Publications by authors named "Robert Greenstein"

48 Publications

Crohn's disease: failure of a proprietary fluorescent in situ hybridization assay to detect M. avium subspecies paratuberculosis in archived frozen intestine from patients with Crohn's disease.

BMC Res Notes 2020 Feb 24;13(1):96. Epub 2020 Feb 24.

Infectious Disease Section, James J. Peters Veterans Affairs Medical Center Bronx, New York, USA.

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.
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http://dx.doi.org/10.1186/s13104-020-04947-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038517PMC
February 2020

A Data-Based Hypothesis That Inflammatory Bowel Disease Unclassified (IBD-U) May Indicate That IBD Is a Spectrum of a Single Infectious Intestinal Disease.

Inflamm Bowel Dis 2019 04;25(5):e48

Department of Infectious Diseases, James J. Peters Veterans Affair Medical Center, Bronx, New York.

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http://dx.doi.org/10.1093/ibd/izy301DOI Listing
April 2019

Failure to detect M. avium subspecies paratuberculosis in Johne's disease using a proprietary fluorescent in situ hybridization assay.

BMC Res Notes 2018 Jul 21;11(1):498. Epub 2018 Jul 21.

Infectious Disease Section, James J. Peters Veterans Affairs Medical Center, Bronx, NY, USA.

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.
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http://dx.doi.org/10.1186/s13104-018-3601-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054717PMC
July 2018

A data-based hypothesis explicating the observations that "Smoking is associated with risk for developing inflammatory bowel disease including late onset ulcerative colitis: a prospective study".

Scand J Gastroenterol 2018 04 15;53(4):505. Epub 2018 Feb 15.

b Department of Medicine , James J. Peters Veterans Affairs Medical Center , Bronx , NY , USA.

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http://dx.doi.org/10.1080/00365521.2018.1440003DOI Listing
April 2018

A Data-Based Hypothesis Explicating Thiopurine Therapeutic Failure in Biologic-Naive UC.

Dig Dis Sci 2017 01 3;62(1):282-283. Epub 2016 Nov 3.

James J Peters VA Medical Center, Bronx, NY, USA.

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http://dx.doi.org/10.1007/s10620-016-4360-6DOI Listing
January 2017

Rejecting Kentucky Medicaid proposal would reaffirm commitment to full coverage.

Mod Healthc 2016 Sep;46(39):25

Kentucky Gov. Matt Bevin, representing a state where the uninsured rate has plummeted from 14.3% to 6% since adopting the Medicaid expansion under the Affordable Care Act, is proposing sweeping Medicaid changes that would make it harder for Kentuckians to keep their coverage. He threatens to cancel the Medicaid expansion if HHS doesn't approve his plan.
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September 2016

Clinical course of sly syndrome (mucopolysaccharidosis type VII).

J Med Genet 2016 06 23;53(6):403-18. Epub 2016 Feb 23.

Edward A. Doisy Department of Biochemistry and Molecular Biology, School of Medicine, Saint Louis University, St. Louis, Missouri, USA.

Background: Mucopolysaccharidosis VII (MPS VII) is an ultra-rare disease characterised by the deficiency of β-glucuronidase (GUS). Patients' phenotypes vary from severe forms with hydrops fetalis, skeletal dysplasia and mental retardation to milder forms with fewer manifestations and mild skeletal abnormalities. Accurate assessments on the frequency and clinical characteristics of the disease have been scarce. The aim of this study was to collect such data.

Methods: We have conducted a survey of physicians to document the medical history of patients with MPS VII. The survey included anonymous information on patient demographics, family history, mode of diagnosis, age of onset, signs and symptoms, severity, management, clinical features and natural progression of the disease.

Results: We collected information on 56 patients from 11 countries. Patients with MPS VII were classified based on their phenotype into three different groups: (1) neonatal non-immune hydrops fetalis (NIHF) (n=10), (2) Infantile or adolescent form with history of hydrops fetalis (n=13) and (3) Infantile or adolescent form without known hydrops fetalis (n=33). Thirteen patients with MPS VII who had the infantile form with history of hydrops fetalis and survived childhood, had a wide range of clinical manifestations from mild to severe. Five patients underwent bone marrow transplantation and one patient underwent enzyme replacement therapy with recombinant human GUS.

Conclusions: MPS VII is a pan-ethnic inherited lysosomal storage disease with considerable phenotypical heterogeneity. Most patients have short stature, skeletal dysplasia, hepatosplenomegaly, hernias, cardiac involvement, pulmonary insufficiency and cognitive impairment. In these respects it resembles MPS I and MPS II. In MPS VII, however, one unique and distinguishing clinical feature is the unexpectedly high proportion of patients (41%) that had a history of NIHF. Presence of NIHF does not, by itself, predict the eventual severity of the clinical course, if the patient survives infancy.
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http://dx.doi.org/10.1136/jmedgenet-2015-103322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4893087PMC
June 2016

Yet Another Flawed "Placebo Controlled" Study in Crohn's Disease?

Foodborne Pathog Dis 2015 Sep 25;12(9):812. Epub 2015 Jun 25.

3 Division of Infections Diseases, James J. Peters Veterans Affairs Medical Center , Bronx, New York.

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http://dx.doi.org/10.1089/fpd.2015.1999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4556337PMC
September 2015

Unanticipated Mycobacterium tuberculosis complex culture inhibition by immune modulators, immune suppressants, a growth enhancer, and vitamins A and D: clinical implications.

Int J Infect Dis 2014 Sep 1;26:37-43. Epub 2014 Jul 1.

Infectious Disease Section, James J. Peters Veterans Affairs Medical Center, Bronx, New York, USA; Department of Medicine, Icahn School of Medicine at Mt. Sinai, New York, USA.

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.
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http://dx.doi.org/10.1016/j.ijid.2014.01.026DOI Listing
September 2014

"Add-on" is scientifically more accurate than "placebo control" in multiple inflammatory bowel disease (IBD) trials.

J Crohns Colitis 2014 Oct 24;8(10):1334-5. Epub 2014 Apr 24.

James J. Peters Veterans Administration Medical Center, United States.

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http://dx.doi.org/10.1016/j.crohns.2014.03.015DOI Listing
October 2014

On the zoonosis of M. avium subspecies paratuberculosis (MAP).

J Crohns Colitis 2012 May 26;6(4):504; author reply 505. Epub 2012 Jan 26.

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http://dx.doi.org/10.1016/j.crohns.2012.01.006DOI Listing
May 2012

Vitamins A & D inhibit the growth of mycobacteria in radiometric culture.

PLoS One 2012 3;7(1):e29631. Epub 2012 Jan 3.

Department of Surgery, James J. Peters Veterans Affairs Medical Center, Bronx, New York, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029631PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3250462PMC
May 2012

A mosaic activating mutation in AKT1 associated with the Proteus syndrome.

N Engl J Med 2011 Aug 27;365(7):611-9. Epub 2011 Jul 27.

National Human Genome Research Institute, Bethesda, Maryland, USA.

Background: The Proteus syndrome is characterized by the overgrowth of skin, connective tissue, brain, and other tissues. It has been hypothesized that the syndrome is caused by somatic mosaicism for a mutation that is lethal in the nonmosaic state.

Methods: We performed exome sequencing of DNA from biopsy samples obtained from patients with the Proteus syndrome and compared the resultant DNA sequences with those of unaffected tissues obtained from the same patients. We confirmed and extended an observed association, using a custom restriction-enzyme assay to analyze the DNA in 158 samples from 29 patients with the Proteus syndrome. We then assayed activation of the AKT protein in affected tissues, using phosphorylation-specific antibodies on Western blots.

Results: Of 29 patients with the Proteus syndrome, 26 had a somatic activating mutation (c.49G→A, p.Glu17Lys) in the oncogene AKT1, encoding the AKT1 kinase, an enzyme known to mediate processes such as cell proliferation and apoptosis. Tissues and cell lines from patients with the Proteus syndrome harbored admixtures of mutant alleles that ranged from 1% to approximately 50%. Mutant cell lines showed greater AKT phosphorylation than did control cell lines. A pair of single-cell clones that were established from the same starting culture and differed with respect to their mutation status had different levels of AKT phosphorylation.

Conclusions: The Proteus syndrome is caused by a somatic activating mutation in AKT1, proving the hypothesis of somatic mosaicism and implicating activation of the PI3K-AKT pathway in the characteristic clinical findings of overgrowth and tumor susceptibility in this disorder. (Funded by the Intramural Research Program of the National Human Genome Research Institute.).
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http://dx.doi.org/10.1056/NEJMoa1104017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3170413PMC
August 2011

Attitudes toward direct-to-consumer advertisements and online genetic testing among high-risk women participating in a hereditary cancer clinic.

J Health Commun 2011 Jul 24;16(6):607-28. Epub 2011 May 24.

Department of Psychology, University of Connecticut, Storrs, Connecticut 06269-1020, USA.

Genetic testing for the breast cancer genes 1/2 (BRCA 1/2) has helped women determine their risk of developing breast and ovarian cancer. As interest in genetic testing has grown, companies have created strategies to disseminate information about testing, including direct-to-consumer advertising (DTCA) and online genetic testing. This study examined attitudes toward DTCA and online testing for BRCA among 84 women at a high-risk clinic as well as additional factors that may be associated with these attitudes, such as personal and familial cancer history, cancer worry and risk perception, and history with genetic testing/counseling. Results showed that the majority of the women held favorable attitudes toward DTCA for BRCA testing but did not support online testing. Factors such as familial ovarian cancer, cancer worry, and satisfaction with genetic counseling/testing were associated with positive attitudes toward DTCA, whereas personal breast cancer history was related to negative attitudes. The findings suggest that women may view DTCA as informational but rely on physicians for help in their decision to undergo testing, and also suggest that cancer history may affect women's acceptance of DTCA and genetic testing.
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http://dx.doi.org/10.1080/10810730.2011.551993DOI Listing
July 2011

Growth of M. avium subspecies paratuberculosis in culture is enhanced by nicotinic acid, nicotinamide, and α and β nicotinamide adenine dinucleotide.

Dig Dis Sci 2011 Feb 29;56(2):368-75. Epub 2010 Jun 29.

Department of Surgery, James J. Peters VAMC, Bronx, NY 10468, USA.

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.
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http://dx.doi.org/10.1007/s10620-010-1301-7DOI Listing
February 2011

The thioamides methimazole and thiourea inhibit growth of M. avium Subspecies paratuberculosis in culture.

PLoS One 2010 Jun 14;5(6):e11099. Epub 2010 Jun 14.

Department of Surgery, James J Peters VA Medical Center, Bronx, New York, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011099PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2885409PMC
June 2010

Genomewide association study of leprosy.

N Engl J Med 2010 Apr;362(15):1447; author reply 1447-8

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April 2010

Clinical and mutational spectrum of neurofibromatosis type 1-like syndrome.

JAMA 2009 Nov;302(19):2111-8

Medical Genomics Laboratory, Department of Genetics, University of Alabama at Birmingham, 720 20th St S, Birmingham, AL 35294, USA.

Context: Autosomal dominant inactivating sprouty-related EVH1 domain-containing protein 1 (SPRED1) mutations have recently been described in individuals presenting mainly with café au lait macules (CALMs), axillary freckling, and macrocephaly. The extent of the clinical spectrum of this new disorder needs further delineation.

Objective: To determine the frequency, mutational spectrum, and phenotype of neurofibromatosis type 1-like syndrome (NFLS) in a large cohort of patients.

Design, Setting, And Participants: In a cross-sectional study, 23 unrelated probands carrying a SPRED1 mutation identified through clinical testing participated with their families in a genotype-phenotype study (2007-2008). In a second cross-sectional study, 1318 unrelated anonymous samples collected in 2003-2007 from patients with a broad range of signs typically found in neurofibromatosis type 1 (NF1) but no detectable NF1 germline mutation underwent SPRED1 mutation analysis.

Main Outcome Measures: Comparison of aggregated clinical features in patients with or without a SPRED1 or NF1 mutation. Functional assays were used to evaluate the pathogenicity of missense mutations.

Results: Among 42 SPRED1-positive individuals from the clinical cohort, 20 (48%; 95% confidence interval [CI], 32%-64%) fulfilled National Institutes of Health (NIH) NF1 diagnostic criteria based on the presence of more than 5 CALMs with or without freckling or an NF1-compatible family history. None of the 42 SPRED1-positive individuals (0%; 95% CI, 0%-7%) had discrete cutaneous or plexiform neurofibromas, typical NF1 osseous lesions, or symptomatic optic pathway gliomas. In the anonymous cohort of 1318 individuals, 34 different SPRED1 mutations in 43 probands were identified: 27 pathogenic mutations in 34 probands and 7 probable nonpathogenic missense mutations in 9 probands. Of 94 probands with familial CALMs with or without freckling and no other NF1 features, 69 (73%; 95% CI, 63%-80%) had an NF1 mutation and 18 (19%; 95% CI, 12%-29%) had a pathogenic SPRED1 mutation. In the anonymous cohort, 1.9% (95% CI, 1.2%-2.9%) of individuals with the clinical diagnosis of NF1 according to the NIH criteria had NFLS.

Conclusions: A high SPRED1 mutation detection rate was found in NF1 mutation-negative families with an autosomal dominant phenotype of CALMs with or without freckling and no other NF1 features. Among individuals in this study, NFLS was not associated with the peripheral and central nervous system tumors seen in NF1.
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http://dx.doi.org/10.1001/jama.2009.1663DOI Listing
November 2009

Monensin causes dose dependent inhibition of Mycobacterium avium subspecies paratuberculosis in radiometric culture.

Gut Pathog 2009 Feb 9;1(1). Epub 2009 Feb 9.

Laboratory of Molecular Surgical Research, VAMC Bronx, NY (112), 130 West Kingsbridge Road, Bronx, NY 10468, USA.

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.
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http://dx.doi.org/10.1186/1757-4749-1-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2664324PMC
February 2009

On the effect of thalidomide on Mycobacterium avium subspecies paratuberculosis in culture.

Int J Infect Dis 2009 Sep 20;13(5):e254-63. Epub 2009 Mar 20.

VAMC Bronx NY (112), 130 West Kingsbridge Rd, Bronx, NY 10468, USA.

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.
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http://dx.doi.org/10.1016/j.ijid.2008.10.016DOI Listing
September 2009

On the prevalence of M. avium subspecies paratuberculosis DNA in the blood of healthy individuals and patients with inflammatory bowel disease.

PLoS One 2008 Jul 2;3(7):e2537. Epub 2008 Jul 2.

Departamento de Producción y Sanidad Animal, Instituto Vasco de Investigación y Desarrollo Agrario (NEIKER-Tecnalia), Derio, Bizkaia, Spain.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0002537PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2434204PMC
July 2008

On the action of cyclosporine A, rapamycin and tacrolimus on M. avium including subspecies paratuberculosis.

PLoS One 2008 Jun 25;3(6):e2496. Epub 2008 Jun 25.

Department of Surgery, Veterans Affairs Medical Center, Bronx, New York, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0002496PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2427180PMC
June 2008

Improved pulmonary and growth outcomes in cystic fibrosis by newborn screening.

Pediatr Pulmonol 2008 Jul;43(7):648-55

Department of Pediatrics, Connecticut Children's Medical Center, Hartford, Connecticut 06106, USA.

Background: Newborn screening for cystic fibrosis (CF) is effective in improving long-term growth outcomes. However, there is conflicting evidence that early diagnosis maintains normal pulmonary function. Our goal was to determine if newborn screening results in improved longitudinal growth and maintenance of normal pulmonary function.

Methods: A retrospective study of individuals with CF born in Connecticut between 1983 and 1997 was conducted by medical record and CF Foundation Registry review. Growth, pulmonary function and bacterial acquisition/colonization data, from diagnosis through July 1, 2005, were compared in those diagnosed by newborn screen (n = 34) to those diagnosed by sweat test after symptom appearance (n = 21).

Results: Screened individuals demonstrated greater weight and height for age at diagnosis (P = 0.01 and 0.01) and through 15 years of age (P = 0.0002 and 0.01). Body mass index was higher in screened individuals (21 vs. 18 kg/m(2)) at 15 years of age (P = 0.01). At 15 years of age, screened individuals had a clinically higher forced expiratory volume in 1 second (FEV(1)) and forced vital capacity (FVC; 90% and 104% predicted) than non-screened individuals (74% and 91% predicted; P = 0.08 and 0.10). Over a 9-year period, from ages 6 to 15, percent predicted FEV(1) and FVC increased by 4% and 13% in screened individuals; and declined by 14% and 5% respectively in non-screened individuals (P = 0.01 and 0.02). Acquisition/colonization of Pseudomonas aeruginosa was similar between groups (P = 0.23).

Conclusions: In this CF cohort, individuals diagnosed by newborn screening have improved growth and preservation of normal pulmonary function without increased risk of Pseudomonas aeruginosa colonization.
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http://dx.doi.org/10.1002/ppul.20842DOI Listing
July 2008

Weight loss reporting.

Obes Surg 2008 Jun 12;18(6):761-2. Epub 2008 Apr 12.

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http://dx.doi.org/10.1007/s11695-008-9450-xDOI Listing
June 2008

Reporting weight loss.

Obes Surg 2007 Sep;17(9):1275; author reply 1276

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http://dx.doi.org/10.1007/s11695-007-9218-8DOI Listing
September 2007

Motion analysis of a child with Niemann-Pick disease type C treated with miglustat.

Mov Disord 2008 Jan;23(1):124-8

Division of Human Genetics, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Connecticut, USA.

Niemann-Pick disease type C (NPC) is a progressive neurodegenerative disorder for which there is no effective treatment other than supportive therapy. Recently, the oral medication miglustat has been offered as a possible therapy aimed at reducing pathological substrate accumulation. This article describes the use of computerized three-dimensional motion analysis to evaluate a 3-year-old child with NPC treated with miglustat for 12 months. Motion analysis provided quantitative data on the patient's gait. However, dementia and motor dysfunction progressed despite the treatment, and the patient lost the ability to walk between 9 and 12 months of the study. Motion analysis should be considered among the tools for measuring functional outcomes in future therapeutical trials of patients with neurodegenerative diseases. It is not possible to draw conclusions about miglustat therapy in NPC from a single patient experience.
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http://dx.doi.org/10.1002/mds.21779DOI Listing
January 2008

Treating morbid obesity with laparoscopic adjustable gastric banding.

Am J Surg 2007 Sep;194(3):333-43; discussion 344-8

Weight Management Center, Louisiana State University Health Sciences Center, 533 Bolivar St, Rm 508, New Orleans, LA 70112, USA.

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.
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http://dx.doi.org/10.1016/j.amjsurg.2007.03.002DOI Listing
September 2007

When is enlargement of the subarachnoid spaces not benign? A genetic perspective.

Pediatr Neurol 2007 Jul;37(1):1-7

Department of Genetics and Developmental Biology, Division of Human Genetics, University of Connecticut Health Center, West Hartford, Connecticut 06119, USA.

Enlargement of the subarachnoid spaces is occasionally encountered during neuroimaging of children. This enlargement is generally regarded as a nonpathologic process that resolves uneventfully. However, there are several genetic disorders in which enlargement of the subarachnoid spaces can be an early sign, or the feature of an associated syndrome, that may aid in the underlying diagnosis. Recognizing subarachnoid space enlargement in these circumstances requires an understanding of the normal physiology of the subarachnoid space at different time points in a child's neurodevelopment. This article reviews the events shaping the subarachnoid space, both during normal physiologic maturation and in specific genetic disorders.
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http://dx.doi.org/10.1016/j.pediatrneurol.2007.04.001DOI Listing
July 2007

On the action of 5-amino-salicylic acid and sulfapyridine on M. avium including subspecies paratuberculosis.

PLoS One 2007 Jun 13;2(6):e516. Epub 2007 Jun 13.

Departments of Surgery and Laboratory of Molecular Surgical Research, VA Medical Center, Bronx, New York, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000516PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1885215PMC
June 2007