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Background: Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease (or paratuberculosis), a chronic wasting disease of ruminants and other animals resulting from granulomatous enteritis. There are increasing concerns that MAP is zoonotic. The prevalence of Johne's disease is increasing worldwide. In an attempt to control an epidemic of ovine Johne's disease (OJD) in New South Wales (NSW), a government/industry sponsored voluntary vaccination/on-farm management program commenced in 2000. We report herein an observational study of changes in disease prevalence as vaccination progressed, based on abattoir surveillance data for OJD from 1999 to 2009. We also discuss the epidemiological, policy, regulatory, research, economic and sociological elements that contributed to the development of a mature control program, whose aim was to halt the epidemic spread of OJD in a naïve sheep population., Methods: NSW was divided into areas of "High" (HPA), "Medium" (MPA) and "Low" (LPA) OJD prevalence. A killed whole cell vaccine (Gudair®) was administered to sheep from 2000 to 2009. Trained examiners evaluated the viscera of adult sheep carcasses at slaughter for gross evidence of OJD. MAP infection was confirmed by histopathology., Principal Findings: From 2000-2009, 12 million vaccine doses were administered in NSW (91%; 10.9 million in the HPA). Many of the vaccinated flocks were suffering > 5% annual mortality in adult sheep, with some individual flocks with 10-15% losses attributable to OJD. A total of 7.6 million carcasses were examined (38%; 2.9 million from the HPA). Overall, 16% of slaughter consignments (sheep consigned to the abattoir from a single vendor) were positive for OJD, of which 94% were from the HPA. In the HPA, the percentage of animals with lesions attributable to OJD at slaughter fell progressively from 2.4% (10,406/432,860) at commencement of vaccination in 2000 to 0.8% (1,573/189,564) by 2009. Herd immunity from vaccination in the HPA was estimated at 70% by 2009, the target commonly espoused for an effective control program based on vaccination. This coincided with a progressive decrease in reports of clinical disease and mortalities in vaccinated flocks., Significance: We show a decrease in the prevalence of lesions attributable to OJD in NSW concomitant with initiation of voluntary vaccination, on-farm management plans, abattoir monitoring and feedback of animal prevalence data to sheep producers. We conclude that a target of ≤ 1% regional prevalence of OJD affected sheep at slaughter is achievable using these interventions., Competing Interests: The authors have read the journal’s policy, and the authors of the manuscript have the following competing interests to declare: CSL Limited and Pfizer Animal Health provided material support in the form of vaccine supplies; sales data: and strong promotion of safe vaccination procedures. The following three relevant patents have been issued to RG: US Patent # 7,846,420: Issue Date Dec 7, 2010. Mycobacterium Avium Subspecies Paratuberculosis Vaccines and Methods for Using the Same. US Continuation-in-part Application entitled “Combination Vaccines Against Mycobacterium Species and Methods of Using Same.” Serial #12/956,064 Filing Date; November 30, 2010. Issued: June 18, 2013. US Patent # 7,902,350: Issue Date March 8, 2011. Method for Monitoring the Efficacy of a Mycobacterium Avium Subspecies Paratuberculosis Therapy. US Patent # 8,507,251: Issue Date August 13, 2013 “Medium and Method for Culturing Mycobacterium Avium Subspecies Paratuberculosis” Serial No: 12/892,039 Filing Date September 28, 2010. This does not alter any of our adherence to all the PLoS ONE policies on sharing data and materials. There are no other patents, products in development or marketed products associated with this research to declare. IL, LD, ME, and LK have no competing interests to declare.

Background: Mycobacterium avium subspecies paratuberculosis (MAP), the cause of Johne disease, is a slow growing mycobacterium. Viable MAP detection is difficult, inconstant and time-consuming. The purpose of this study was to compare a rapid phage/qPCR assay performed on peripheral blood mononuclear cells (PBMCs) with three standard methods of MAP detection: fecal MAP PCR; plasma antigen-specific IFN-γ & serum MAP ELISA hypothesizing that, if sensitive and specific, Johne animals would be positive and Control animals negative. We studied a well characterized herd of Holstein cattle that were naturally infected with MAP and their Controls., Results: With phage/qPCR 72% (23/32) of Johne and 35% (6/17) of Controls were MAP positive. With fecal PCR 75% (24/32) of Johne and 0% (0/17) of Controls were MAP positive. With plasma antigen-specific IFN-γ 69% (22/32) of Johne and 12% (2/17) of Controls were MAP positive. With serum MAP ELISA, 31% (10/32) of Johne and 0% (0/17) of Controls were MAP positive. When phage / qPCR and fecal PCR results were combined, 100% (32/32) Johne and 35% (6/17) of Control animals were MAP positive. Younger Control animals (1-3 years) had significantly fewer plaques (25 ± 17 SEM) than older Controls (4-12 years) (309 ± 134 p = 0.04). The same trend was not observed in the Johne animals (p = 0.19)., Conclusions: In contrast to our hypothesis, using the phage/qPCR assay we find that viable circulating MAP can rapidly be detected from the blood of animals infected with, as well as those in the Control group evidently colonized by MAP. These data indicate that the presence of viable MAP in blood does not necessarily signify that an animal must of necessity be demonstrably ill or be MAP positive by standard diagnostic methods.

Objectives: Although controversial, there is increasing concern that Crohn's disease may be a zoonotic infectious disease consequent to a mycobacterial infection. The most plausible candidate is M. avium subspecies paratuberculosis (MAP) that is unequivocally responsible for Johne's disease in ruminants. The purpose of this study was to evaluate a proprietary (Affymetrix™ RNA view ® ) fluorescent in situ hybridization (FISH) assay for MAP RNA. Non-identifiable intestine from patients with documented Crohn's disease was assayed according to the manufacturer's instructions and with suggested modifications. Probes were custom designed for MAP and human β-actin (as the eukaryotic housekeeping gene) from published genomes., Results: Repetitively, false positive signal was observed in our "No-Probe" negative control. Attempts were made to correct this according to the manufacturer's suggestions (by modifying wash solutions, using recommended hydrochloric acid titration and different fluorescent filters). None prevented false positive signal in the "No-Probe" control. It is concluded that when performed according to manufactures instruction and with multiple variations on the manufactures recommended suggestions to correct for false positive signal, that the Affymetrix™ RNA view ® cannot be used to detect MAP in pre-frozen resected intestine of humans with Crohn's disease.

Objectives: Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants. The "gold standard" of MAP detection is by culture, DNA sequencing possibly supplemented by identification of Ziehl-Neelsen positive mycobacteria. The purpose of this study was to evaluate a proprietary (Affymetrix™ RNA view ® ) fluorescent in situ hybridization (FISH) assay for MAP RNA. Intestine from a steer with documented Johne's disease was assayed according to the manufacturer's instructions. Probes were custom designed for MAP and bovine β-actin (as the eukaryotic housekeeping gene) from published genomes. We attempt to prevent false positive signal in the "no-probe" control, by modifying wash solutions, using recommended hydrochloric acid titration and different fluorescent filters (TritC for Texas Red and "Hope" for Cy-5)., Results: Repetitively, false positive signal was observed in our "no probe" negative control. Attempts to correct this according to the manufacturers suggestions, and with multiple derivative techniques have been unsuccessful. It is concluded that when performed according to manufactures instruction and with multiple variations on the manufactures recommended suggestions to correct for false positive signal, that the Affymetrix™ RNA view ® cannot be used to detect MAP in pre-frozen intestine of cattle with Johne's disease.

Background: The development of novel antibiotics to treat multidrug-resistant (MDR) tuberculosis is time-consuming and expensive. Multiple immune modulators, immune suppressants, anti-inflammatories, and growth enhancers, and vitamins A and D, inhibit Mycobacterium avium subspecies paratuberculosis (MAP) in culture. We studied the culture inhibition of Mycobacterium tuberculosis complex by these agents., Methods: Biosafety level two M. tuberculosis complex (ATCC 19015 and ATCC 25177) was studied in radiometric Bactec or MGIT culture. Agents evaluated included clofazimine, methotrexate, 6-mercaptopurine, cyclosporine A, rapamycin, tacrolimus, monensin, and vitamins A and D., Results: All the agents mentioned above caused dose-dependent inhibition of the M. tuberculosis complex. There was no inhibition by the anti-inflammatory 5-aminosalicylic acid, which causes bacteriostatic inhibition of MAP., Conclusions: We conclude that, at a minimum, studies with virulent M. tuberculosis are indicated with the agents mentioned above, as well as with the thioamide 5-propothiouricil, which has previously been shown to inhibit the M. tuberculosis complex in culture. Our data additionally emphasize the importance of vitamins A and D in treating mycobacterial diseases., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The role of vitamins in the combat of disease is usually conceptualized as acting by modulating the immune response of an infected, eukaryotic host. We hypothesized that some vitamins may directly influence the growth of prokaryotes, particularly mycobacteria., Methods: The effect of four fat-soluble vitamins was studied in radiometric Bactec® culture. The vitamins were A (including a precursor and three metabolites,) D, E and K. We evaluated eight strains of three mycobacterial species (four of M. avium subspecies paratuberculosis (MAP), two of M. avium and two of M. tb. complex)., Principal Findings: Vitamins A and D cause dose-dependent inhibition of all three mycobacterial species studied. Vitamin A is consistently more inhibitory than vitamin D. The vitamin A precursor, β-carotene, is not inhibitory, whereas three vitamin A metabolites cause inhibition. Vitamin K has no effect. Vitamin E causes negligible inhibition in a single strain., Significance: We show that vitamin A, its metabolites Retinyl acetate, Retinoic acid and 13-cis Retinoic acid and vitamin D directly inhibit mycobacterial growth in culture. These data are compatible with the hypothesis that complementing the immune response of multicellular organisms, vitamins A and D may have heretofore unproven, unrecognized, independent and probable synergistic, direct antimycobacterial inhibitory activity.

Background: Without known mechanisms of action, Crohn's disease is exacerbated, and ulcerative colitis is improved, by the use of tobacco. Mycobacterium avium subspecies paratuberculosis (MAP) may be zoonotic. We hypothesized that tobacco components might alter the growth kinetics of MAP, explaining these divergent clinical observations., Methods: The effect of nicotine, nicotinic acid, nicotinamide and α and β nicotinamide adenine dinucleotide (α and β NAD) were studied on eight strains of three mycobacterial species (MAP, M. avium and M. tb. complex). Data are obtained as "cumulative growth index," (cGI) and presented as "percent increase in cumulative GI" (% + ΔcGI)., Results: Nicotinic acid enhances the two human MAP isolates (Dominic; 225% + ΔcGI and UCF-4; 92% + ΔcGI) and M. avium (ATCC 25291; 175% + ΔcGI). Nicotinamide (at 6.4 µg/ml) enhances the human MAP isolates (Dominic; 156% + ΔcGI and UCF-4; 79% + ΔcGI) and M. avium (ATCC 25291; 144% + ΔcGI.) Both α and β NAD enhance Dominic; (135 and 150 % + ΔcGI) and UCF-4; (81 and 79% + ΔcGI). At the doses tested, nicotine has no effect on any strain studied., Conclusions: We show enhancement of MAP growth by nicotinic acid, one of ≥4,000 tobacco-related molecules, its amide, nicotinamide and α and β NAD. Pure nicotine has no enhancing effect at the doses studies.

Background: Thyrotoxicosis is conceptualized as an "autoimmune" disease with no accepted infectious etiology. There are increasingly compelling data that another "autoimmune" affliction, Crohn disease, may be caused by Mycobacterium avium subspecies paratuberculosis (MAP). Like M. tb, MAP is systemic. We hypothesized that some cases of thyrotoxicosis may be initiated by a MAP infection. Because other thioamides treat tuberculosis, leprosy and M. avium complex, we hypothesized that a mode of action of some thioamide anti-thyrotoxicosis medications may include MAP growth inhibition., Methods: The effect of the thioamides, thiourea, methimazole and 6-propo-2-thiouracil (6-PTU) were studied in radiometric Bactec culture, on ten strains of three mycobacterial species (six of MAP, two of M. avium and two of M. tb. complex). Data are presented as "cumulative growth index," (cGI) or "percent decrease in cumulative GI" (%-DeltacGI)., Principal Findings: Methimazole was the most effective thioamide at inhibiting MAP growth. At 128microg/ml: MAP UCF-4; 65%-DeltacGI & MAP ATCC 19698; 90%-DeltacGI. Thiourea inhibited MAP "Ben" maximally; 70%-DeltacGI. Neither methimazole nor thiourea inhibited M. avium or M. tb. at the doses tested. 6-PTU has no inhibition on any strain studied, although a structurally analogous control, 5-PTU, was the most inhibitory thioamide tested., Significance: We show inhibition of MAP growth by the thioamides, thiourea and methimazole in culture. These data are compatible with the hypothesis that these thioamides may have anti-prokaryotic in addition to their well-established eukaryotic actions in thyrotoxic individuals.

Background: Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants, a chronic enteritis evocative of human inflammatory bowel disease. In industrialized countries MAP has been cultured from pasteurized milk, compounding the increasing concern that MAP may be zoonotic. The purpose of this study was to evaluate commercially available unpasteurized and pasteurized milk and its products for the presence of viable MAP or MAP DNA from an area of northern India with a population of 150 million people., Methods: We studied 43 samples (16 unpasteurized, 27 pasteurized) purchased in Mathura, Agra, or New Delhi, for the presence of MAP by culture or by PCR for IS900 MAP DNA. Positives results were confirmed as MAP by restriction endonuclease analysis and/or DNA sequencing., Results: Colonies appeared in 1.5-20 months post-inoculation. Of the unpasteurized samples, 44% (7/16) were MAP culture-positive and 6% (1/16) were positive for IS900 MAP DNA. Of the pasteurized samples, 67% (18/27) were MAP culture-positive and 33% (9/27) were IS900-positive. Subsequently, 100% (25/25) of the cultured colonies were IS900 and IS1311 MAP DNA-positive., Conclusions: This is the first report from a developing country of MAP cultured from both pasteurized and unpasteurized milk and milk products. Thus we corroborate the presence of viable MAP in the food chain reported from industrialized countries. With the increasing concern that MAP may be zoonotic, these findings have major implications for healthcare in India. The decreased sensitivity in detecting MAP DNA by PCR directly from milk should be ascribed to our employing only one set of PCR primers., (Copyright 2009 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: Without known mechanisms of action, thalidomide is used to treat a variety of non-malignant 'idiopathic' diseases. There is increasing concern that Mycobacterium avium subspecies paratuberculosis (MAP) may be zoonotic. Recently, methotrexate, azathioprine, 6-mercaptopurine (6-MP), 5-aminosalicylic acid (5-ASA), cyclosporine A, rapamycin, and tacrolimus have been shown to inhibit MAP growth in culture, indicating that, unknowingly, MAP infections may have been treated for decades. We herein test the hypothesis that thalidomide may inhibit MAP growth., Methods: Using the radiometric 14CO2 (Bactec) system we quantified growth kinetics of thalidomide (+/-), (+), and (-) and two components for thalidomide, phthalimide and 1-hydroxypiperidine-2,6-dione (HPD). We studied four MAP strains (three human isolates, 'Ben', 'Dominic', and UCF-4, and a bovine MAP isolate 19698) and three mycobacterial controls (Mycobacterium avium and bacillus Calmette-Guérin (BCG)). Growth was quantified as growth index (GI) and inhibition as percent decrease in cumulative GI (%-DeltacGI)., Results: Phthalimide had no dose-dependent inhibition on any strain. Neither thalidomide nor HPD inhibited M. avium or BCG. MAP inhibition varied; at 64 microg/ml, amongst human isolates, Dominic was most susceptible: thalidomide (+)=58%-DeltacGI and HPD=46%-DeltacGI. UCF-4 was next: thalidomide (-)=37%-DeltacGI and HPD=40%-DeltacGI. Ben was least susceptible: HPD=24%-DeltacGI., Conclusions: We have shown, in culture, the heretofore-undescribed inhibition of MAP growth by thalidomide and its enantiomers. Phthalimide was found to have no anti-MAP activity, whereas HPD was found to inhibit MAP growth. These data are compatible with the hypothesis that thalidomide, like other 'anti-inflammatories' and 'immunomodulators' may act, in part, as an anti-MAP antibiotic.

Background: Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic wasting diarrheal disease in ruminants called Johne's disease, that is evocative of human inflammatory bowel disease (IBD). Agents used to treat IBD, called "anti-inflammatories", immuno-modulators" and "immuno-suppressants" inhibit MAP growth in culture. We concluded that, unknowingly, the medical profession has been treating MAP since sulfasalazine's introduction in 1942. Monensin, called a "Growth Enhancer" in cattle, ameliorates Johne's disease without a documented mechanism of action. We hypothesized that Monensin would inhibit MAP in culture., Methods: Using the radiometric 14CO2 Bactec system, that expresses mycobacterial growth in arbitrary growth index (GI) units, we studied the effect of Monensin on the growth kinetic of MAP isolated from humans with IBD ("Dominic", "Ben" & UCF-4) and cattle with Johne's disease (303 & ATCC 19698.) Results are expressed as percent inhibition of cumulative GI (%-Delta cGI)., Results: The positive control Clofazimine inhibits every strain tested. The negative controls Cycloheximide & Phthalimide, have no inhibition on any MAP strain. Monensin has dose dependent inhibition on every MAP strain tested. The most susceptible human isolate was UCF-4 (73% - Delta cGI at 1 microg/ml) and bovine isolate was 303 (73% - Delta cGI at 4 microg/ml.) Monensin additionally inhibits M. avium ATCC 25291 (87% - Delta cGI at 64 microg/ml) & BCG (92% - Delta cGI at 16 microg/ml)., Discussion: We show that in radiometric culture the "Growth Enhancer" Monensin causes dose dependent inhibition of mycobacteria including MAP. We posit that the "Growth Enhancer" effect of Monensin may, at least in part, be due to inhibition of MAP in clinical or sub-clinical Johne's disease.

Background: Mycobacteria, such as M. leprae and M. tuberculosis infect billions of humans. However, because of appropriate immune responses and antibiotic therapy, overt mycobacterial diseases occur far less frequently. M. avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants, an affliction evocative of inflammatory bowel disease (IBD). Several agents used to treat IBD (5-ASA, methotrexate, azathioprine and its metabolite 6-MP) have recently been shown to be antiMAP antibiotics. We herein evaluate the prevalence of MAP DNA in healthy individuals and compare them with IBD patients on antiMAP antibiotics., Methods: We studied 100 healthy individuals (90 blood donors) and 246 patients with IBD. IS900 MAP DNA was identified using a nested primer PCR in the buffy coat of blood. Positive signal was confirmed as MAP by DNA sequence analysis. PCR positive results frequencies were compared according to medications used. Significance was accepted at p<0.05., Results: 47% (47/100) healthy controls and 16% (40/246) IBD patients were IS900 positive (p<0.0001). MAP DNA was identified in 17% of 143 patients receiving mesalamine and 6% of 16 receiving sulfasalazine. None of the IBD patients receiving methotrexate (n = 9), 6-MP (n = 3), ciprofloxacin (n = 5) or Tacrolimus (n = 3) had MAP DNA detectable in their blood., Discussion: We found a disquietingly large percentage of healthy individuals have MAP DNA in their blood, the significance of which remains to be determined. Counter-intuitively, the incidence of MAP DNA was significantly lower in patients with IBD. Agents with the most potent in vitro antiMAP activity were associated with clearance of blood MAP DNA. We posit that the use antiMAP antibiotics was responsible for the decreased prevalence of MAP DNA in patients with IBD.

Background: Mycobacterium avium subspecies paratuberculosis (MAP) may be zoonotic. Recently the "immuno-modulators" methotrexate, azathioprine and 6-MP and the "anti-inflammatory" 5-ASA have been shown to inhibit MAP growth in vitro. We concluded that their most plausible mechanism of action is as antiMAP antibiotics. The "immunosuppressants" Cyclosporine A, Rapamycin and Tacrolimus (FK 506) treat a variety of "autoimmune" and "inflammatory" diseases. Rapamycin and Tacrolimus are macrolides. We hypothesized that their mode of action may simply be to inhibit MAP growth., Methodology: The effect on radiometric MAP (14)CO(2) growth kinetics of Cyclosporine A, Rapamycin and Tacrolimus on MAP cultured from humans (Dominic & UCF 4) or ruminants (ATCC 19698 & 303) and M. avium subspecies avium (ATCC 25291 & 101) are presented as "percent decrease in cumulative GI" (%-DeltacGI.), Principal Findings: The positive control clofazimine has 99%-DeltacGI at 0.5 microg/ml (Dominic). Phthalimide, a negative control has no dose dependent inhibition on any strain. Against MAP there is dose dependent inhibition by the immunosuppressants. Cyclosporine has 97%-DeltacGI by 32 microg/ml (Dominic), Rapamycin has 74%-DeltacGI by 64 microg/ml (UCF 4) and Tacrolimus 43%-DeltacGI by 64 microg/ml (UCF 4), Conclusions: We show heretofore-undescribed inhibition of MAP growth in vitro by "immunosuppressants;" the cyclic undecapeptide Cyclosporine A, and the macrolides Rapamycin and Tacrolimus. These data are compatible with our thesis that, unknowingly, the medical profession has been treating MAP infections since 1942 when 5-ASA and subsequently azathioprine, 6-MP and methotrexate were introduced in the therapy of some "autoimmune" and "inflammatory" diseases.

Background: Morbid obesity results in multiple comorbidities and an increased mortality rate. The National Institutes of Health has stated that surgery is the most effective long-term therapy; therefore, we evaluated a laparoscopically implantable adjustable gastric band., Methods: We reviewed 2 multicenter prospective, open-label, single-arm surgical trials--trial A (3 years) and trial B (1 year)--with ongoing safety follow-up. These trials were conducted in United States community and university hospitals (trial A = 8 sites and trial B = 12 sites). Trial A comprised 292 subjects (mean +/- SD preoperative weight: 133 kg +/- 24.4), and trial B comprised 193 subjects (129 kg +/- 20.8). Intervention included placement of a constrictive, adjustable band around the upper stomach to limit food intake and induce weight loss. Main outcome measures were the primary efficacy end point of weight loss. Secondary end-points were change in quality-of-life, safety parameters, and complications, including band slippage, reoperation, and device explantation., Results: In the 2 trials, 485 devices were implanted (92% laparoscopically), and no deaths occurred. Of the patients in trial A, 206 (70.5%) completed the 3-year follow-up, and 142 (73.6%) of patients in trial B completed the 1-year follow-up. Weight-loss results, using the last value carried forward, for all 292 patients in trial A and all 193 patients in trial B demonstrated a change in mean body mass index (kg/m2) +/- SD from 47.4 +/- 7.0 to 39.0 +/- 7.3 in trial A and from 46.7 +/- 7.8 to 38.4 +/- 7.6 in trial B subjects at 1 year (P < .001 for both trials A and B), with minimal further change at 3 years (39.0 +/- 8.5) in trial A subjects. The percentage of initial body weight lost at 1 year was 17.7% +/- 9.4% for trial A subjects and 18.2% +/- 8.9% for trial B subjects, whereas the 3-year total for trial A subjects was 18.3% +/- 13.1%. At 1 year, 76% of patients in trial A and 66% of patients in trial B had complications, mostly related to upper gastrointestinal symptoms. By 9 years after surgery, 33% (96 of 292) of trial A subjects had their devices explanted because of complications or inadequate weight loss., Conclusions: These first-generation implantable adjustable gastric band results suggest that this is a viable bariatric surgery therapeutic option for the treatment of obesity.

Background: Introduced in 1942, sulfasalazine (a conjugate of 5-aminosalicylic acid (5-ASA) and sulfapyridine) is the most prescribed medication used to treat "inflammatory" bowel disease (IBD.) Although controversial, there are increasingly compelling data that Mycobacterium avium subspecies paratuberculosis (MAP) may be an etiological agent in some or all of IBD. We have shown that two other agents used in the therapy of IBD (methotrexate and 6-MP) profoundly inhibit MAP growth. We concluded that their most plausible mechanism of action is as antiMAP antibiotics. We herein hypothesize that the mechanism of action of 5-ASA and/or sulfapyridine may also simply be to inhibit MAP growth., Methodology: The effect on MAP growth kinetics by sulfasalazine and its components were evaluated in bacterial culture of two strains each of MAP and M. avium, using a radiometric ((14)CO(2) BACTEC(R)) detection system that quantifies mycobacterial growth as arbitrary "growth index units" (GI). Efficacy data are presented as "percent decrease in cumulative GI" (%-DeltacGI)., Principal Findings: There are disparate responses to 5-ASA and sulfapyridine in the two subspecies. Against MAP, 5-ASA is inhibitory in a dose-dependent manner (MAP ATCC 19698 46%-DeltacGI at 64 microg/ml), whereas sulfapyridine has virtually no effect. In contrast, against M. avium ATCC 25291, 5-ASA has no effect, whereas sulfapyridine (88%-DeltacGI at 4 microg/ml) is as effective as methotrexate, our positive control (88%-DeltacGI at 4 microg/ml)., Conclusions: 5-ASA inhibits MAP growth in culture. We posit that, unknowingly, the medical profession has been treating MAP infections since sulfasalazine's introduction in 1942. These observations may explain, in part, why MAP has not previously been identified as a human pathogen. We conclude that henceforth in clinical trials evaluating antiMAP agents in IBD, if considered ethical, the use of 5-ASA (as well as methotrexate and 6-MP) should be excluded from control groups.

Background: Clinical improvement in inflammatory bowel disease (IBD) treated with methotrexate and 6-mercaptopurine (6-MP) is associated with a decrease in pro-inflammatory cytokines. This has been presumed to indicate the mechanism of action of methotrexate and 6-MP. Although controversial, there are increasingly compelling data that Mycobacterium avium subspecies paratuberculosis (MAP) may be an etiological agent in some or all of IBD. We hypothesized that the clinical efficacy of methotrexate and 6-MP in IBD may be to simply inhibit the growth of MAP., Methodology: The effect on MAP growth kinetics by methotrexate and 6-MP were evaluated in cell culture of two strains each of MAP and M. avium using a radiometric ((14)CO(2) BACTEC detection system that quantifies mycobacterial growth as arbitrary "growth index units" (GI). Efficacy data are presented as "percent decrease in cumulative GI" (% -DeltacGI)., Principal Findings: The positive control antibiotic (clarithromycin) has >or=85% -DeltacGI at a concentration of 0.5 microg/ml. The negative control (ampicillin) has minimal inhibition at 64 microg/ml. MAP ATCC 19698 shows >or=80% -DeltacGI for both agents by 4 microg/ml. With the other three isolates, although more effective than ampicillin, 6-MP is consistently less effective than methotrexate., Conclusions: We show that methotrexate and 6-MP inhibit MAP growth in vitro. Each of the four isolates manifests different % -DeltacGI. These data are compatible with the hypothesis that the clinical improvement in patients with IBD treated with methotrexate and 6-MP could be due to treating a MAP infection. The decrease in pro-inflammatory cytokines, thought to be the primary mechanism of action, may simply be a normal, secondary, physiological response. We conclude that henceforth, in clinical studies that evaluate the effect of anti-MAP agents in IBD, the use of methotrexate and 6-MP should be excluded from any control groups.

Objective: To establish a database that permits description and analysis of the evolving role, patterns of use, and costs of critical care medicine (CCM) in the United States from 1985 to 2000., Design: Retrospective study combining data from federal (Hospital Cost Report Information System, Center for Medicare and Medicaid Services, Baltimore, MD) and private (Hospital Statistics, American Hospital Association, Chicago, IL) databases to analyze U.S. hospitals, hospital and CCM beds, and occupancy. CCM costs were calculated by the Russell equation and compared with national health care and financial indexes., Setting: Nonfederal, acute care hospitals with CCM units in the United States., Subjects: None., Interventions: None., Measurements and Main Results: We analyzed hospitals with CCM units and focused on hospital and CCM beds, CCM occupancy, and CCM costs. CCM costs were compared with national cost indexes. Between 1985 and 2000, the total number of U.S. hospitals decreased by 8.9% (6,032 to 5,494) and acute care hospitals offering CCM decreased by 13.7% (4,150 to 3,581). The total number of beds in hospitals with CCM units decreased by 26.4% (889,600 to 654,400). In contrast, CCM beds increased by 26.2% (69,300 to 87,400). CCM occupancy was constant at 65%. CCM bed costs per day increased by 126% (1,185 to 2,674 US dollars). Although CCM costs increased by 190.4% (19.1 billion to 55.5 billion US dollars), the proportion of national health expenditures allocated to CCM decreased by 5.4%. In 2000, CCM costs represented 13.3% of hospital costs, 4.2% of national health expenditures, and 0.56% of the gross domestic product., Conclusions: CCM is increasingly used and prominent in a shrinking U.S. hospital system. CCM occupancy is lower than expected. Despite its increasing use and cost, CCM is using proportionally less of national health expenses and the gross domestic product than previously estimated.

Although Crohn's disease is considered to be autoimmune in origin, there is increasing evidence that it may have an infectious cause. The most plausible candidate is Mycobacterium avium subspecies paratuberculosis (MAP). Intriguingly, Koch's postulates may have been fulfilled for MAP and Crohn's disease, even though they still have not been met for Mycobacterium leprae and leprosy. In animals MAP causes Johne's disease, a chronic wasting intestinal diarrhoeal disease evocative of Crohn's disease. Johne's disease occurs in wild and domesticated animals, including dairy herds. Viable MAP is found in human and cow milk, and is not reliably killed by standard pasteurisation. MAP is ubiquitous in the environment including in potable water. Since cell-wall-deficient MAP usually cannot be identified by Ziehl-Neelsen staining, identification of MAP in human beings requires culture or detection of MAP DNA or RNA. If infectious in origin, Crohn's disease should be curable with appropriate antibiotics. Many studies that argue against a causative role for MAP in Crohn's disease have used antibiotics that are inactive against MAP. However, trials that include macrolide antibiotics indicate that a cure for Crohn's disease is possible. The necessary length of therapy remains to be determined. Mycobacterial diseases have protean clinical manifestations, as does Crohn's disease. The necessity of stratifying Crohn's disease into two clinical manifestations (perforating and non-perforating) when interpreting the results of antibiotic therapy is discussed. Rational studies to evaluate appropriate therapies to cure Crohn's disease are proposed.

In this study we evaluate both proximal and more distal transcriptional regulation of the 5' flanking region of the rat cholecystokinin gene in transfected GH3 (rat pituitary tumor) cells. Transcriptional activity was measured on the intact (-400 to +73) 5' flanking region of cholecystokinin (CCK), as well as with DNA constructs, which were deleted in both the conventional 5' to 3', as well as an unconventional 3' to 5' direction. Our in vivo studies indicate complex phorbol ester and forskolin interactions in the 10-base pair region between -130 and -140. We conclude, there are at least two transcriptional factors involved in regulation of the rat CCK transcription in this region. In vitro studies utilizing heterologous nuclear (HeLa) extract, as well as purified transcription factors AP-2 and NF-kappa B, identify overlapped AP-2- and NF-kappa B-responsive elements within the 17-base pair sequence between -149 and -134 of the distal 5' flanking region. In this region complex transcriptional regulation occurs, which indicates inhibition of AP-2 CCK promoter complexing by NF-kappa B. Six-point mutations introduced into this sequence prevent AP-2 and NF-kappa B binding to CCK promoter, as well as its transcriptional activation by phorbol ester and forskolin in GH3 cells.

Background: Cationic liposomes may be used in gene transfer. However, different liposome configurations have varying efficiency in different tissues., Aims: To compare multiple lipids during gene transfer into the intestinal mucosa, liver and central nervous system in the adult rat. We evaluate different lipid aliphatic and polar head domains., Materials and Methods: Nine cationic or neutral phospholipids, combined with a cationic cholesterol derivative, have been compared to Lipofectin. Transfection was into GH3 cells and the adult rat brain, liver or intestinal mucosa. Results Optimum DNA:lipid ratio was lowest (1:2) in the intestinal mucosa and highest in GH3 cells (1:40). Lipofectin ", was most effective in brain and GH3 cells but had no activity in intestinal mucosa. Saturated cationic lipids transfect differently in GH3 cells and GI mucosa than in liver and brain. However, with saturated neutral phospholipids, GH3 cells, intestinal mucosa and liver transfect similarly. DOTAP the longest unsaturated cationic lipid (18:1) was most effective in the intestine, whereas DMEPC the shortest saturated neutral lipid (14:0) was optimal in the liver., Conclusions: In this study we propose a rational approach, based on systematic variations of lipids, to optimize liposome mediated gene transfer into the ventricular system of the brain, the liver and gastro-intestinal tract in the adult rat. Additionally, we demonstrate the feasibility of gene transfer into the mucosal cells of the gastro-intestinal tract as well as throughout the ventricular system of the rat brain. This requires liposomes which contain a cationic cholesterol derivative.

Liposome mediated gene transfer has a great potential in gene therapy. In this review we discuss the physical and chemical properties of cationic liposomes that affect their abilities to mediate gene transfer into eukaryotic cells. The specific focus is on functional domains of cationic lipids. We address polar head variations, counterions, linker bonds, acyl chain variations, as well as composition of liposomes. We additionally discuss different functional groups of lipids affecting lipid bilayer packing, lipid association with DNA, fusion with the cellular membranes and the release of transferred DNA from endosomes into the cytoplasm.

Background: The purpose of this study was to assess factors of clinical importance in morbidly obese patients having a laparoscopically adjustable gastric band (LAP-BAND) implanted in order to achieve weight loss., Methods: Preoperative evaluation of hiatus hernia and esophageal (dys)motility were compared with the need for reoperation. Results are presented for the first 50 consecutive patients entered., Results: Nine of the first 50 patients required reoperation (18%). Five (10%) were for LAP-BAND slippage on the stomach. Of these five, reoperation was required in four of 12 (33%) with hiatus hernia (P = 0.0093); three of nine (33%) with a motility disorder (P = 0.025); and three of six (50%) with both hiatus hernia and a motility disorder (P = 0.0076)., Conclusions: We identify two factors, hiatus hernia and esophageal dysmotility, which are associated, both independently as well as in combination, with reoperation for LAP-BAND slippage. Both patients and their physicians should consider these data when considering the LAP-BAND as possible therapy for morbid obesity.

Brain/gut cholecystokinin (CCK) has well-established translational and post-translational differences. By contrast, CCK transcription regulation is reported to be the same in brain and gut. Accordingly, the rat CCK gene has been evaluated for differential brain/gut transcription initiation. Using the 5'-flanking region of the rat CCK gene, DNase I protection assays were performed. We evaluated reporter constructs deleted of the conventional transcription start site in cell culture. Finally, brain and gut mRNA was evaluated using both a reverse transcription serial primer polymerase chain reaction assay as well as rapid amplification of cDNA ends (RACE) analysis. TFIID protein protects against DNase I digestion between -177 and -196 bp. In tissue culture, spontaneous 5'-flanking region transcription initiation occurs until deletion proceeds upstream of -140 bp. RACE analysis performed on mRNA from the rat brain identifies heretofore undescribed transcription initiation at -43 bp 5' as well as at +1,212 (in exon II). These alternative transcription initiation sites are utilized in brain, but not gut. The rat CCK gene has alternative 5'-flanking region transcription initiation sites. These alternative sites are utilized only in the brain. These data may provide insights into how brain and gut respond to their differing physiological demands.

Crohn disease (CD) is a chronic, panenteric intestinal inflammatory disease. Its etiology is unknown. Analogous to the tuberculoid and lepromatous forms of leprosy, CD may have two clinical manifestations. One is aggressive and fistulizing (perforating), and the other is contained, indolent, and obstructive (nonperforating) [Gi]-berts, E. C. A. M., Greenstein, A. J., Katsel, P., Harpaz, N. & Greenstein, R. J. (1994) Proc. Natl. Acad. Sci. USA 91, 12721-127241. The etiology, if infections, may be due to Mycobacterium paratuberculosis. We employed reverse transcription PCR using M. paratuberculosis subspecies-specific primers (IS 900) on total RNA from 12 ileal mucosal specimens (CD, n = 8; controls, n = 4, 2 with ulcerative colitis and 2 with colonic cancer). As a negative control, we used Myobacterium avium DNA, originally cultured from the drinking water of a major city in the United States. cDNA sequence analysis shows that all eight cases of Crohn's disease and both samples from the patients with ulcerative colitis contained M. paratuberculosis RNA. Additionally, the M. avium control has the DNA sequence of M. paratuberculosis. We demonstrate the DNA sequence of M. paratuberculosis from mucosal specimens from humans with CD. The potable water supply may be a reservoir of infection. Although M. paratuberculosis signal in CD has been previously reported, a cause and effect relationship has not been established. In part, this is due to conflicting data from studies with empirical antimycobacterial therapy. We conclude that clinical trials with anti-M. paratuberculosis therapy are indicated in patients with CD who have been stratified into the aggressive (perforating) and contained (nonperforating) forms.

Objective: To evaluate acid-base and electrolyte changes in high volume cell saver autologous blood transfusion when normal saline (0.9% NaCl) is used as the wash solution., Design: Open-label study., Materials and Methods: Nine anesthetized and anticoagulated mongrel dogs underwent 15 cycles of cell saver autologous blood transfusion. Eight percent of the circulating blood volume (125 mL) was withdrawn, washed with normal saline, and retransfused for each cycle., Measurements and Main Results: Analyses of acid-base, electrolyte, and hematologic parameters were performed on both systemic and the washed blood. The washed blood had increased levels of sodium and chloride. There were decreased levels in pH, Pco2, total CO2 (bicarbonate), lactic acid, potassium, total and ionized calcium, magnesium, inorganic phosphorus, total protein, and albumin. Systemically, in the animals, by the end of the study, there were significant increases in the levels of chloride, inorganic phosphorus, hemoglobin, and hematocrit and significant decreases in the levels of pH, total CO2, total and ionized calcium, magnesium, total protein, and albumin., Conclusions: Acid-base, electrolyte, and hematologic changes occur when normal saline is used as the wash solution in high volume cell saver autologous blood transfusion. The washed blood with its elevation of sodium and chloride appears to reflect the constituents of the wash solution, normal saline. The depletion in the washed blood of PCO2, total CO2, potassium, total calcium, ionized calcium, magnesium, phosphorus, total protein, and albumin we feel is because of the absence of these electrolytes in the wash solution and their physical removal during salvaged blood separation and washing. The systemic acid-base and electrolyte changes primarily reflect the electrolyte pattern of the reinfused washed blood except for inorganic phosphorus. Inorganic phosphorus was maintained systemically, despite its wash out in the cell salvage process. This paradoxical finding may be caused by intracellular to extracellular inorganic phosphorus flux caused by the progressive systemic metabolic acidosis.

Crohn's disease is an idiopathic chronic panenteric intestinal inflammatory disease. Data concerning the pathogenesis of, and the immune responses occurring in, Crohn's disease are often conflicting. Current therapy is empirical and either non-specifically immunosuppressive or surgically ablative in nature. Although controversial, Crohn's disease may be thought of as having two different presentations, an aggressive fistulizing form and an indolent obstructive form. This is analogous to the tuberculoid and lepromatous manifestations of leprosy. If correct, this subclassification may provide key insights into the pathogenesis and differing host immune responses in Crohn's disease and also allow the development of more rational therapies.

BACKGROUND: There is a paucity of information about adolescent morbid obesity and bariatric surgery. METHODS: We interviewed 78% of an adolescent (

Recent epidemiological evidence suggests that there are two forms of Crohn disease (CD): perforating and nonperforating. We hypothesized that, just as with tuberculoid and lepromatous leprosy, differences in the two forms of CD would be both identified and determined by differences in the host immune response. Resected intestinal tissue from control patients as well as perforating and nonperforating CD patients was evaluated for mRNA levels. We employed 32P PCR amplification with published or custom-designed primers of a housekeeping gene (beta-actin); a human T-cell marker (CD3-delta); and the cytokines tumor necrosis factor alpha, transforming growth factor beta, granulocyte/macrophage colony-stimulating factor, interleukin (IL) 1 beta, IL-1ra, and IL-6. Differences were identified with IL-1 beta (control = 162 +/- 57 vs. perforating = 464 +/- 154 vs. nonperforating = 12,582 +/- 4733; P < or = 0.02) and IL-1ra (control = 1337 +/- 622 vs. perforating = 2194 +/- 775 vs. nonperforating = 9715 +/- 2988; P < or = 0.02). These data corroborate the epidemiological observation that there are two forms of CD. Nonperforating CD, the more benign form, is associated with increased IL-1 beta and IL-1ra mRNA expression. We conclude that it is the host immune response that determines which form of CD becomes manifest in any given individual and discuss the investigative, diagnostic, and therapeutic implications of these observations.

Weight loss and psychosocial events have been compared between low calorie conventional diet (n = 11) or following obesity surgery (n = 17). Interviews were >/= 9 months following initiation of treatment. After surgery significantly less hunger was experienced (surgery 76% [13/17] vs diet 18% [2/11] p < 0.01) and less will-power was required to stop eating (surgery 88% [15/17] vs diet 27% [3/11] p < 0.001). More dieters stopped eating because of 'figure and health' (surgery 12 % [2/17] vs diet 64 % [7/11 ] p < 0.01) whereas postoperative patients stopped due to vomit avoidance (surgery 53% [9/17] vs diet 0% [0/11] p /= 9 months following surgery or beginning a conventional diet, the morbidly obese have a more positive response to surgery.

The relevance of a post-gastroplasty physician-and dietitian-supervised support group for weight loss, dietary habits, dietary knowledge, and exercise has been assessed. Two post vertical banded gastroplasty groups were compared: those who did (n = 10) and those who did not (n = 11) attend a monthly post-gastroplasty support session. A 55-point questionnaire plus a 24-hour dietary recall and food frequency analysis were employed. Addressed were dietary and exercise history, eating habits, and factual dietary knowledge. At the time following surgery of this study (support: mean 29.7 vs no support: mean 34.9 months, NS), there was no difference in weight loss between the two groups. There was no difference in the two groups between preoperative, length of time from surgery to interview or weight at Interview. Those who attended the support group consumed significantly fewer calories (1559 +/- 320 vs 2278 +/- 271, p

Psychosocial and dietary habits have been compared in patients considering (Pre n = 33) or who had (Post n = 32) surgery for morbid obesity. Failure of conventional diets was attributed to lack of self-discipline (Pre 92% [22/24] vs. Post 87% [20/23], NS). Consumption of fast foods fell (Pre 70% [23/33] vs. Post 16% [5/32], p < 0.0001). Preoperative patients had unrealistic social expectations. They exaggerated the prospect of improved friendship (Pre 67% [22/32] vs. Post 34% [11/21], p < 0.05), erroneously anticipated better sex (Pre 78% [25/32] vs. Post 50% [15/30], p < or = 0.05), predicted better acceptance at work (Pre 85% [23/27] vs. Post 50% [15/30], p < or = 0.05), and misanticipated improved relationship with their partner (Pre 77% [20/26] vs. Post [47% 8/17], p < or = 0.05). Two factors predicted becoming employed following surgery: age (became employed [n = 5] 28 +/- 2 years vs. remained unemployed [n = 12] 44 +/- 4 years, p < 0.05) and percentage of excess weight lost (became employed 76 +/- 11 vs. remained unemployed 51 +/- 7, p < 0.05). The free support group was "useful" (17/17), yet only 5% attended regularly. Patients considering obesity surgery had specific unrealistic psychosocial expectations. They infrequently availed themselves of postoperative professional help. We identify the features associated with gaining employment.

This is a retrospective review of 260 consecutive patients, of one surgeon, operated on for morbid obesity. There were 245 primary, and 15 referred secondary, patients. For primary cases, there was a 0.8% (2/245) mortality, and 4.4% (11/245) emergency reexploration rate. Of these 2.5% (6/245) were at < 30 days (all < 5 days) and 1.6% (41245) > 30 days (all > 2 years). Ten (4.1 %) were electively reexplored because of vertical partition staple-line disruptions. Of the three staplers used, we show one (USS Corp. TA 90B) has demonstrably fewer disruptions. Identifiable causes of long-term morbidity include cigarette smoking in young females (2/245), a full set of upper dentures (2/245) and preexisting esophageal dysmotility (1/245).

The prototypical brain/gut peptide cholecystokinin (CCK) has been used to assess brain and gut repair kinetics following cytotoxic injury in the rat. Studies addressed the effect of repetitive injury as well as aging. Injury was induced by one of the two alkylating agents, one active in the brain, the other systematically. Consistently the responses differ between brain and intestine. Total RNA falls (as predicted) in the intestine (control 1.5 +/- 1.4 versus cytotoxic 0.21 +/- 0.06 tRNA mg/organ, P < or = 0.0001), but rises (unexpectedly) in the brain (control 0.79 +/- 0.04 versus cytotoxic 1.02 +/- 0.03 tRNA mg/organ, P < or = 0.001). CCK mRNA concentration falls in the brain (predicted) (control 27 +/- 1 versus cytotoxic 11 +/- 1 pg CCK mRNA/micrograms tRNA, P < or = 0.001), but rises in the intestine (unexpectedly) (control 0.18 +/- 0.02 versus cytotoxic 0.3 +/- 0.04 pg CCK mRNA/micrograms tRNA, P < or = 0.001). CCK peptides do not change in the brain (control 39 +/- 4 versus cytotoxic 34 +/- 4 nmol/g, P < or = NS), but rise (unexpectedly) in the intestine (control 43 +/- 4 versus cytotoxic 250 +/- 27 nmol/g, P < or = 0.001). We ascribe these observations to differing brain/gut cell turnover kinetics. These data indicate that a rebound phenomenon occurs during gut recovery from cytotoxic injury. We additionally show a differential age-related response to cytotoxic injury. Younger animals tolerated the injury better than old ones (mortality: young 27% (3/11) versus old 66% (8/12), P < or = 0.001). Additionally, intestinal recovery is more rapid in younger animals. These data suggest that with increasing age, chemotherapeutic dosages may need to be modulated. It is additionally possible that clinically applicable algorithms may be developed using our animal model.