M. C. Thomas

Luminal Bacteria and Small-Intestinal Permeability

BACKGROUND The influence of luminal bacteria on small-intestinal permeability has not been fully assessed. This study addressed this issue. METHODS Thirty-four subjects (mean age 64 years; range 22-95 years) were investigated for possible small-intestinal bacterial overgrowth (SIBO) with culture of a small-intestinal aspirate. A lactulose/mannitol small-intestinal permeability test was performed, small-intestinal histology assessed and serum vitamin B12 concentrations measured in all subjects. Permeability was also assessed in a control group of 34 asymptomatic volunteers. RESULTS Urinary lactulose/mannitol ratios were significantly increased in subjects with SIBO with colonic-type flora (P < 0.0005), even in the absence of villous atrophy. Urinary lactulose/mannitol ratios were increased in this group due to significantly increased urinary lactulose concentrations (P < 0.0005) rather than reduced urinary mannitol levels, after correcting for inter-subject variations in renal function. Counts of intraepithelial lymphocytes of CD8 phenotype were significantly increased in this group (P = 0.003). Although a significant correlation was found between intraepithelial lymphocyte counts and small-intestinal permeability overall (P < 0.002), these counts were not significantly different in subjects with SIBO with colonic-type flora whose permeability values were < or = > 0.028, the upper limit of normal in asymptomatic controls. Serum vitamin B12 concentrations did not differ significantly between groups (P > 0.5). Ageing did not independently influence small-intestinal permeability (P > 0.5). CONCLUSIONS Small-intestinal permeability is increased in subjects with SIBO with colonic-type bacteria. This effect is independent of ageing and not mediated by vitamin B12 deficiency. Although counts of intraepithelial lymphocytes of CD8 phenotype are increased in this disorder, it is also unlikely that these cells play an important causative role in this process. Routine light microscopic assessment underestimates the prevalence of small-intestinal functional disturbance in this disorder.

Small intestinal mucosal immunity and morphometry in luminal overgrowth of indigenous gut flora.

OBJECTIVE:The aim of this study was to investigate the separate effects of indigenous oropharyngeal- and colonic-type flora on small intestinal mucosal immunity and morphometry in small intestinal bacterial overgrowth (SIBO).METHODS:A duodenal aspirate and random biopsies of underlying mucosa were obtained from 52 adult subjects (age range, 18–90 yr; median, 60 yr) without disorders that may otherwise disturb small intestinal histology or mucosal immunity. Villus height, crypt depth, villus/crypt ratios, counts of intraepithelial lymphocytes (IELs) and lamina propria total mononuclear cells, IgA, IgM, and IgG plasma cells, mast cells, and B and T lymphocytes were determined in relation to the presence or absence of SIBO and the nature of the overgrowth flora in all subjects. CD4+ve and CD8+ve T-cell counts were determined in 24 subjects.RESULTS:SIBO was present in 26 of 52 (50%) subjects. Overgrowth flora included colonic-type bacteria in 20 subjects and oropharyngeal-type flora alone in 6 subjects. Lamina propria IgA plasma cell counts were significantly increased in subjects with SIBO, irrespective of whether the overgrowth flora comprised oropharyngeal-type flora alone or included colonic-type bacteria. Neither villus height, crypt depth, villus/crypt ratios, nor total or other mononuclear cell counts in lamina propria differed significantly between subjects with and without SIBO, irrespective of the nature of the overgrowth flora. IEL counts were significantly higher than in culture-negative subjects only when the overgrowth flora included colonic-type bacteria. Even then, IEL counts were within a range currently considered normal. A significant, inverse correlation between advancing age and IEL counts became apparent after adjusting for the effect of SIBO of colonic-type flora.CONCLUSIONS:SIBO of oropharyngeal- and colonic-type flora are associated with differing disturbances of local duodenal mucosa. Nonetheless, these would not be readily apparent during routine histological assessment. Old age independently influences duodenal IEL counts.

Effects of ethanol, acetaldehyde and cholesteryl esters on pancreatic lysosomes.

Recent studies indicate that altered lysosomal function may be involved in the early stages of pancreatic injury. Chronic consumption of ethanol increases rat pancreatic lysosomal fragility. The aim of this study is to determine whether the lysosomal fragility observed after chronic ethanol consumption is mediated by ethanol per se, its oxidative metabolite acetaldehyde or cholesteryl esters (substances which accumulate in the pancreas after ethanol consumption). Pancreatic lysosomes from chow fed rats were incubated for 30 minutes at 37 degrees C with ethanol, acetaldehyde or phosphatidylcholine vesicles containing cholesteryl oleate. Lysosomal stability was then assessed by determination of: (a) Latency--that is, the per cent increase in lysosomal enzyme activity after addition of Triton X-100 and (b) Supernatant activity--that is, the proportion of lysosomal enzyme remaining in the supernatant after resedimentation of lysosomes. Acid phosphatase, N-acetyl glucosaminidase, beta-glucuronidase and cathepsin B were assayed as lysosomal marker enzymes. Lysosomes incubated with homogenising medium alone or equivalent volumes of phosphatidylcholine vesicles without cholesteryl oleate were used as controls. Cholesteryl oleate at concentrations of 15 and 20 mM increased pancreatic lysosomal fragility as shown by decreased latency and increased supernatant enzyme. In contrast, ethanol (150 mM) and acetaldehyde (5 mM) had no effect on lysosomal stability in vitro. These results suggest that increased pancreatic lysosomal fragility observed with ethanol may be mediated by cholesteryl ester accumulation rather than by ethanol or acetaldehyde.

Alpha 1 antitrypsin phenotypes and alcoholic pancreatitis.

Altered frequencies of alpha 1 antitrypsin phenotypes have been reported in patients with chronic pancreatitis, suggesting a possible genetic basis for individual susceptibility to this disease. Alpha 1 antitrypsin phenotypes, with particular regard to alcoholic pancreatitis, were studied. Patients with alcoholic pancreatitis were compared with alcoholic control subjects with no history of pancreatic disease. Serum alpha 1 antitrypsin concentrations were raised in pancreatitis patients sampled within one month of an acute attack of pancreatitis, but otherwise values were similar to those of control subjects. There were no significant differences in alpha 1 antitrypsin phenotypes between alcoholics with pancreatitis and alcoholic control subjects. This study of alpha 1 antitrypsin phenotypes provides no evidence of an inherited susceptibility to alcoholic pancreatitis.

Small intestinal bacterial overgrowth and the irritable bowel syndrome

for overgrowth. Secondly, Mishkinet al. principally tested subjects with functional dyspepsia. Although a proportion of their subjects also had IBS, we included patients on the basis of their meeting only the criteria for IBS. Because functional dyspepsia represents a heterogeneous collection of conditions including problems such as gastroesophageal reflux disease (3), the prevalence of SIBO in this group of patients may well be lower than that in IBS alone. What is most interesting is that, in addition to Mishkin et al. (1), Galatolaet al. (4) report a positiveC-xylose breath test in up to 56% of subjects with IBS. The difference in prevalence between all three studies (1, 2, 4) could be based on the technique used. However, in our study we took this one step further by demonstrating that normalization of the abnormal breath test finding with antibiotics was associated with symptomatic improvement in IBS. This point is very important in the argument that the breath test findings do indeed represent overgrowth (as opposed to a false positive test result) because clinical outcomes improve and are linked to the abnormality of the test.

Small intestinal bacterial overgrowth, intestinal permeability, and non-alcoholic steatohepatitis

In a recent issue, Wigg and colleagues ( Gut 2001; 48 :206–11) reported that small intestinal bacterial overgrowth (SIBO), as diagnosed by a combined 14C-d-xylose/lactulose breath test, is significantly more common in patients with non-alcoholic steatohepatitis (NASH) than in control subjects without liver disease. The authors investigated the possible pathogenic significance of this observation by examining whether intestinal permeability and circulating levels of endotoxin and tumour necrosis factor α are increased in NASH patients with SIBO compared with those without. No significant differences in any of these parameters could be demonstrated in the two groups. An important factor influencing the validity or otherwise of these findings is the diagnostic accuracy of the 14C-d-xylose and lactulose breath tests for SIBO. Our experience, using a sterile endoscopic technique to sample small intestinal secretions under direct vision, is that these breath tests lack sensitivity and specificity for culture proven SIBO.1,2 Endogenous CO2 production and colonic metabolism of d-xylose are important factors inherently limiting the accuracy of the 14C-d-xylose breath test for SIBO.1 Furthermore, reliance on the finding of “double peaks” in serial breath hydrogen or methane levels after ingestion of lactulose to improve the accuracy of the 14C-d-xylose breath test, or as a diagnostic marker in its own right, is problematic. In a study in which a scintigraphic tracer was administered concurrently with lactulose, we found that each of the double peaks in breath hydrogen values may occur after the arrival of the test meal at the caecum, paralleling delivery patterns of fermentable substrate to caecal bacteria. A caecal source of each peak was suggested on 50% of occasions, rather than the first peak necessarily reflecting small intestinal metabolism …

Evaluation of the rice breath hydrogen test for small intestinal bacterial overgrowth

OBJECTIVES:The aims of this study were 1) to document the sensitivity, specificity, and predictive values of the rice breath hydrogen test for small intestinal bacterial overgrowth; 2) to determine the possible influence of concurrent gastric bacterial overgrowth and gastroduodenal pH on the efficacy of this test; and 3) to investigate whether reliability is limited by an inability of small intestinal luminal flora to ferment rice or its product of hydrolysis, maltose.METHODS:Twenty adult subjects were investigated with microbiological culture of proximal small intestinal aspirate and a 3-g/kg rice breath hydrogen test. Gastroduodenal pH, the presence or absence of gastric bacterial overgrowth, and the in vitro capability of small intestinal luminal flora to ferment rice and maltose, its product of hydrolysis, were determined.RESULTS:Sensitivity of the rice breath hydrogen test for small intestinal bacterial overgrowth was 33% and remained low even when subjects with small intestinal overgrowth with oropharyngeal-type (38%) and colonic-type flora (20%) and those with concurrent small intestinal and gastric bacterial overgrowth (40%) were considered separately. Sensitivity remained suboptimal despite favorable gastroduodenal luminal pH and documented ability of bacterial isolates to ferment rice and maltose in vitro. Specificity of the rice breath hydrogen test for small intestinal bacterial overgrowth was 91%. Positive predictive value, negative predictive value, and predictive accuracy were 75%, 63%, and 65%, respectively.CONCLUSIONS:Clinical value of the rice breath hydrogen test for detecting small intestinal bacterial overgrowth is limited. The rice breath hydrogen test is not a suitable alternative to small intestinal intubation and culture of secretions for the detection of small intestinal bacterial overgrowth.

Mucosal Cytokine Production in Small-Intestinal Bacterial Overgrowth

BACKGROUND Mucosal production of interferon-gamma, interleukin-6, and tumour necrosis factor-alpha is increased in inflammatory bowel disease and parallels disease activity. Interferon-gamma production is also increased in coeliac disease. Conversely, local cytokine profiles have not been investigated in small-intestinal bacterial overgrowth. This study addressed this issue. METHODS Eighteen adult subjects were studies with culture of proximal small-intestinal luminal secretion and measurement of luminal interferon-gamma, interleukin-6, and tumour necrosis factor-alpha concentrations by enzyme-linked immunosorbent assay. Small-intestinal histology was assessed by light microscopy. RESULTS Interferon-gamma, interleukin-6, and tumour necrosis factor-alpha were measurable in proximal small-intestinal luminal secretions of all subjects, even in the absence of light microscopic evidence of enteropathy. Small-intestinal bacterial overgrowth was present in 12 of 18 (66.7%) subjects. Luminal concentrations of neither interferon-gamma nor tumour necrosis factor-alpha differed significantly in subjects with and without small-intestinal bacterial overgrowth (P + 0.06 and P = 1.0, respectively). Conversely, luminal interleukin-6 concentrations were significantly increased in subjects with this disorder (P = 0.02). Multivariate linear regression analysis suggested that colonic-type rather than salivary-type flora mediated this increased interleukin-6 response (P = 0.02 and P = 0.64, respectively). No correlation was found between luminal interleukin-6 and tumour necrosis factor-alpha concentrations, even after the confounding influence of colonic-type bacteria was excluded (P = 0.60). CONCLUSIONS These findings suggest that increased mucosal production of interleukin-6 occurs in small-intestinal bacterial overgrowth, particularly when the overgrowth flora includes colonic-type bacteria. Conversely, luminal levels of neither interferon-gamma nor tumour necrosis factor-alpha are increased in the circumstance, distinguishing the local cytokine profile in this disorder from those that occur in coeliac disease and inflammatory bowel disease.

Serum immunoglobulin and soluble IL-2 receptor levels in small intestinal overgrowth with indigenous gut flora.

Murine studies have demonstrated that thepresence of indigenous gut flora is crucial for theinduction of systemic immune hyporesponsiveness toantigens initially encountered within thegastrointestinal lumen. This study investigated whetherincreased titers of such flora, as occur in human smallintestinal bacterial overgrowth, may be associated withincreased suppression of systemic immune responsiveness and the possible relation between systemic andmucosal immunity in this setting. Serum totalimmunoglobulin (Ig), immunoglobulin subclass, andsoluble interleukin-2 receptor levels and lamina propriaIgA plasma cell counts were determined in 50consecutive subjects with (N = 30) and without (N = 20)small intestinal bacterial overgrowth. Luminal IgAlevels were measured in 35 of these subjects. Serumconcentrations of IgG3, but not of otherimmunoglobulin isotypes or soluble interleukin-2receptors, were significantly reduced in subjects withbacterial overgrowth (P < 0.0005). Small intestinallamina propria IgA plasma cell counts (P < 0.0005) andluminal IgA concentrations (P = 0.001) weresignificantly increased in this group. SerumIgG3 levels were significantly inverselycorrelated with luminal IgA levels (P < 0.01) and fell below the lower limit ofnormal (0.41 g/liter) in 17/30 (56.7%) subjects withbacterial overgrowth compared to 1/20 (5.0%) subjectswithout (P < 0.0005). These findings document an association between small intestinalbacterial overgrowth with indigenous gut flora andreduced serum IgG3 reactivity in humans,possibly via an interaction with mucosa-relatedimmunoregulatory mechanisms. The possibility of underlying small intestinalbacterial overgrowth should be considered in patientswith serum IgG3 deficiency, especially thosewith compatible symptoms and/or knownpredisposition.

Luminal Immunity in Small-Intestinal Bacterial Overgrowth and Old Age

BACKGROUND The independent influences of small-intestinal bacterial overgrowth and old age on mucosal immunoglobulin production and secretion have not been assessed. This is an important issue, since luminal IgA deficiency may exacerbate small-intestinal bacterial overgrowth, the prevalence of which is high in selected elderly populations. METHODS Proximal small-intestinal aspirates were obtained from 33 subjects for bacteriologic analysis and measurement of total IgA, IgM, total IgG. IgG subclass, and IgD concentrations. IgA subclasses were measured in 24 unselected subjects. Serum immunoglobulin and salivary IgA concentrations were measured in all subjects. RESULTS IgA2 and IgG3 were predominant IgA and IgG subclasses in proximal small-intestinal luminal secretions. Luminal concentrations of IgA2 and IgM, but not IgG3 or any other IgG subclass, were significantly increased in small-intestinal bacterial overgrowth, which was present in 19 of 33 (57.6%) subjects. Old age did not influence these levels. Luminal immunoglobulin concentrations did not correlate significantly with either serum or salivary values. IgD was not measureable in proximal small-intestinal secretions. CONCLUSIONS Increased luminal concentrations of the secretory immunoglobulins, IgA2 and IgM, occur in small-intestinal bacterial overgrowth. Local investigation is mandatory when assessing the mucosal immunopathology of this disorder. Luminal IgG3 is unlikely to be predominantly derived from serum. Old age does not independently influence luminal immunity.