Walter Morales

Cedars-Sinai Medical Center, Los Angeles, California, United States

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Publications (26)215.02 Total impact

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    ABSTRACT: Acute gastroenteritis can precipitate irritable bowel syndrome (IBS) in humans. Cytolethal distending toxin is common to all pathogens causing gastroenteritis. Its active subunit, CdtB, is associated with post-infectious bowel changes in a rat model of Campylobacter jejuni infection, including small intestinal bacterial overgrowth (SIBO). To evaluate the role of host antibodies to CdtB in contributing to post-infectious functional sequelae in this rat model. Ileal tissues from non-IBS human subjects, C. jejuni-infected and control rats were immunostained with antibodies to CdtB, c-Kit, S-100, PGP 9.5 and vinculin. Cytosolic and membrane proteins from mouse enteric neuronal cell lysates were immunoprecipitated with anti-CdtB and analyzed by mass spectrometry. ELISAs were performed on rat cardiac serum using CdtB or vinculin as antigens. Anti-CdtB antibodies bound to a cytosolic protein in interstitial cells of Cajal (ICC) and myenteric ganglia in C. jejuni-infected and naïve rats and human subjects. Mass spectrometry identified vinculin, confirmed by co-localization and ELISAs. Anti-CdtB antibodies were higher in C. jejuni-infected rats (1.27 ± 0.15) than controls (1.76 ± 0.12) (P < 0.05), and rats that developed SIBO (2.01 ± 0.18) vs. rats that did not (1.44 ± 0.11) (P = 0.019). Vinculin expression levels were reduced in C. jejuni-infected rats (0.058 ± 0.053) versus controls (0.087 ± 0.023) (P = 0.0001), with greater reductions in rats with two C. jejuni infections (P = 0.0001) and rats that developed SIBO (P = 0.001). Host anti-CdtB antibodies cross-react with vinculin in ICC and myenteric ganglia, required for normal gut motility. Circulating antibody levels and loss of vinculin expression correlate with number of C. jejuni exposures and SIBO, suggesting that effects on vinculin are important in the effects of C. jejuni infection on the host gut.
    Digestive Diseases and Sciences 11/2014; DOI:10.1007/s10620-014-3435-5 · 2.55 Impact Factor
  • Gastroenterology 05/2014; 146(5):S-465-S-466. DOI:10.1016/S0016-5085(14)61667-X · 13.93 Impact Factor
  • Gastroenterology 05/2014; 146(5):S-524-S-525. DOI:10.1016/S0016-5085(14)61899-0 · 13.93 Impact Factor
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    ABSTRACT: AIM: Rifaximin is a non-absorbed antibiotic relative of rifampicin. The location of effect and staphylococcal resistance are two recent potential concerns with rifaximin. In this study we evaluate the location of effect of rifaximin as well as the development of staphylococcal rifampicin resistance. METHODS: Rats were divided into three groups. Group 1 gavaged for 10 days with PBS, group 2 gavaged with rifaximin for 10 days, and group 3 gavaged with rifaximin for 10 days and housed for 30 days. In each group, stool was collected daily for quantitative culture of Staphylococcus spp. and coliforms. After euthanasia luminal bacterial counts were determined at multiple gut locations by qPCR. Rifampicin susceptibility was tested on Staphylococcus pre and post rifaximin. RESULTS: At baseline, rats had a median of 2.90 × 10(6) cfu/ml Staphylococcus spp. in stool. After 10 days of rifaximin, this dropped to 1.20 × 10(5) cfu/ml (P < 0.01). With coliform counts, rats had a median of 1.86 × 10(4) cfu/ml at baseline which dropped to 2.2 × 10(3) cfu/ml (P < 0.01) after rifaximin. After cessation of rifaximin, coliform counts recovered within 3 days. When examining the total bacterial counts by qPCR, rifaximin reduced small bowel bacterial levels, but not colon. This reduction was sustained for 30 days. No colonies of Staphylococcus became resistant and only one colony was intermediate. The mean inhibitory concentration for rifampicin was not different before and after rifaximin. CONCLUSION: Staphylococcal spp. fail to demonstrate resistance to rifampicin after rifaximin. The transient reductions in stool coliform counts recover while rifaximin appears to produce durable reductions in duodenal bacteria.
    Digestive Diseases and Sciences 04/2013; 58(6). DOI:10.1007/s10620-013-2675-0 · 2.55 Impact Factor
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    ABSTRACT: It is increasingly understood that gastrointestinal (GI) methanogens, including Methanobrevibacter smithii, influence host metabolism. OBJECTIVE: Therefore, we compared M. smithii colonization and weight gain in a rat model under different dietary conditions. DESIGN AND METHODS: Sprague-Dawley rats were inoculated with M. smithii or vehicle (N = 10/group), fed normal chow until day 112 postinoculation, high-fat chow until day 182, then normal chow until day 253. Thereafter, five rats from each group were fed high-fat and normal chow until euthanasia. RESULTS: Both groups exhibited M. smithii colonization, which increased following inoculation only for the first 9 days. Change to high-fat chow correlated with significant increases in weight (P < 0.00001) and stool M. smithii (P < 0.01) in all rats, with stool M. smithi decreasing on return to normal chow. Rats switched back to high-fat on day 253 further increased weight (P < 0.001) and stool M. smithii (P = 0.039). Euthanasia revealed all animals had higher M. smithii, but not total bacteria, in the small intestine than in the colon. Rats switched back to high-fat chow had higher M. smithii levels in the duodenum, ileum, and cecum than those fed normal chow; total bacteria did not differ in any bowel segment. Rats which gained more weight had more bowel segments colonized, and the lowest weight recorded was in a rat on high-fat chow which had minimal M. smithii colonization. CONCLUSIONS: We conclude that M. smithii colonization occurs in the small bowel as well as in the colon, and that the level and extent of M. smithii colonization is predictive of degree of weight gain in this animal model.
    Obesity 04/2013; 21(4):748-754. DOI:10.1002/oby.20277 · 4.39 Impact Factor
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    ABSTRACT: BACKGROUND: Campylobacter jejuni infection is a leading cause of gastroenteritis and post infectious irritable bowel syndrome (PI-IBS). Unanswered questions include the role of cytokines, effects on gut flora, and why IBS is not more prevalent in countries with higher gastroenteritis rates. Therefore, we determined the effects of early and repeat C. jejuni infections on post infectious phenotypes, gut flora, and cytokine levels in a rat model of functional bowel and microbial changes. METHODS: Sprague-Dawley rats were gavaged with 10(8) cfu C. jejuni as juveniles and again as adults (J+/A+), as adults only (J-/A+), or vehicle (controls). Stool consistency during acute colonization, post infectious stool wet weight, total bacteria and Methanobrevibacter smithii levels in bowel segments, and ileal cytokines were evaluated. KEY RESULTS: C. jejuni colonization was longer for first exposures as juveniles (43.4 ± 1.7 days) vs. adults (30.4 ± 3.5 days) (P < 0.01) and shortest for second exposures (10.5 ± 1.7 days, P < 0.05). Small intestinal bacterial overgrowth (SIBO) was more prevalent in J+/A+ (47%) than J-/A+ rats (26%) (P = 0.019), but J-/A+ rats had greater stool consistency alterations (P < 0.01). Ileal β-defensin 2, TLR-4, IL-8, and β-defensin 6 levels were increased in J-/A+ rats and further increased in J+/A+ rats; TNF-α was highest and IL6 lowest in J-/A+ rats. Total bacteria increased, and M. smithii decreased, with successive infections. CONCLUSIONS & INFERENCES: We conclude that C. jejuni infection results in long-term alterations in small bowel flora, including methanogens. Mucosal defense mediators appear related to the number of infections, but not to SIBO development or the development of functional bowel phenotypes.
    Neurogastroenterology and Motility 03/2013; DOI:10.1111/nmo.12118 · 3.42 Impact Factor
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    ABSTRACT: Campylobacter jejuni infection is a leading cause of acute gastroenteritis, which is a trigger for post-infectious irritable bowel syndrome (PI-IBS). Cytolethal distending toxin (CDT) is expressed by enteric pathogens that cause PI-IBS. We used a rat model of PI-IBS to investigate the role of CDT in long-term altered stool form and bowel phenotypes. Adult Sprague-Dawley rats were gavaged with wildtype C. jejuni (C+), a C. jejunicdtB knockout (CDT-) or saline vehicle (controls). Four months after gavage, stool from 3 consecutive days was assessed for stool form and percent wet weight. Rectal tissue was analyzed for intraepithelial lymphocytes, and small intestinal tissue was stained with anti-c-kit for deep muscular plexus interstitial cells of Cajal (DMP-ICC). All 3 groups showed similar colonization and clearance parameters. Average 3-day stool dry weights were similar in all 3 groups, but day-to-day variability in stool form and stool dry weight were significantly different in the C+ group vs both controls (P < 0.01) and the CDT- roup (P < 0.01), but were not different in the CDT- vs controls. Similarly, rectal lymphocytes were significantly higher after C. jejuni (C+) infection vs both controls (P < 0.01) and CDT-exposed rats (P < 0.05). The counts in the latter 2 groups were not significantly different. Finally, c-kit staining revealed that DMP-ICC were reduced only in rats exposed to wildtype C. jejuni. In this rat model of PI-IBS, CDT appears to play a role in the development of chronic altered bowel patterns, mild chronic rectal inflammation and reduction in DMP-ICC.
    Journal of neurogastroenterology and motility 10/2012; 18(4):434-42. DOI:10.5056/jnm.2012.18.4.434 · 2.70 Impact Factor
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    ABSTRACT: PURPOSE: Among irritable bowel syndrome (IBS) patients, breath methane producers overwhelmingly have constipation predominance (C-IBS). Although the most common methanogen in humans is Methanobrevibacter smithii, incidence and type of methanogenic bacteria in C-IBS patients are unknown. METHODS: By use of a questionnaire and lactulose breath testing, subjects with Rome II C-IBS and methane (>3 ppm) were selected (n = 9). The control group included subjects with IBS who had no breath methane (n = 10). Presence of bacterial DNA was assessed in a stool sample of each subject by quantitative-PCR using universal 16S rDNA primer. M. smithii was quantified by use of a specific rpoB gene primer. RESULTS: M. smithii was detected in both methane and non-methane subjects. However, counts and relative proportion of M. smithii were significantly higher for methane-positive than for methane-negative subjects (1.8 × 10(7) ± 3.0 × 10(7) vs 3.2 × 10(5) ± 7.6 × 10(5) copies/g wet stool, P < 0.001; and 7.1 ± 6.3 % vs 0.24 ± 0.47 %, P = 0.02 respectively). The minimum threshold of M. smithii resulting in positive lactulose breath testing for methane was 4.2 × 10(5) copies/g wet stool or 1.2 % of total stool bacteria. Finally, area-under-curve for breath methane correlated significantly with both absolute quantity and percentage of M. smithii in stool (R = 0.76; P < 0.001 and R = 0.77; P < 0.001 respectively). CONCLUSIONS: M. smithii is the predominant methanogen in C-IBS patients with methane on breath testing. The number and proportion of M. smithii in stool correlate well with amount of breath methane.
    Digestive Diseases and Sciences 05/2012; 57(12). DOI:10.1007/s10620-012-2197-1 · 2.55 Impact Factor
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    ABSTRACT: A recent post-infectious rat model with Campylobacter jejuni 81-176 has replicated the events noted in humans with post-infectious irritable bowel syndrome (IBS). In this study, we test whether prophylactic treatment with the antibiotic rifaximin will prevent the development of long-term altered bowel function in this model. Sprague-Dawley rats were divided into two groups. Both groups were gavaged with a 1 mL solution of 10(8) cfu/mL of C. jejuni. However, one group was also prophylactically gavaged with a solution of rifaximin 200 mg per day for 3 days (the day before gavage, the day of gavage, and the day after gavage with C. jejuni). Fresh stool was collected from rats daily until two consecutive stool cultures were negative for C. jejuni. The rats were then housed for 3 months. At the end of 3 months, fresh stool was collected on three consecutive days to determine stool % wet weight and stool consistency on a stool score. Rats that received rifaximin antibiotic prophylaxis had a greater rate of stool shedding of C. jejuni. However, the mean duration of colonization was shorter in the rifaximin-treated group (10.3 ± 7.1 days) compared to rats receiving no prophylaxis (12.6 ± 5.9 days) (P < 0.01). After 3 months, rats that did not receive rifaximin had a greater variability in stool % wet weight (P < 0.01). Furthermore, the average stool consistency over 3 days of measurement was closer to normal in the rifaximin-treated rats, with a consistency of 1.1 ± 0.3, compared to 1.5 ± 0.4 in rats receiving no prophylaxis (P < 0.00001). Prophylactic treatment of rats with the antibiotic rifaximin in a new animal model of post-infectious IBS with C. jejuni mitigated the development of long-term altered stool form and function.
    Digestive Diseases and Sciences 07/2011; 56(7):1962-6. DOI:10.1007/s10620-010-1548-z · 2.55 Impact Factor
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    ABSTRACT: Recent evidence suggests a role for gut bacteria and antibiotics in the pathophysiology and treatment of irritable bowel syndrome (IBS), respectively. While the benefits of the antibiotic rifaximin have demonstrated efficacy and durable improvement in symptoms over 3 months, the long-term need for retreatment using this approach is mostly unknown. In this retrospective study, subjects with nonconstipated IBS who were retreated with rifaximin were examined. Charts of patients who were seen at a tertiary care medical center between 2007 and 2011 were reviewed. After exclusion criteria were applied, subjects who had received rifaximin and were seen for retreatment were fully reviewed. During review, demographic information, duration of response, and success of treatment and retreatment were evaluated. A total of 522 charts were reviewed. Of these 522 charts, 71 subjects were nonconstipated IBS subjects who had received at least one retreatment. Of these, 48 had a second, 22 had a third, 7 had a fourth, and 4 had a fifth treatment. More than 75% of subjects who initially responded to rifaximin also responded to any further retreatment, with no significant reduction in benefit for successive retreatments. Furthermore, there was no change in the duration of benefit (median time between treatments) for successive retreatments. Retreatment with rifaximin for subjects with nonconstipated IBS in a real-world clinical practice was successful up to five times without decrease in duration or effect.
    Digestive Diseases and Sciences 07/2011; 56(7):2067-72. DOI:10.1007/s10620-011-1728-5 · 2.55 Impact Factor
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    ABSTRACT: Campylobacter jejuni has been implicated in the pathogenesis of post-infectious irritable bowel syndrome (PI-IBS) in humans, effects which may be because of cytolethal distending toxin (CDT). In this study, we characterized both acute and chronic-phase histological changes of the small bowel in rats exposed to wild-type C. jejuni 81-176, or a strain that does not produce CDT, by using a validated rat model of PI-IBS. Sprague-Dawley rats were given 1.0 × 10(8) CFU of either wild-type C. jejuni 81-176 (C+, PI/C+) or the CDT-negative strain (CDT-), or vehicle alone (Control). Acute-phase rats (C+, CDT-) were euthanized on days 2, 4, 8, 16, and 32. Chronic-phase rats (PI/C+, Control) were euthanized 3 months after clearing the initial infection. Segments of duodenum, jejunum, and ileum were resected and the contents plated for C. jejuni culture, and tissue sections were stained for histology. We observed preferential infection of the ileum and jejunum by Campylobacter jejuni. Compared with controls, epithelial cell basal membrane ballooning, villous tip disruption, and reduced villous-to-crypt ratios were observed for both C+ and CDT- rats. Villous widening, the only result significantly different in C+ vs. CDT- rats, was greatest at day 4 (134.1 ± 21.12 μm vs. 109.9 ± 10.6 μm for CDT-, P < 0.01). Little or no cellular inflammatory changes were seen during acute C. jejuni infection. Three months after clearing the initial infection, no histological changes remained. Significant histological changes, with the absence of inflammatory cells, are seen in the duodenum, jejunum, and ileum of rats during acute infection with C. jejuni. These changes occurred irrespective of the presence or absence of the CDT toxin.
    Digestive Diseases and Sciences 03/2011; 56(9):2575-84. DOI:10.1007/s10620-011-1662-6 · 2.55 Impact Factor
  • Gastroenterology 01/2011; 140(5). DOI:10.1016/S0016-5085(11)61516-3 · 13.93 Impact Factor
  • Gastroenterology 01/2011; 140(5). DOI:10.1016/S0016-5085(11)62164-1 · 13.93 Impact Factor
  • Gastroenterology 01/2011; 140(5). DOI:10.1016/S0016-5085(11)62495-5 · 13.93 Impact Factor
  • Gastroenterology 01/2011; 140(5). DOI:10.1016/S0016-5085(11)62494-3 · 13.93 Impact Factor
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    ABSTRACT: To investigate the interstitial cells of Cajal (ICC) number using a new rat model. Sprague-Dawley rats were assigned to two groups. The first group received gavage with Campylobacter jejuni (C. jejuni) 81-176. The second group was gavaged with placebo. Three months after clearance of Campylobacter from the stool, precise segments of duodenum, jejunum, and ileum were ligated in self-contained loops of bowel that were preserved in anaerobic bags. Deep muscular plexus ICC (DMP-ICC) were quantified by two blinded readers assessing the tissue in a random, coded order. The number of ICC per villus was compared among controls, Campylobacter recovered rats without small intestinal bacterial overgrowth (SIBO), and Campylobacter recovered rats with SIBO. Three months after recovery, 27% of rats gavaged with C. jejuni had SIBO. The rats with SIBO had a lower number of DMP-ICC than controls in the jejunum and ileum. Additionally there appeared to be a density threshold of 0.12 DMP-ICC/villus that was associated with SIBO. If ileal density of DMP-ICC was < 0.12 ICC/villus, 54% of rats had SIBO compared to 9% among ileal sections with > 0.12 (P < 0.05). If the density of ICC was < 0.12 DMP-ICC/villus in more than one location of the bowel, 88% of these had SIBO compared to 6% in those who did not (P < 0.001). In this post-infectious rat model, the development of SIBO appears to be associated with a reduction in DMP-ICC. Further study of this rat model might help understand the pathophysiology of post-infectious irritable bowel syndrome.
    World Journal of Gastroenterology 08/2010; 16(29):3680-6. DOI:10.3748/wjg.v16.i29.3680 · 2.43 Impact Factor
  • Gastroenterology 05/2010; 138(5). DOI:10.1016/S0016-5085(10)63447-6 · 13.93 Impact Factor
  • Gastroenterology 05/2010; 138(5). DOI:10.1016/S0016-5085(10)63551-2 · 13.93 Impact Factor
  • Gastroenterology 05/2010; 138(5). DOI:10.1016/S0016-5085(10)63457-9 · 13.93 Impact Factor
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    ABSTRACT: There is a growing interest in methane and its association with constipation in functional bowel disease. Neomycin-based treatment of methane-positive subjects has resulted in improvement of constipation. Rifaximin, although superior for the treatment of irritable bowel syndrome compared with other antibiotics, seems less effective in methane-positive subjects. In this study, we evaluate 3 different antibiotic treatments in patients who have a methane-positive breath test: rifaximin only, neomycin only, and the combination of neomycin and rifaximin. A retrospective chart review was conducted on patients with methane on their lactulose breath test (> or =3 ppm of methane) who received one of the following antibiotic treatments: 500 mg b.i.d. for 10 days of neomycin alone, 400 mg t.i.d. for 10 days of rifaximin alone, or a combination of both rifaximin and neomycin for 10 days. All patients must have received antibiotic treatment after their initial consultation at the medical center and, in addition, had at least 1 follow-up to evaluate the effects of the treatment. After inclusion/exclusion criteria were met, all charts were evaluated to determine if the subject was a responder to the antibiotic therapy. This included clinical symptom improvement and eradication of methane on their breath test. Of the subjects receiving the treatment of rifaximin and neomycin (n=27), 85% had a clinical response, compared with 63% of subjects in the neomycin only group (n=8) (P=0.15) and 56% of subjects in the rifaximin only group (n=39) (P=0.01). When comparing the neomycin group with the rifaximin group, the difference was nonsignificant. When evaluating methane eradication results, 87% of subjects taking the rifaximin and neomycin combination eradicated the methane on their breath test. This is compared with 33% of subjects in the neomycin group that eradicated the methane (P=0.001), and only 28% of subjects in the rifaximin group (P=0.001). Of the patients who did not eliminate the methane with only rifaximin treatment, 66% of those who subsequently used the rifaximin and neomycin treatment were able to normalize their breath test. The combination of rifaximin and neomycin is more effective in treating methane-producing subjects-in both clinical response and methane elimination.
    Journal of clinical gastroenterology 12/2009; 44(8):547-50. DOI:10.1097/MCG.0b013e3181c64c90 · 3.19 Impact Factor