 0.23-0.49], P<0.0001). The percentage of Bifidobacterium in faeces was also significantly higher during lactulose (25.3±1.4) than placebo (18.2±1.4) treatment (Δ7.1 [95% CI 2.9-11.4], P=0.0014). Finally, straining at defaecation (/defaecation) during lactulose (3.62±0.24) treatment was significantly lower than during placebo (3.97±0.24) treatment (Δ0.35 [95% CI -0.69 - -0.02], P=0.037). No significant difference was observed between lactulose and placebo with regard to flatulence. Severe adverse effects did not occur. Thus, oral ingestion of 2 g/day lactulose had a prebiotic effect, increasing the number and percentage of bifidobacteria in faeces, softening the faeces, and increasing defaecation frequency, but without increasing flatulence.

Low frequency of bcl-2 expression in large non-polypoid colonic neoplasms.

Large non-polypoid colorectal adenomas that spread over the mucosa and morphologically flat lesions are included in a group called granule-aggregating tumours. These are uncommon in the West. We aimed to clarify the biological differences between granule-aggregating tumours and colorectal polypoid adenomas. We evaluated the extent of apoptotic cell loss and expression of bcl-2 and p53 oncoproteins in 26 granule-aggregating tumours and 19 polypoid adenomas. The mean apoptosis index value of granule-aggregating tumours was significantly higher than that of polypoid adenomas. Only two (7.7%) granule-aggregating tumours and 13 (68.4%) polypoid colorectal adenomas expressed bcl-2, while 12 (46.2%) granule-aggregating tumours and six (31.6%) polypoid colorectal adenomas were p53-positive. Our results show that the higher apoptosis index and frequent expression of bcl-2 oncoprotein differentiates granule-aggregating tumours from polypoid colorectal adenomas. We propose that large non-polypoid granule-aggregating tumours of the colorectum are a biologically distinct entity.

Clinicopathological characteristics in large, non-polypoid colorectal adenomas with granule-aggregating appearance.

Endoscopically, most large, non-polypoid colorectal adenomas have a granule-aggregating appearance, made up of elevations of small, flat lesions projecting above the surrounding mucosa. In this study we investigated the clinicopathological characteristics in 26 cases of such tumours and proposed the term 'granule-aggregating tumour' (GAT). Patients with GATs (16 men and 10 women) had a mean age of 64.3 years (range, 38-83 years). The mean diameter of the GATs was 28 mm (range, 12-62 mm). Histologically, GATs were diagnosed as tubular (15 cases) or tubulovillous (11 cases) adenomas without submucosal invasion. These tumours had a surface morphology comprised mainly of small, flat elevations or granular structures, measuring 1.2 +/- 0.3 mm in diameter. Of the 26 patients with GATs, 19 (73.1%) were asymptomatic and 11 of these 19 cases (57.9%) were detected by a faecal occult blood test. In nine of the 26 patients (34.6%) GATs were accompanied by colorectal cancers arising at other sites. GATs show typical endoscopic features, often lack symptoms and are frequently accompanied by other cancers in other organs. An awareness of the existence of GATs should assist in the screening, prevention and therapy of colorectal tumours.

[Two patients with Bartonella henselae infection from a dog].

Two patients were reported as having been infected with Bartonella henselae after having contact with a dog. Both of the patients owned a dog, but had no contact with cats. One patient was a 10-year-old boy who had experienced a fever of 38-39 degrees C for 11 days, as well as having bilateral cervical lymphadenopathy. The boy's serum IgM antibodies to B. henselae were negative on the 6th and 16th day of his illness, whereas his IgG value, using indirect fluorescence antibody (IFA) method, was found to be elevated from 1:256 to 1:1,024. B. henselae DNA was detected, by PCR method, in swabs from the gingiva and buccal membrane of the dog with which the boy had been in contact. The boy was first treated with cefdinir (300 mg daily) for 6 days without beneficial effect. He responded, however, to minocycline (100 mg daily) with symptom resolution in four days. The other patient was a 64-year-old man who had experienced a fever of 38-39 degrees C for 27 days, as well as having right inguinal lymphadenopathy. The man's serum IgM antibody to B. henselae was negative, although his IgG value, determined by IFA, was 1:1,024. In addition, B. henselae DNA was detected, by PCR method, in parafin-embedded tissue obtained from the biopsied inguinal lymph nodes. The man was treated with cefazolin (2 g daily). His fever resolved, but his lymph nodes remained swollen. After a regimen of erythromycin (1,200 mg daily), the swelling in his inguinal lymphnodes gradually disappeared. Careful review of suspected CSD victims' history of contact with animals is important in making a prompt diagnosis of B. henselae infection.

[Prognostic significance of myocardial imaging with iodine-123 beta-methyl-p-iodophenyl-pentadecanoic acid in patients with angina pectoris].

OBJECTIVES: To assess the clinical significance of iodine-123 beta-methyl-p-iodophenyl-pentadecanoic acid(BMIPP) single photon emission computed tomography(SPECT), the predictive value of BMIPP imaging in patients with angina pectoris was evaluated. METHODS: One hundred seventy-four patients who underwent BMIPP imaging in our institution were aged 61.8 +/- 11 years. One hundred thirty-five patients had stable angina and 39 had unstable angina at the time of examination. Patients with previous myocardial infarction or myocardial disorders were excluded. Early and delayed images were acquired in BMIPP SPECT, and the images were analyzed visually. Cardiac events were classified into hard and soft events: the former consisted of cardiac death and nonfatal myocardial infarction, and the latter included coronary revascularization and heart failure. RESULTS: The findings of BMIPP imaging were normal in 82 patients and abnormal in 92. During follow-up of 15.5 +/- 9.5 months, hard events were observed in 4 patients and soft events in 53. In patients with normal BMIPP imaging, soft events were observed in nine patients, but no hard event was encountered. Furthermore, in patients with both normal BMIPP and stress thallium imagings, no cardiac event was observed during 2 years. In contrast, 4 hard events and 44 soft events occurred in patients with abnormal BMIPP imaging. Patients with abnormal BMIPP imaging had a higher incidence of soft events than those with normal BMIPP imaging, regardless of the type of angina(16/62 vs 3/73, p < 0.0005 for stable angina; 28/30 vs 6/9, p < 0.0001 for unstable angina). CONCLUSIONS: The finding of BMIPP imaging correlates well with the mid-term prognosis of patients with angina pectoris. Since BMIPP SPECT is performed without stress to the patient, this imaging modality is important in evaluating patients with stable or unstable angina.

[An adult case of systemic cat-scratch disease with hepatosplenic involvement].

A previously healthy 25-year-old female was admitted to our hospital in November, 1997, for treatment of a spike-fever of 2 weeks' duration. She had a cat in her house but reported no history of cat bites or scratches. No peripheral lymphadenopathy was detected. White blood cell count was within normal limits, but an increased C-reactive protein level of 11.4 mg/dl was noted. Infectious disease was suspected but ruled out as blood cultures were negative. Empiric therapy with clarithromyoin, isoniazid, and rifampicin was ineffective. In January, 1998, abdominal ultrasonogram revealed multiple hypoechoic mass lesions in the spleen and liver, and a splenectomy was performed in March. Histopathologic examination showed numerous necrotizing and caseating granulomas, which tested positive for Bartonella henselae DNA by PCR. Furthermore, the patient tested positive for B. henselae antibody by immunofluorescence assay. A diagnosis of systemic cat-scratch disease with hepatospnenic involvement was made. Combination therapy with minocycline, sulbactam/cefoperazone, and tosufloxacin was administered and her inflammatory findings improved gradually. We report an adult case of systemic cat-scratch disease with liver and spleen involvement in the non-immunocompromised host.

Effects of brewer's yeast cell wall on constipation and defecation in experimentally constipated rats.

Brewer's yeast cell wall (BYC) was tested on constipated male Sprague-Dawley rats that had been induced by loperamide (2 mg/kg of body weight). The preventive effect of BYC on constipation was examined and compared with that of a non-fiber diet (NF) as the control. The dose-response of BYC and the effect on defecation by constipated experimental rats were also compared with the characteristics of cellulose diet (CE) group which served as a control. Defecation was observed to be greater by the rats fed with BYC than by those fed with NF or CE. The fecal water content and level of volatile fatty acids (VFA) in the cecal contents were likewise higher in the rats fed with BYC. These results indicate that the administration of BYC was effective for improving defecation and other parameters related to defecation. These favorable effects of BYC supplemented to the diet are attributed to the fermentation ability, water holding capacity and swelling force in the large intestine.

[Serological cross-reaction among Bartonella henselae, Chlamydia pneumoniae and Coxiella burnetii by indirect fluorescence antibody method].

We studied the serological cross-reactions among Bartonella henselae, Chlamydia pneumoniae and Coxiella burnetii by indirect fluorescence antibody (IFA) method, using sera from 8 patients with cat scratch disease (CSD), 13 patients with C. pneumoniae infection and 12 patients with acute Q fever. B. henselae IgG antibody was negative in 13 patients with C. pneumoniae infection, and was positive in 3 (titers being 1:64) of 12 patients with Q fever, whereas B. henselae IgM antibody was negative in all the patients with C. pneumoniae infection or Q fever. C. burnetii IgG antibody was removed by absorption of these 3 sera with C. burnetii antigens, whereas B. henselae IgG antibody did not change. C. pneumoniae IgG antibody was positive in 3 (titers being 1:125 in two, 1:32 in one) of 8 patients with CSD. Both C. pneumoniae and B. henselae IgG antibody titers were significantly reduced by absorption of these 3 sera with B. henselae antigens. C. burnetii IgG or IgM antibodies were negative in all patients with CSD. In conclusion, no serological cross-reaction between B. henselae and C. burnetii was observed. On the other hand. B. henselae IgG antibody cross-reacted to C. pneumoniae antigens, whereas C. pneumoniae IgG antibody did not cross-react to B. henselae antigens. Our findings suggest that determination of B. henselae IgG or IgM antibodies were not influenced by C. pneumoniae and C. burnetii antigens.

[Bartonella henselae infection in domestic cat and dog fleas].

We studied on the infection of domestic cat and dog fleas with Bartonella henselae by polymerase chain reaction (PCR). A total of 62 fleas (36 Ctenocephalidis felis from cats, 24 C. felis from dogs and 2 Ctenocephalidis canis from dogs), stored in 70% ethanol, were analyzed by PCR for B. henselae specific DNA. Of the 62 fleas, C. felis from cats and dogs were positive for B. henselae specific DNA in 12 of the 36 (33.3%) and in 5 of the 24 (20.8%), respectively, and C. canis from dogs was positive in 2 of the 2 (100%). Our results demonstrated that pet fleas were infected with B. henselae, and suggest that flea transmission of B. henselae between cats or dogs may occur, and direct transmission of B. henselae from pet fleas to human may cause cat scratch disease.

Evaluation of apoptosis as a factor affecting the growth of non-polypoid colorectal adenomas.

Some colorectal adenomas show non-polypoid features endoscopically, e.g. nodule-aggregation or lateral spreading. To understand the developmental mechanism of non-polypoid adenomas better, we evaluated apoptosis indices in the upper and lower parts of 26 colorectal adenomas of non-polypoid appearance using TUNEL or single-stranded DNA (ssDNA) immunohistochemical staining. Regression analysis showed an extremely strong positive correlation between the apoptosis index (AI) values generated using each method. The AI of the lower section of lesion (4.54 +/- 1.86) was significantly higher than that of the upper part (1.21 +/- 1.47). In conclusion, ssDNA immunostaining is as useful as the TUNEL method in the evaluation of apoptosis in colorectal adenomas. It seems that vertical growth in non-polypoid adenomas is inhibited by apoptosis allowing horizontal growth to dominate and resulting in the characteristic, laterally spreading morphology.

Effects of yogurt supplemented with brewer's yeast cell wall on constipation and intestinal microflora in rats.

Brewer's yeast cell wall (BYC) is a valuable foodstuff that prevents constipation in rats. In this study, the effects of yogurt supplemented with brewer's yeast cell wall (BYC-Y) on constipated male Sprague-Dawley rats induced by loperamide (2 mg/kg body weight) were investigated. The preventive effect of BYC-Y on experimentally constipated rats was examined and compared with that of nonfiber diet (Control) and standard yogurt (Yogurt) as the control diets. The number of feces and fecal dry weight were greater in rats fed with BYC-Y than in those fed with Control or Yogurt. Moreover, the effect of BYC on the improvement of cecal microflora in constipated experimental model rats was evaluated. The number of Lactobacillus, Eubacterium, and total anaerobes per unit cecal contents increased (p<0.03) as a result of BYC ingestion. These findings indicate that BYC-Y or BYC is effective for the alleviation of constipation and bowel environment.

[Clinical evaluation of commercial serological test for Bartonella infection].

We evaluated the usefulness of a serological diagnostic kit (Bartonella IFA IgG, IgM; MRL Diagnostics) for Bartonella henselae infection. Of the 110 healthy individuals, 107 (97.3%) were with titers being less than 1:64 for IgG antibody to B. henselae, 2 were with titers being 1:64 and 1 with 1:128, IgM antibody to B. henselae was negative in all individuals. Serological diagnosis of cat scratch disease (CSD) using indirect fluorescence antibody (IFA) methods (in-house and diagnostic kit) was made in either elevated titers of IgM (> or = 1:20) or IgG (> or = 1:256) antibodies, or a four-fold rise in IgG titer between acute and convalescent sera. Of the 18 individuals with serological diagnosis of CSD by in-house IFA method in 26 CSD clinical diagnosed patients, 15 (83%) were compatible with the results of the diagnostic kit, whereas 3 (17%) were not compatible. Of the 8 without serological diagnosis, 1 (13%) was serologically diagnosed as CSD, and the others were negative. Overall, the serological diagnosis was made in 16 of 26 (62%). The specificity and sensitivity of this kit were 100% and 62%, respectively. The cross-reaction between B. henselae and Bartonella quintana was observed in sera from controls and patients. Our results show that the diagnostic kit as well as in-house method is an useful tool for the serological diagnosis of cat scratch disease.

DNA microsatellite instability and mismatch repair protein loss in adenomas presenting in hereditary non-polyposis colorectal cancer.

BACKGROUND AND AIM: Hereditary non-polyposis colorectal cancer (HNPCC), as its name implies, is associated with few adenomas, and the early evolution of colorectal neoplasia is poorly understood. In this study our aim was to clarify the genetic profiles of benign polyps in subjects with HNPCC using a combined molecular and immunohistochemical approach. METHODS: Thirty adenomas and 17 hyperplastic polyps were obtained from 24 affected HNPCC subjects. DNA was extracted from paraffin embedded tissue by microdissection and analysed for the presence of microsatellite instability (MSI) and mutations in five genes known to be targets in mismatch repair deficiency (TGFbetaRII, IGF2R, BAX, hMSH3, and hMSH6). Serial sections were stained by immunohistochemistry for hMLH1 and hMSH2. RESULTS: Twenty four (80%) of 30 adenomas showed MSI. Of MSI positive adenomas, 66.7% showed MSI at more than 40% of markers (high level of MSI (MSI-H)). Two of 17 hyperplastic polyps revealed MSI at one marker (low level of MSI (MSI-L)). A significant association was found between MSI-H and high grade dysplasia in adenomas (p=0.004). Eight of nine adenomas with mutations of coding sequences revealed high grade dysplasia and all nine were MSI-H. Four of the nine ranged in size from 2 to 5 mm. The presence of the hMSH6 mutation was significantly correlated with high levels of MSI (80% of markers) (p<0.02). Twenty four adenomas gave evaluable results with immunohistochemistry. One of six (17%) microsatellite stable, six of seven (86%) MSI-L, and 11 of 11 (100%) MSI-H adenomas showed loss of either hMLH1 or hMSH2. CONCLUSIONS: Most adenomas in subjects with a definite diagnosis of HNPCC show MSI (80%). The finding of MSI-L is usually associated with loss of expression of hMLH1 or hMSH2, unlike the situation in MSI-L sporadic colorectal cancer. The transition from MSI-L to MSI-H correlated with the finding of high grade dysplasia and mutation of coding sequences and may be driven by mutation of secondary mutators such as hMSH3 and hMSH6. Advanced genetic changes may be present in adenomas of minute size.

Neoplastic progression occurs through mutator pathways in hyperplastic polyposis of the colorectum.

AIM: Colorectal cancer has been described in association with hyperplastic polyposis but the mechanism underlying this observation is unknown. The aim of this study was to characterise foci of dysplasia developing in the polyps of subjects with hyperplastic polyposis on the basis of DNA microsatellite status and expression of the DNA mismatch repair proteins hMLH1, hMSH2, and hMSH6. MATERIALS AND METHODS: The material was derived from four patients with hyperplastic polyposis and between one and six synchronous colorectal cancers. Normal (four), hyperplastic (13), dysplastic (13), and malignant (11) samples were microdissected and a PCR based approach was used to identify mutations at 10 microsatellite loci, TGFbetaIIR, IGF2R, BAX, MSH3, and MSH6. Microsatellite instability-high (MSI-H) was diagnosed when 40% or more of the microsatellite loci showed mutational bandshifts. Serial sections were stained for hMLH1, hMSH2, and hMSH6. RESULTS: DNA microsatellite instability was found in 1/13 (8%) hyperplastic samples, in 7/13 (54%) dysplastic foci, and in 8/11 (73%) cancers. None of the MSI-low (MSI-L) samples (one hyperplastic, three dysplastic, two cancers) showed loss of hMLH1 expression. All four MSI-H dysplastic foci and six MSI-H cancers showed loss of hMLH1 expression. Loss of hMLH1 in MSI-H but not in MSI-L lesions showing dysplasia or cancer was significant (p<0.001, Fisher's exact test). Loss of hMSH6 occurred in one MSI-H cancer and one MSS focus of dysplasia which also showed loss of hMLH1 staining. CONCLUSION: Neoplastic changes in hyperplastic polyposis may occur within a hyperplastic polyp. Neoplasia may be driven by DNA instability that is present to a low (MSI-L) or high (MSI-H) degree. MSI-H but not MSI-L dysplastic foci are associated with loss of hMLH1 expression. At least two mutator pathways drive neoplasia in hyperplastic polyposis. The role of the hyperplastic polyp in the histogenesis of sporadic DNA microsatellite unstable colorectal cancer should be examined.

Bartonella henselae infection as a cause of fever of unknown origin.

Fourteen of 41 patients (34%) with a serological diagnosis of Bartonella henselae infection were found to have prolonged fever or fever of unknown origin, suggesting that generalized systemic B. henselae infection is not rare in immunocompetent healthy individuals.

DNA microsatellite instability in hyperplastic polyps, serrated adenomas, and mixed polyps: a mild mutator pathway for colorectal cancer?

AIM: To investigate the distribution of DNA microsatellite instability (MSI) in a series of hyperplastic polyps, serrated adenomas, and mixed polyps of the colorectum. METHODS: DNA was extracted from samples of 73 colorectal polyps comprising tubular adenomas (23), hyperplastic polyps (21), serrated adenomas (17), and mixed polyps (12). The presence of MSI was investigated at six loci: MYCL, D2S123, F13B, BAT-40, BAT-26, and c-myb T22, using polymerase chain reaction based methodology. MSI cases were classified as MSI-Low (MSI-L) and MSI-High (MSI-H), based on the number of affected loci. RESULTS: The frequency of MSI increased in tubular adenomas (13%), hyperplastic polyps (29%), serrated adenomas (53%), and mixed polyps (83%) (Wilcoxon rank sum statistic, p < 0.001). Hyperplastic epithelium was present in nine of 12 mixed polyps and showed MSI in eight of these. MSI was mostly MSI-L. MSI-H occurred in two serrated adenomas and three mixed polyps. Clonal relations were demonstrated between hyperplastic and dysplastic epithelium in four of eight informative mixed polyps. CONCLUSIONS: The findings support the view that hyperplastic polyps may be fundamentally neoplastic rather than hyperplastic. A proportion of hyperplastic polyps may serve as a precursor of a subset (10%) of colorectal cancers showing the MSI-L phenotype, albeit through the intermediate step of serrated dysplasia. This represents a novel and distinct morphogenetic pathway for colorectal cancer.

[Three children with systemic cat scratch disease].

Three girls with systemic cat scratch disease, aged 10, 13 and 9 years, were reported. They presented a prolonged fever and back pain in the early stage of the disease, and had no regional lymphadenopathy. Two of them had hepatosplenic granulomas, one with multiple 5 mm hypoechoic lesions in the liver and spleen, and the other with a single 2.5 cm hypodense lesion in the left hepatic lobe. The latter patient underwent a partial left hepatic lobectomy. All patients had elevated titers of antibodies to Bartonella henselae. Polymerase chain reaction detected B. henselae DNA in tissue specimens of the patient who underwent a hepatic lobectomy. Cat scratch disease should be recognized as a cause of fever of unknown origin because the prevalence of B henselae infection might be higher in Japan.