What the early pathologists got wrong, and right, about the pathology of Crohn's disease: a historical perspective.

Surgeons, who documented what they had seen and felt in the abdomen of the patient, made the earliest descriptions of Crohn's disease (CD). Dalziel wrote the first pathology description in 1913. Crohn and his coworkers reinvented what Dalziel had written about and called it by a different name, 'regional enteritis'. Later others elaborated on the histologic features, at first the lymphoid follicles, later the granulomas. Some thought the latter were comprised of lymphatic endothelial cells and that endothelial plugs obstructed the lymphatics. Tonelli and others recognized that lymphedema was important and caused by obstructions to lymphatic vasculature. Some lymphatics they described contained lymphocyte plugs and others granulomas. Immunohistochemistry (IHC) has now shown that endothelial cells are not the cause of lymphatic obstruction, but rather CD68-positive macrophages, concluding that the 'lymphocyte thrombi' are passive, caught upstream of granuloma-obstructed lymphatics. Numerous authors recognized that transmural edema was the most significant change in Crohn's disease and that this was later followed by fibrosis and contracture of the diseased segment. Key descriptive papers spoke of the segmental lymphedema. Most recently, attention has been given to attachments of the intralymphatic CD68+ granulomas to a focal point where endothelial damage occurred, damage suggesting infectious penetration of the mucosa, necrosis of lymphatic endothelium and then granulomatous response, both inside and outside the lymphatics, of submucosa, muscularis, and subserosa. D2-40 IHC outlines the endothelium, and anti-CD68 shows the granulomas. IHC adds a valuable perspective when reviewing CD resections.

Canine circoviral hemorrhagic enteritis in a dog in Connecticut.

A 5-mo-old Bassett Hound-Labrador Retriever cross was autopsied following a bout of lethargy, inappetence, and bleeding gums. Mucous membranes were white, and the small intestine was blue-black; the colon contained black feces. The spleen was swollen, and multiple lymph nodes were enlarged and hemorrhagic. Microscopically, the small intestine had focal crypt cell necrosis and circumferential transmural vasculitis, the latter the cause of infarction and the blue-black coloration. Lymphocytes were necrotic in spleen and lymph nodes, and erythrophagocytosis was present in some nodes. Vasculitis was present in brain, meninges, lung, liver, and kidneys. Electron microscopy revealed aggregates of 15-18 nm round viral particles in damaged crypt cells and in the endothelium of small blood vessels. Electron-dense intracytoplasmic inclusions consisting of paracrystalline-arrayed virus were demonstrated in macrophages in medullary lymph node sinuses. These virions were identified as circovirus, which was confirmed by real-time PCR and sequencing.

Correction: A PRRSV GP5-Mosaic vaccine: Protection of pigs from challenge and ex vivo detection of IFNγ responses against several genotype 2 strains.

[This corrects the article DOI: 10.1371/journal.pone.0208801.].

A PRRSV GP5-Mosaic vaccine: Protection of pigs from challenge and ex vivo detection of IFNγ responses against several genotype 2 strains.

Porcine reproductive and respiratory syndrome virus (PRRSV), is a highly mutable RNA virus that affects swine worldwide and its control is very challenging due to its formidable heterogeneity in the field. In the present study, DNA vaccines constructed with PRRSV GP5-Mosaic sequences were complexed to cationic liposomes and administered to experimental pigs by intradermal and intramuscular injection, followed by three boosters 14, 28 and 42 days later. The GP5-Mosaic vaccine thus formulated was immunogenic and induced protection from challenge in vaccinated pigs comparable to that induced by a wild type (VR2332) GP5 DNA vaccine (GP5-WT). Periodic sampling of blood and testing of vaccine-induced responses followed. Interferon-γ (IFN-γ) mRNA expression by virus-stimulated peripheral blood mononuclear cells (PBMCs) of GP5-Mosaic-vaccinated pigs was significantly higher compared to pigs vaccinated with either GP5-WT or empty vector at 21, 35 and 48 days after vaccination. Cross-reactive cellular responses were also demonstrated in GP5-Mosaic vaccinated pigs after stimulation of PBMCs with divergent strains of PRRSV. Thus, significantly higher levels of IFN-γ mRNA were detected when PBMCs from GP5-Mosaic-vaccinated pigs were stimulated by four Genotype 2 strains (VR2332, NADC9, NADC30 and SDSU73), which have at least 10% difference in GP5 amino acid sequences, while such responses were recorded only upon VR2332 stimulation in GP5-WT-vaccinated pigs. In addition, the levels of virus-specific neutralizing antibodies were higher in GP5-Mosaic or GP5-WT vaccinated pigs than those in vector-control pigs. The experimental pigs vaccinated with either the GP5-Mosaic vaccine or the GP5-WT vaccine were partially protected from challenge with VR2332, as measured by significantly lower viral loads in sera and tissues and lower lung lesion scores than the vector control group. These data demonstrate that the GP5-Mosaic vaccine can induce cross-reactive cellular responses to diverse strains, neutralizing antibodies, and protection in pigs.

ELISA Serology for Antibodies Against Chlamydia trachomatis in Crohn's Disease.

BACKGROUND: Recently we reported IgA anti-Chlamydia antibodies in patients with Crohn's disease (CD), in particular in four patients from a single family of six with CD. METHODS: We studied sera from four cohorts from the north of France. These were identified as: EPIMAD (80 pediatric onset CD and 20 pediatric onset ulcerative colitis), MINOTOR (148 adult onset sporadic CD and 50 adult onset ulcerative colitis), Grande Famillies (50) and matched controls for the Grande Famillies cohort (49). Sera were tested using commercial anti-Chlamydia trachomatis (LGV2:434) IgG and IgA human enzyme-linked immunosorbent assay (ELISA) kits. Cutoff for positivity was 11.0 standard units. RESULTS: Patients with sporadic CD, unaffected first degree relatives from multiplex families and ulcerative colitis patients had no greater serologic reactivity than controls. However, multiplex families' patients had twice as many positives as the other groups: for IgG 20% vs. 8%; for IgA 20% vs. 10%. CONCLUSIONS: Though not attaining statistical significance, the data showed that familial CD patients had greater exposure to C. trachomatis than sporadic CD patients, supporting our earlier results from one family from the north of France. More specific serologic tests based on outer membrane proteins will need to be employed against the various Chlamydia species with zoonotic potential.

An Infectious Pig Model of Crohn's Disease.

BACKGROUND: Over the last several years, we have demonstrated that intestinal lymphangitis and lymphatic obstruction are fundamental lesions in Crohn's disease, for which no therapy is currently available. There is an infectious enteritis of young pigs that offers an opportunity to understand how the lymphangitis of CD may have been initiated. The pathology of chlamydial enteritis was described earlier, from 1987 to 2009. MATERIALS AND METHODS: Tissue blocks and hematoxylin and eosin-stained slides from Chlamydia suis-inoculated young pigs were provided by D. Rogers and F. Guscetti. Experimental animals, gnotobiotic and conventional, had been autopsied 4, 7, and 10 days postinoculation. Serial sections of intestine were immunostained with a Chlamydia trachomatis antibody, which cross-reacted with C. suis antigen, and compared with hematoxylin and eosin preparations. RESULTS: Immunohistochemistry revealed antigen in villous epithelial cells of jejunum and ileum and in the endothelium of lacteals and lymphatics by day 4. This was accompanied by lymphatic endothelial necrosis, lymphangitis, and inflammatory lymphatic obstruction, through several layers of the affected intestinal segments, days 4 through 10. CONCLUSIONS: Although the original authors documented lesions to define the porcine disease, here the author characterizes the lymphangitis as a model for understanding Crohn's disease and suggests a chlamydial origin for the latter.

Antibiotics associated with increased risk of new-onset Crohn's disease but not ulcerative colitis: a meta-analysis.

OBJECTIVES: The objective of this study was to perform a meta-analysis investigating antibiotic exposure as a risk factor for developing inflammatory bowel disease (IBD). METHODS: A literature search using Medline, Embase, and Cochrane databases was performed to identify studies providing data on the association between antibiotic use and newly diagnosed IBD. Included studies reported Crohn's disease (CD), ulcerative colitis (UC), or a composite of both (IBD) as the primary outcome and evaluated antibiotic exposure before being diagnosed with IBD. A random-effects meta-analysis was conducted to determine overall pooled estimates and 95% confidence intervals (CIs). RESULTS: A total of 11 observational studies (8 case-control and 3 cohort) including 7,208 patients diagnosed with IBD were analyzed. The pooled odds ratio (OR) for IBD among patients exposed to any antibiotic was 1.57 (95% CI 1.27-1.94). Antibiotic exposure was significantly associated with CD (OR 1.74, 95% CI 1.35-2.23) but was not significant for UC (OR 1.08, 95% CI 0.91-1.27). Exposure to antibiotics most markedly increased the risk of CD in children (OR 2.75, 95% CI 1.72-4.38). All antibiotics were associated with IBD, with the exception of penicillin. Exposure to metronidazole (OR 5.01, 95% CI 1.65-15.25) or fluoroquinolones (OR 1.79, 95% CI 1.03-3.12) was most strongly associated with new-onset IBD. CONCLUSIONS: Exposure to antibiotics appears to increase the odds of being newly diagnosed with CD but not UC. This risk is most marked in children diagnosed with CD.

Granulomas obstruct lymphatics in all layers of the intestine in Crohn's disease.

After an earlier study defining immunolabeled lymphoid follicles, obstructed lymphatics, and granulomas of the diseased ilea of 24 Crohn's disease patients, we chose to trace the lymphatics of these cases and 10 additional by serial sectioning. Particular attention was given to establishing physical continuity between granuloma-obstructed lymphatics and lymphatics with 'lymphocytic thrombi'. Formalin-fixed paraffin-embedded tissue blocks from resected diseased ilea and proximal colons from 34 patients were reviewed. Patients were 13 men and 21 women, aged 14-60 years. Duration of disease ranged from 1 month to 10 years. Immunohistochemistry employed D2-40 antibody to label lymphatics and anti-CD68 to label granulomas. Twenty-nine of the 34 (85%) resection tissues had lymphangectasia, in mucosa, submucosa, and subserosa. In 53% of the specimens, lymphatics of the various layers were obstructed by granulomas that filled the lumina. In 44%, 15/34, there were also distended lymphatics that were totally plugged with lymphocytes. In 10 of the 15, serial sections revealed continuity between the lymphocyte-plugged lymphatics and the endolymphatic granulomatous obstruction downstream. In 5 of the cases, D2-40 immunostaining revealed redundant lymphatic endothelium interwoven with the granuloma cells. Granulomas totally obstruct lymphatics in all layers of the intestine in Crohn's disease. Upstream of these obstructions, lymphatics are distended with lymphocytes. The degree and extent of this potentially irreversible 'lymphangitis nodosa' have undoubtedly confounded treatment regimens and clinical trials. There currently are no imaging methods to demonstrate the lymphangitis, nor treatments to resolve it.

Immunohistochemical search for viral and bacterial antigens in Crohn's disease.

BACKGROUND: Recent studies show that diseased intestinal tissues of patients with Crohn's disease (CD) contain obstructed lymphatics, granulomas, and tertiary lymphoid organs, representing responses to persistent antigen. METHODS: Forty-seven tissue sections from 28 CD patients and 20 tissue sections from 17 control patients were studied. Tissues were immunostained with antibody directed against adenovirus, Epstein-Barr virus, herpes simplex virus I, parvovirus B19, Listeria monocytogenes, Escherichia coli, Clostridium perfringens, and Mycobacterium avium subspecies paratuberculosis. RESULTS: There was no evidence of adenovirus, Epstein-Barr virus, parvovirus B19, or M. avium subsp. paratuberculosis in the tissues. Clostridia were positively stained in the mucus of 18.5% of CD patients versus 35.3% of controls and in the tissue of 11.1% of CD patients but in no controls. Immunoreactivity to listeria antibody occurred in the mucus of 3.7% of CD patients and in 5.9% of controls while it occurred in the tissue of 37.0% of CD patients and 29.4% of controls. E. coli occurred in the mucus of 48.1% CD and 64.7% controls and in the tissue of 18.5% and 5.9% respectively. CONCLUSIONS: Of the agents demonstrated in this search, none was located in granulomas or inflamed lymphatics. Finding the common gut microbes, E. coli and clostridia, in the mucus of patients and controls was not unexpected. The minor focal staining of E. coli and clostridia does not suggest a primary role for these pathogens in CD. Positive staining for listeria in patients and controls may very well represent cross reactivity rather than specific identification.

Human herpesvirus-6 in patients with Crohn's disease.

Human herpesvirus-6 (HHV-6) infections are usually asymptomatic reactivations in immunocompetent persons, but may be severe in immunocompromised individuals. Although primary HHV-6 infection is mainly associated with roseola infantum, it has also been associated with gastroenteritis, diarrhea, and nausea in children. In this study, we investigated the potential role of HHV-6 in Crohn's disease (CD). Evidence of HHV-6 infection in CD patients and controls was determined by immunohistochemistry (IHC), polymerase chain reaction (PCR), and quantitative real-time PCR (qPCR). Fifty-one tissue blocks from 23 CD patients and 20 tissue blocks from 20 controls were examined. Quantitativereal-time PCR was used to assess HHV-6 viral loads. IHC, PCR and qPCR indicated the presence of HHV-6 in both CD patients and controls. Immunohistochemistry of tissues revealed an almost equal frequency and distribution of positive cells; however, non-specific immunostaining confounded interpretation. HHV-6 DNA was detected in 52% (12/23) of CD and 55% (11/20) of control patients by PCR and in 69.5% (16/23) of CD cases and 65% (13/20) of controls by qPCR. Mean viral load in intestinal tissues was similar in CD and controls (33.4 and 57.9 copies microg(-1) DNA, respectively). Finding equal evidence of HHV-6 in patients and controls by multiple methods suggests that this virus is ubiquitous and probably not a cause of CD.

Clostridial abomasal disease in Connecticut dairy calves.

Over 2 years, 24 dairy calves died of emphysematous abomasitis and abomasal bloat. Anaerobic cultures of necrotic abomasal mucosa yielded Clostridium perfringens from 10 of 15 calves. Sarcina were observed in 17 of 22 examined histologically. A change in the antibiotic regimen for newborns and improved sanitizing of feeding utensils eliminated further losses.

Candida albicans colonization and ASCA in familial Crohn's disease.

OBJECTIVES: Anti-Saccharomyces cerevisiae antibodies (ASCAs) are present in 50-60% of patients with Crohn's disease (CD) and in 20-25% of their healthy relatives (HRs). The yeast, Candida albicans, has been shown to generate ASCAs, but the presence of C. albicans in the digestive tract of CD patients and their HRs has never been investigated. Therefore, we studied C. albicans carriage in familial CD and its correlation with ASCAs. METHODS: Study groups consisted of 41 CD families composed of 129 patients and 113 HRs, and 14 control families composed of 76 individuals. Mouth swabs and stool specimens were collected for isolation, identification, and quantification of yeasts. Serum samples were collected for detection of ASCAs and anti-C. albicans mannan antibodies (ACMAs). RESULTS: C. albicans was isolated significantly more frequently from stool samples from CD patients (44%) and their HRs (38%) than from controls (22%) (P<0.05). The prevalence of ACMAs was similar between CD patients, their HRs, and controls (22, 19, and 21%, respectively, P=0.845), whereas the prevalence of ASCAs was significantly increased in CD families (72 and 34% in CD and HRs, respectively, in contrast to 4% in controls, P<0.0001). AMCA levels correlated with C. albicans colonization in all populations. ASCA levels correlated with C. albicans colonization in HRs but not in CD patients. CONCLUSIONS: CD patients and their first-degree HRs are more frequently and more heavily colonized by C. albicans than are controls. ASCAs correlate with C. albicans colonization in HRs but not in CD. In HRs, ASCAs could result from an altered immune response to C. albicans. In CD, a subsequent alteration in sensing C. albicans colonization could occur with disease onset.

Pseudomonas-associated discospondylitis in a two-month-old llama.

A 2-month-old female llama with a history of tetraparesis was presented for necropsy. This cria was apparently normal until it became ataxic during its second week of life. It had diminished flexor reflexes of the left forelimb and both hind limbs. Hematology and serum biochemistry revealed neutrophilia, elevated alkaline phosphatase, elevated phosphorus, and modest hyperglycemia. Radiography of the cervical spine demonstrated an abnormal C3-C4 intervertebral disc and bony proliferation of the vertebral end plates. The llama was treated with antibiotics and corticosteroids but failed to respond. Postmortem examination revealed cervical intervertebral disc disease and vertebral exostosis. Microscopically, there was necrosis of intervertebral connective tissue and focal malacia of the spinal cord. Pseudomonas aeruginosa was isolated from the intervertebral disc space and from cerebrospinal fluid. To the authors' knowledge, this is the first description of Pseudomonas-associated discospondylitis in a llama.

Suppurative polyarthritis in striped skunks (Mephitis mephitis) from Cape Cod, Massachusetts: detection of mycoplasma DNA.

Striped skunks (Mephitis mephitis) from Cape Cod, Massachusetts, U.S.A. were necropsied (n=34; 1995-1997) or clinically evaluated (n=25, 2002-2003) to characterize a lameness and polyarthritis, reported by wildlife veterinarians and rehabilitators, and unsuccessfully treated with antibiotics. Overall, 22 affected skunks had one or multiple swollen joints, swollen paws, and subcutaneous abscesses. Purulent exudate was located in joint spaces, in periarticular connective tissue between muscle fascicles and tendons, and between and along flexor and extensor tendons of the paws. Histologic examination revealed suppurative arthritis, with necrosis and erosion of articular cartilage, and suppurative osteomyelitis. Special stains failed to reveal a causative microorganism within affected joints, and routine bacteriologic cultures failed to isolate a pathogen with any significant frequency or consistency. Polymerase chain reaction (PCR) experiments were performed using DNA extracted from archived, formalin-fixed joint samples of 11 affected skunks, and DNA from joints of 7 of 11 affected skunks yielded amplicons with sequences highly similar to sequences of Mycoplasma fermentans within the Mycoplasma bovis cluster, whereas DNA samples from joints of four unaffected skunks were negative by PCR. Skunks from Connecticut, U.S.A. (n=21; 1995-2003) were similarly examined and were found not to have suppurative polyarthritis, suggesting a unique geographic distribution of this condition. Concurrent pathologic conditions in adult skunks from both Cape Cod and Connecticut included verminous pneumonia, gastric nematodiasis, arthropod ectoparasitism, and canine distemper. Amyloidosis was present in skunks with and without suppurative polyarthritis, and the amyloid was immunohistochemically identified as AA-amyloid. This is the first report of suppurative polyarthritis in wild skunks with evidence of a mycoplasmal etiology.