The 2000 Ogura Lecture: Olfactory neural cells: An untapped diagnostic and therapeutic resource

Objective. This is an over-view of the cellular biology of upper nasal mucosal cells that have special characteristics that enable them to be used to diagnose and study congenital neurological diseases and to aid neural repair. Study Design: After mapping the distribution of neural cells in the upper nose, the authors' investigations moved to the use of olfactory neurones to diagnose neurological diseases of development, especially schizophrenia. Olfactory-ensheating glial cells (OEGs) from the cranial cavity promote axonal penetration of the central nervous system and aid spinal cord repair in rodents. The authors sought to isolate these cells from the more accessible upper nasal cavity in rats and in humans and prove they could likewise promote neural regeneration, making these cells suitable for human spinal repair investigations. Methods: The schizophrenia-diagnosis aspect of the study entailed the biopsy of the olfactory areas of 10 schizophrenic patients and 10 control subjects. The tissue samples were sliced and grown in culture medium. The ease of cell attachment to fibronectin (artificial epithelial basement membrane), as well as the mitotic and apoptotic indices, was studied in the presence and absence of dopamine in those cell cultures. The neural repair part of the study entailed a harvesting and insertion of first rat olfactory lamina propria rich in OEGs between cut ends of the spinal cords and then later the microinjection of an OEG-rich suspension into rat spinal cords previously transected by open laminectomy. Further studies were done in which OEG insertion was performed up to 1 month after rat cord transection and also in monkeys. Results: Schizophrenic patients' olfactory tissues do not easily attach to basement membrane compared with control subjects, adding evidence to the theory that cell wall anomalies are part of the schizophrenic lesion of neurones. Schizophrenic patient cell cultures had higher mitotic and apoptotic indices compared with control subjects. The addition of dopamine altered these indices enough to allow accurate differentiation of schizophrenics from control patients, leading to, possibly for the first time, an early objective diagnosis of schizophrenia and possible assessment of preventive strategies. OEGs from the nose were shown to be as effective as those from the olfactory bulb in promoting axonal growth across transected spinal cords even when added I month after injury in the rat. These otherwise paraplegic rats grew motor and proprioceptive and fine touch fibers with corresponding behavioral improvement. Conclusions. The tissues of the olfactory mucosa are readily available to the otolaryngologist. Being surface cells, they must regenerate (called neurogenesis). Biopsy of this area and amplification of cells in culture gives the scientist a window to the developing brain, including early diagnosis of schizophrenia. The Holy Grail of neurological disease is the cure of traumatic paraplegia and OEGs from the nose promote that repair. The otolaryngologist may become the necessary partner of the neurophysiologist and spinal surgeon to take the laboratory potential of paraplegic cure into the day-to-day realm of clinical reality.

Th1 cytokines in oral lichen planus

Background: Cell-mediated immune responses in oral lichen planus (OLP) may be regulated by cytokines and their receptors. Methods: In situ cytokine expression and in vitro cytokine secretion in OLP were determined by immunohistochemistry and ELISA. Resulults: The majority of subepithelial and intraepithelial mononuclear cells in OLP were CD8(+) . In some cases, intraepithelial CD8(+) cells were adjacent to degenerating keratinocytes. CD4(+) cells were observed mainly in the deep lamina propria with occasional CD4(+) cells close to basal keratinocytes. Mononuclear cells expressed IFN-gamma in the superficial lamina propria and TNF-alpha adjacent to basal keratinocytes. Basal keratinocytes expressed TNF-alpha as a continuous band. TNF R1 was expressed by mononuclear cells and basal and suprabasal keratinocytes. There was variable expression of TGF-beta1 in the subepithelial infiltrate while all intraepithelial mononuclear cells were TGF-beta1(-) . Keratinocytes in OLP stained weakly for TGF-beta1. Unstimulated OLP lesional T cells secreted IFN-gammain vitro . TNF-alpha stimulation down-regulated IFN-gamma secretion and up-regulated TNF-alpha secretion. IL-4, IL-10 and TGF-beta1 secretion were not detected. Conclusions: These data suggest the development of a T helper 1 immune response that may promote CD8(+) cytotoxic T-cell activity in OLP.

The histopathological approach to inflammatory bowel disease: a practice guide

Inflammatory bowel diseases (IBDs) are lifelong disorders predominantly present in developed countries. In their pathogenesis, an interaction between genetic and environmental factors is involved. This practice guide, prepared on behalf of the European Society of Pathology and the European Crohn's and Colitis Organisation, intends to provide a thorough basis for the histological evaluation of resection specimens and biopsy samples from patients with ulcerative colitis or Crohn's disease. Histopathologically, these diseases are characterised by the extent and the distribution of mucosal architectural abnormality, the cellularity of the lamina propria and the cell types present, but these features frequently overlap. If a definitive diagnosis is not possible, the term indeterminate colitis is used for resection specimens and the term inflammatory bowel disease unclassified for biopsies. Activity of disease is reflected by neutrophil granulocyte infiltration and epithelial damage. The evolution of the histological features that are useful for diagnosis is time- and disease-activity dependent: early disease and long-standing disease show different microscopic aspects. Likewise, the histopathology of childhood-onset IBD is distinctly different from adult-onset IBD. In the differential diagnosis of severe colitis refractory to immunosuppressive therapy, reactivation of latent cytomegalovirus (CMV) infection should be considered and CMV should be tested for in all patients. Finally, patients with longstanding IBD have an increased risk for the development of adenocarcinoma. Dysplasia is the universally used marker of an increased cancer risk, but inter-observer agreement is poor for the categories low-grade dysplasia and indefinite for dysplasia. A diagnosis of dysplasia should not be made by a single pathologist but needs to be confirmed by a pathologist with expertise in gastrointestinal pathology.

Megabladder in experimental Chagas disease: pathological features of the bladder wall

Mega-organs, primarily in the digestive tract, are well known to occur in chronic Chagas disease. Acute experimental infection with Trypanosoma cruzi results in parasitism of a wide range of cells, tissues, and organs, including the urinary bladder. Infection of BALB/c mice with 100,000 bloodstream forms of the Y strain of T. cruzi induced acute infection with intense parasitism of all layers of the urinary bladder. Parasites were found in the mucosa, lamina propria, muscular, adventitial connective, and fat tissue. Desquamate epithelial cells with amastigotes in the bladder lumen were also found. After 60 days of infection, mice inoculated with 50 bloodstream forms developed dilated, thin-walled bladders that had inflammatory infiltrates and foci of fibrosis replacing areas of damaged muscular layer. These lesions result from direct damage to the muscle fibers by the T. cruzi, leading to myosites, muscle damage, and scarring. Direct damage of paraganglia cells secondary to parasitism, leading to dilatation, damage of muscle fibers, and scarring with replacement of muscular tissue with connective tissue, should also be considered as a cause of functional disturbance of the urinary bladder.

Schellackia (Apicomplexa: Eimeriidae) of the brazilian tree-frog, Phrynohyas venulosa (Amphibia: Anura) from Amazonian Brazil

Endogenous stages of a Schellackia species are described in histological sections of the intestine of the tree-frog, Phrynohyas venulosa, from North Brazil. Most oocysts sporulate within the epithelial cells of the gut, but a few were detected in the lamina propria.

Agglutinating Secretory IgA Preserves Intestinal Epithelial Cell Integrity during Apical Infection by Shigella flexneri.

Shigella flexneri, by invading intestinal epithelial cells (IECs) and inducing inflammatory responses of the colonic mucosa, causes bacillary dysentery. Although M cells overlying Peyer's patches are commonly considered the primary site of entry of S. flexneri, indirect evidence suggests that bacteria can also use IECs as a portal of entry to the lamina propria. Passive delivery of secretory IgA (SIgA), the major immunoglobulin secreted at mucosal surfaces, has been shown to protect rabbits from experimental shigellosis, but no information exists as to its molecular role in maintaining luminal epithelial integrity. We have established that the interaction of virulent S. flexneri with the apical pole of a model intestinal epithelium consisting of polarized Caco-2 cell monolayers resulted in the progressive disruption of the tight junction network and actin depolymerization, eventually resulting in cell death. The lipopolysaccharide (LPS)-specific agglutinating SIgAC5 monoclonal antibody (MAb), but not monomeric IgAC5 or IgGC20 MAbs of the same specificity, achieved protective functions through combined mechanisms, including limitation of the interaction between S. flexneri and epithelial cells, maintenance of the tight junction seal, preservation of the cell morphology, reduction of NF-κB nuclear translocation, and inhibition of proinflammatory mediator secretion. Our results add to the understanding of the function of SIgA-mediated immune exclusion by identifying a mode of action whereby the formation of immune complexes translates into maintenance of the integrity of epithelial cells lining the mucosa. This novel mechanism of protection mediated by SIgA is important to extend the arsenal of effective strategies to fight against S. flexneri mucosal invasion.

MHC-II-independent CD4+ T cells induce colitis in immunodeficient RAG-/- hosts.

CD4(+) alpha beta T cells from either normal C57BL/6 (B6) or MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice engrafted into congenic immunodeficient RAG1(-/-) B6 hosts induced an aggressive inflammatory bowel disease (IBD). Furthermore, CD4(+) T cells from CD1d(-/-) knockout (KO) B6 donor mice but not those from MHC-I(-/-) (homozygous transgenic mice deficient for beta(2)-microglobulin) KO B6 mice induced a colitis in RAG(-/-) hosts. Abundant numbers of in vivo activated (CD69(high)CD44(high)CD28(high)) NK1(+) and NK1(-) CD4(+) T cells were isolated from the inflamed colonic lamina propria (cLP) of transplanted mice with IBD that produced large amounts of TNF-alpha and IFN-gamma but low amounts of IL-4 and IL-10. IBD-associated cLP Th1 CD4(+) T cell populations were polyclonal and MHC-II-restricted when derived from normal B6 donor mice, but oligoclonal and apparently MHC-I-restricted when derived from MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice. cLP CD4(+) T cell populations from homozygous transgenic mice deficient for beta(2)-microglobulin KO B6 donor mice engrafted into RAG(-/-) hosts were Th2 and MHC-II restricted. These data indicate that MHC-II-dependent as well as MHC-II-independent CD4(+) T cells can induce a severe and lethal IBD in congenic, immunodeficient hosts, but that the former need the latter to express its IBD-inducing potential.

Characterization of human fibroleukin, a fibrinogen-like protein secreted by T lymphocytes.

We have recently cloned the human homologue of the murine pT49 cDNA (hpT49h), a transcript encoding a protein homologous to the beta- and gamma-chains of fibrinogen. Here, we report the identification of the hpT49h gene product using mAbs generated against a peptide corresponding to the carboxyl-terminal end of the deduced protein and a recombinant protein fragment expressed in Escherichia coli. mAbs 23A6, 7B12, and 3F4 specifically recognized a protein of 70 kDa in reducing SDS-PAGE in the culture supernatant of 293T cells transiently transfected with the full length hpT49h cDNA and freshly isolated PBMC. Under nonreducing conditions, the material migrated with a molecular mass of 250 to 300 kDa, indicating that the 70-kDa protein forms a disulfide bonded complex. Because of its homology with fibrinogen, we have termed this protein fibroleukin. Fibroleukin is spontaneously secreted in vitro by freshly isolated CD4+ and CD8+ T lymphocytes. RT-PCR analysis revealed preferential expression of fibroleukin mRNA in memory T lymphocytes (CD3+/CD45R0+) compared with naive T lymphocytes (CD3+/CD45RA+). Fibroleukin production by PBMC was rapidly lost in culture. Production could be partially maintained in the presence of IFN-gamma, while T lymphocyte activation had no effect. To demonstrate fibroleukin production in vivo, we analyzed colon mucosa by immunohistology. Fibroleukin staining was detected in the extracellular matrix of the T lymphocyte-rich upper portion of the lamina propria mucosa. While the exact function of fibroleukin remains to be defined, these data suggest that fibroleukin may play a role in physiologic lymphocyte functions at mucosal sites.

Acute inflammatory response in the stomach of BALB/c mice challenged with coccoidal Helicobacter pylori

An experimental murine model was used to verify the viability and pathogenicity of coccoid Helicobacter pylori. For this purpose, 27 BALB/c mice were inoculated intragastrically with 1 ml broth culture (10(8)organisms/ml) of a coccoid H. pylori clinical isolate. The animals were divided into two groups. Nine were infected on a one-time basis (GA1) and 18 were infected on two consecutive days (GA2). Other 27 mice were inoculated with Brucella broth and divided in the same way; they composed the control group. Mice were killed at 2, 3, 7, 14 and 21 days post inoculation (pi). Fragments of stomach and duodenum were collected, fixed with 12% formalin and stained by hematoxilin-eosin and Giemsa for histopathological examination. Until the 14th()day, only reinfected mice had mild-to-moderate inflammatory infiltrate in the stomach. The infiltration was predominantly lymphomonocytic, although plasma cells and eosinophils could be seen. However, at 21st day, severe eosinophilic infiltration was present in the lamina propria and submucosa of gastric corpus. In subgroup GA1, animals presented lymphomonocytic infiltration in the stomach from 14th()day pi. Our results showed that coccoid H. pylori was able to induce an acute inflammatory response in stomach of reinfected mice since the initial periods of infection.

Pharmacological inhibition of interleukin-15 prevents colitis and associated bone loss in IL-10 knockout mice

Bone loss secondary to inflammatory bowel diseases (IBD) is largely explained by activated T cells producing cytokines that trigger osteoclastogenesis and accelerate bone resorptionwhile inhibiting bone formation. In IBD, elevated expression of interleukin (IL)-15, a T cell growth factor, plays a central role in T cell activation, pro-inflammatory cytokine production and the development of colitis. We previously reported that IL-15 enhances RANKL-induced osteoclastogenesis and that an IL-15 antagonist, CRB-15, prevents weight and bone loss in a mousemodel of dextran sulfate sodium-induced colitis.We hypothesized that inhibition of IL-15 signalingmight prevent bone loss in IL-10 deficient (IL10−/−) mice, that develop spontaneous bowel inflammation associatedwith osteopeniawhen they are no longer raised under germ-free conditions.Mice received anIL-15 antagonist (CRB-15, 5 μg/day, n=5) or IgG2a (5 μg/day, n=4) fromweek 10 to 14 of age. The severity of colitis was assessed by histology and bowel cytokine gene expression by real time PCR. Bone mass and architecturewere evaluated by ex vivo DXA on femur and micro-computed tomography on femur and vertebra. Bodyweight gainwas similar in the two groups. After 4 weeks, colonwas 29% shorter in CRB-15 treatedmice (p<0.006), a sign of reduced inflammation. Histological analysis indicated a transmural infiltration of inflammatory cells, lymphoepithelial lesions and increased size of villi (histological score=4/6) in IgG2a treated mice, whereas colon from CRB-15 treated mice exhibited mild infiltration of inflammatory cells of the lamina propria, no mucosal damages and a minimal increased size of villi (histological score=1.6/6). Levels of TNFα, IL-17 and IL-6 mRNA in the colon were significantly reduced in CRB-15 treated mice (p<0.04 vs IgG2), indicating a decrease in colon inflammation. CRB-15 improved femur BMD (+10.6% vs IgG2a, p<0.002), vertebral trabecular bone volume fraction (BV/TV, +19.7% vs IgG2a, p<0.05) and thickness (+11.6% vs IgG2a, p<0.02). A modest but not significant increase in trabecular BV/TV was observed at the distal femur. Cortical thicknesswas also higher at themidshaft femur in CRB-15 treatedmice (+8.3% vs IgG2a, p<0.02). In conclusion, we confirm and extend our results about the effects of CRB-15 in colitis. Antagonizing IL-15 may exert favorable effects on intestinal inflammation and prevent bone loss and microarchitecture alterations induced by colitis. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: B. Brounais-Le Royer Grant / Research Support from Novartis Consumer Health Foundation, S. Ferrari-Lacraz: none declared, D. Velin: none declared, X. Zheng: none declared, S. Ferrari: none declared, D. Pierroz: none declared.

Trichurid nematodes in ring-necked pheasants from backyard flocks of the State of Rio de Janeiro, Brazil: frequency and pathology

The present investigation is related to the frequency of infection and to the gross and microscopic lesions associated to the presence of trichurid worms in 50 ring-necked pheasants (Phasianus colchicus) from backyard flocks in the state of Rio de Janeiro, Brazil. In the investigated birds, the overall infection rate was of 74%, with the presence of Eucoleus perforans with 72% of prevalence and 21.2 of mean intensity, in the esophageal and crop mucosa and rarely in the junction of the proventriculus and esophagus, E. annulatus with 2% and 3 in the crop mucosa, Capillaria phasianina, with 12% and 4.3 in the cecum and small intestine and Baruscapillaria obsignata, for the first time referred in this host, with 2% and 1 in the small intestine. Clinical signs were absent. The gross lesions observed in the crop and esophagus of 14 (38.9%) pheasants parasitized with E. perforans were thickening, small nodules, congestion, and petechial haemorrhages in the mucosa. These birds presented a mean infection of 37.5 and a range of infection of 10-82. The microscopic lesions revealed chronic esophagitis with diffuse inflammatory process in the lamina propria characterized mostly by a mononuclear cell infiltrate and also with the presence of granulocytes. In the case of the parasitism of pheasants with C. phasianina, the gross lesions were absent; microscopic lesions were characterized by chronic typhlitis with mononuclear infiltrate. Gross and microscopic lesions were absent in the pheasants parasitized with E. annulatus and B. obsignata.

How to Assess Histology in Eosinophilic Esophagitis: Standardization by the EEsAI Histopathology Questionnaire

Background and Aims: The international EEsAI study group iscurrently developing the first activity index specific forEosinophilic Esophagitis (EoE). So far histologic assessment inEoE i s not standardized but urgently needed to harmonizehistologic endpoints i n clinical trials. G oal: To develop andevaluate the EEsAI histopathology questionnaire.Methods: B ased o n multiple Delphi r ounds w ith national a ndinternational ( Europe and North America) E oE e xperthistopathologists and gastroenterologists, w e have d evelopedthe EEsAI histopathology questionnaire.Results: T he EEsAI histopathology questionnaire t akes i ntoaccount the following items: number of eosinphils per highpower field, pattern of i nflammation, eosinophil abscesses,basal l ayer e nlargement, and lamina propria f ibrosis. D istinctdefinitions a nd categories f or every item were established byagreement among experts. EoE expert pathologists had amedian of 22 minutes to complete the questionnaire and judgedit as well feasible. The histopathology questionnaire was pilotedin 20 patients and is currently evaluated in a cohort of 150 adultEoE patients.Conclusions: T he EEsAI histopathology questionnaire is thefirst tool that assesses EoE severity according to clearly definedhistologic criteria. These definitions, as well as their categories,established by i nternational E oE e xpert pathologists, w illstandardize h istologic EoE s everity assessment and therebyfacilitate the comparison of histologic outcome measures inclinical trials.

Ulcerative colitis impairs the acylethanolamide-based anti-inflammatory system reversal by 5-aminosalicylic acid and glucocorticoids

Studies in animal models and humans suggest anti-inflammatory roles on the N acylethanolamide (NAE)-peroxisome proliferators activated receptor alpha (PPARα) system in inflammatory bowel diseases. However, the presence and function of NAE-PPARα signaling system in the ulcerative colitis (UC) of humans remain unknown as well as its response to active anti-inflammatory therapies such as 5-aminosalicylic acid (5-ASA) and glucocorticoids. Expression of PPARα receptor and PPARα ligands-biosynthetic (NAPE-PLD) and -degrading (FAAH and NAAA) enzymes were analyzed in untreated active and 5-ASA/glucocorticoids/immunomodulators-treated quiescent UC patients compared to healthy human colonic tissue by RT-PCR and immunohistochemical analyses. PPARα, NAAA, NAPE-PLD and FAAH showed differential distributions in the colonic epithelium, lamina propria, smooth muscle and enteric plexus. Gene expression analysis indicated a decrease of PPARα, PPARγ and NAAA, and an increase of FAAH and iNOS in the active colitis mucosa. Immunohistochemical expression in active colitis epithelium confirmed a PPARα decrease, but showed a sharp NAAA increase and a NAPE-PLD decrease, which were partially restored to control levels after treatment. We also characterized the immune cells of the UC mucosa infiltrate. We detected a decreased number of NAAA-positive and an increased number of FAAH-positive immune cells in active UC, which were partially restored to control levels after treatment. NAE-PPARα signaling system is impaired during active UC and 5-ASA/glucocorticoids treatment restored its normal expression. Since 5-ASA actions may work through PPARα and glucocorticoids through NAE-producing/degrading enzymes, the use of PPARα agonists or FAAH/NAAA blockers that increases endogenous PPARα ligands may yield similar therapeutics advantages.

Polymeric IgA is superior to monomeric IgA and IgG carrying the same variable domain in preventing Clostridium difficile toxin A damaging of T84 monolayers.

The two exotoxins A and B produced by Clostridium difficile are responsible for antibiotic-associated enterocolitis in human and animals. When added apically to human colonic carcinoma-derived T84 cell monolayers, toxin A, but not toxin B, abolished the transepithelial electrical resistance and altered the morphological integrity. Apical addition of suboptimal concentration of toxin A made the cell monolayer sensitive to toxin B. Both toxins induced drastic and rapid epithelial alterations when applied basolaterally with a complete disorganization of tight junctions and vacuolization of the cells. Toxin A-specific IgG2a from hybridoma PCG-4 added apically with toxin A alone or in combination with toxin B abolished the toxin-induced epithelial alterations for up to 8 h. The Ab neutralized basolateral toxin A for 4 h, but not the mixture of the two toxins. Using an identical Ab:Ag ratio, we found that recombinant polymeric IgA (IgAd/p) with the same Fv fragments extended protection against toxin A for at least 24 h in both compartments. In contrast, the recombinant monomeric IgA counterpart behaved as the PCG-4 IgG2a Ab. The direct comparison between different Ig isotype and molecular forms, but of unique specificity, demonstrates that IgAd/p Ab is more efficient in neutralizing toxin A than monomeric IgG and IgA. We conclude that immune protection against C. difficile toxins requires toxin A-specific secretory Abs in the intestinal lumen and IgAd/p specific for both toxins in the lamina propria.

Anti-inflammatory properties of secretory IgA on intestinal epithelial cells during infection by Shigella flexneri

The intestinal immune system hasthe complex task to protect the sterilecore of the organism against invasion.Most of invasive enterobacteria targetintestinal epithelial cells (IEC) inducingmajor damages to the mucosa.Shigella flexneri, by invading IECand inducing inflammatory responsesof the colonic mucosa, causes bacillarydysentery, a bloody diarrhea thatis endemic worldwide. The mechanismof entry of this bacterium is stilla matter of debate. Mcells participatingin sampling antigens from the gutlumen through Peyers patches arecommonly considered as the primarysite of entry of the bacteria. Once inthe lamina propria, Shigella can invadeIEC via their basolateral poleand spread from cell-to-cell leading tomassive tissue destruction. More recently,data are accumulating demonstratingthat bacteria can also enter thelamina propria directly via IEC, underscoringIEC as another gate of entry.In addition, the protective role ofsecretory IgA (SIgA) produced byplasmocytes of the lamina propria hasbeen established in shigellosis contextbut few is known about its role inmaintaining IEC monolayer integrity.Here, the impact of the bacterium wasstudied using polarized CaCo 2 cellmonolayer apically infected with avirulent strain of S. flexneri eitheralone or complexed with its cognateanti LPS SIgA. Parameters associatedwith the infection process includingcytokine measurements (IL-8, IL-18)and laser scanning confocal microscopydetection of Zonula Occludens-1, a tight junction (TJ) protein werestudied.We demonstrate that bacteriaare able to infect IEC through theirluminal-like pole as well, inducingthe complete disruption of TJ and thedestruction of the whole reconstitutedCaCo-2 cell monolayer. SIgA uponneutralization of bacteria led to themaintenance of TJ supporting IEC integrity,and the modulation of cytokinereleases. Together with anti-inflammatoryproperties of SIgA, thefact that apical bacteria can damagethe IEC without the intervention ofother cells such as Mcells offers newpossibilities in understanding thepathogenic mechanisms involved inshigellosis.