Primary head and neck cancer cell cultures are susceptible to proliferation of Epstein-Barr virus infected lymphocytes.

BACKGROUND: New concepts for a more effective anti-cancer therapy are urgently needed. Experimental flaws represent a major counter player of this development and lead to inaccurate and unreproducible data as well as unsuccessful translation of research approaches into clinics. In a previous study we have created epithelial cell cultures from head and neck squamous cell carcinoma (HNSCC) tissue. METHODS: We characterize primary cell populations isolated from human papillomavirus positive HNSCC tissue for their marker expression by RT-qPCR, flow cytometry, and immunofluorescence staining. Their sensitivity to MDM2-inhibition was measured using cell viability assays. RESULTS: Primary HNSCC cell cultures showed the delayed formation of spheroids at higher passages. These spheroids mimicked the morphology and growth characteristics of other established HNSCC spheroid models. However, expression of epithelial and mesenchymal markers could not be detected in these cells despite the presence of the HNSCC stem cell marker aldehyde dehydrogenase 1 family member A1. Instead, strong expression of B- and T-lymphocytes markers was observed. Flow cytometry analysis revealed a heterogeneous mixture of CD3 + /CD25 + T-lymphocytes and CD19 + B-lymphocytes at a ratio of 4:1 at passage 5 and transformed lymphocytes at late passages (≥ passage 12) with CD45 + CD19 + CD20 + , of which around 10 to 20% were CD3 + CD25 + CD56 + . Interestingly, the whole population was FOXP3-positive indicative of regulatory B-cells (Bregs). Expression of transcripts specific for the Epstein-Barr-virus (EBV) was detected to increase in these spheroid cells along late passages, and this population was vulnerable to MDM2 inhibition. HPV + HNSCC cells but not EBV + lymphocytes were detected to engraft into immunodeficient mice. CONCLUSIONS: In this study we present a primary cell culture of EBV-infected tumor-infiltrating B-lymphocytes, which could be used to study the role of these cells in tumor biology in future research projects. Moreover, by describing the detailed characteristics of these cells, we aim to caution other researchers in the HNSCC field to test for EBV-infected lymphocyte contaminations in primary cell cultures ahead of further experiments. Especially researchers who are interested in TIL-based adopted immunotherapy should exclude these cells in their primary tumor models, e.g. by MDM2-inhibitor treatment. BI-12-derived xenograft tumors represent a suitable model for in vivo targeting studies.

Viral and Clinical Oncology of Head and Neck Cancers.

PURPOSE OF REVIEW: This study assesses the current state of knowledge of head and neck squamous cell carcinomas (HNSCC), which are malignancies arising from the orifices and adjacent mucosae of the aerodigestive tracts. These contiguous anatomical areas are unique in that 2 important human oncoviruses, Epstein-Barr virus (EBV) and human papillomavirus (HPV), are causally associated with nasopharyngeal and oropharyngeal cancers, respectively. Mortality rates have remained high over the last 4 decades, and insufficient attention paid to the unique viral and clinical oncology of the different subgroups of HNSCC. RECENT FINDINGS: We have compared and contrasted the 2 double-stranded DNA viruses and the relevant molecular oncogenesis of their respective cancers against other head and neck cancers. Tobacco and alcohol ingestion are also reviewed, as regard the genetic progression/mutation accumulation model of carcinogenesis. The importance of stringent stratification when searching for cancer mutations and biomarkers is discussed. Evidence is presented for a dysplastic/pre-invasive cancerous phase for HPV+ oropharyngeal cancers, and analogous with other HPV+ cancers. This raises the possibility of strategies for cancer screening as early diagnosis will undoubtedly save lives. Staging and prognostication have changed to take into account the distinct biological and prognostic pathways for viral+ and viral- cancers. Diagnosis of pre-cancers and early stage cancers will reduce mortality rates. Multi-modal treatment options for HNSCC are reviewed, especially recent developments with immunotherapies and precision medicine strategies. Knowledge integration of the viral and molecular oncogenic pathways with sound planning, hypothesis generation, and clinical trials will continue to provide therapeutic options in the future.

Bi-allelic Mutations in Phe-tRNA Synthetase Associated with a Multi-system Pulmonary Disease Support Non-translational Function.

The tRNA synthetases catalyze the first step of protein synthesis and have increasingly been studied for their nuclear and extra-cellular ex-translational activities. Human genetic conditions such as Charcot-Marie-Tooth have been attributed to dominant gain-of-function mutations in some tRNA synthetases. Unlike dominantly inherited gain-of-function mutations, recessive loss-of-function mutations can potentially elucidate ex-translational activities. We present here five individuals from four families with a multi-system disease associated with bi-allelic mutations in FARSB that encodes the beta chain of the alpha2beta2 phenylalanine-tRNA synthetase (FARS). Collectively, the mutant alleles encompass a 5'-splice junction non-coding variant (SJV) and six missense variants, one of which is shared by unrelated individuals. The clinical condition is characterized by interstitial lung disease, cerebral aneurysms and brain calcifications, and cirrhosis. For the SJV, we confirmed exon skipping leading to a frameshift associated with noncatalytic activity. While the bi-allelic combination of the SJV with a p.Arg305Gln missense mutation in two individuals led to severe disease, cells from neither the asymptomatic heterozygous carriers nor the compound heterozygous affected individual had any defect in protein synthesis. These results support a disease mechanism independent of tRNA synthetase activities in protein translation and suggest that this FARS activity is essential for normal function in multiple organs.

FARS1-related disorders caused by bi-allelic mutations in cytosolic phenylalanyl-tRNA synthetase genes: Look beyond the lungs!

Aminoacyl-tRNA synthetases (ARSs) catalyze the first step of protein biosynthesis (canonical function) and have additional (non-canonical) functions outside of translation. Bi-allelic pathogenic variants in genes encoding ARSs are associated with various recessive mitochondrial and multisystem disorders. We describe here a multisystem clinical phenotype based on bi-allelic mutations in the two genes (FARSA, FARSB) encoding distinct subunits for tetrameric cytosolic phenylalanyl-tRNA synthetase (FARS1). Interstitial lung disease with cholesterol pneumonitis on histology emerged as an early characteristic feature and significantly determined disease burden. Additional clinical characteristics of the patients included neurological findings, liver dysfunction, and connective tissue, muscular and vascular abnormalities. Structural modeling of newly identified missense mutations in the alpha subunit of FARS1, FARSA, showed exclusive mapping to the enzyme's conserved catalytic domain. Patient-derived mutant cells displayed compromised aminoacylation activity in two cases, while remaining unaffected in another. Collectively, these findings expand current knowledge about the human ARS disease spectrum and support a loss of canonical and non-canonical function in FARS1-associated recessive disease.

Prognostic Factors for Long-Term Survival after Surgical Resection of Primary Cardiac Sarcoma.

BACKGROUND: Primary cardiac sarcoma (CS) is an extremely rare disease. This study aims to identify possible prognostic factors for long-term survival. METHODS: A total of 17 consecutive patients who were treated for primary CS between 2003 und 2018 at two cardiac centers were investigated. Clinical data and histological characteristics of the tumors were analyzed. Long-term follow-up of all patients were performed. RESULTS: The median age was 54 years (range: 23-74). The tumors originated from the left side of the heart in nine patients. Histologically, there were four angiosarcomas, three intimal sarcomas, and three synovial sarcomas. One- and 7-year survivals were 81.9 and 18.2%, respectively. Low expression levels of Ki-67 tended to be associated with increased survival (log-rank p = 0.06). Adjuvant chemotherapy but not radiotherapy regardless of existing metastases was associated with significantly increased survival (log-rank p = 0.001). CONCLUSION: Angiosarcoma was the most common type of CS. The survival of CS patients is poor but prognostic factors, such as Ki-67, may help estimate the course of the disease. Survival could be improved significantly with chemotherapy.

Lung disease caused by ABCA3 mutations.

BACKGROUND: Knowledge about the clinical spectrum of lung disease caused by variations in the ATP binding cassette subfamily A member 3 (ABCA3) gene is limited. Here we describe genotype-phenotype correlations in a European cohort. METHODS: We retrospectively analysed baseline and outcome characteristics of 40 patients with two disease-causing ABCA3 mutations collected between 2001 and 2015. RESULTS: Of 22 homozygous (15 male) and 18 compound heterozygous patients (3 male), 37 presented with neonatal respiratory distress syndrome as term babies. At follow-up, two major phenotypes are documented: patients with (1) early lethal mutations subdivided into (1a) dying within the first 6 months or (1b) before the age of 5 years, and (2) patients with prolonged survival into childhood, adolescence or adulthood. Patients with null/null mutations predicting complete ABCA3 deficiency died within the 1st weeks to months of life, while those with null/other or other/other mutations had a more variable presentation and outcome. Treatment with exogenous surfactant, systemic steroids, hydroxychloroquine and whole lung lavages had apparent but many times transient effects in individual subjects. CONCLUSIONS: Overall long-term (>5 years) survival of subjects with two disease-causing ABCA3 mutations was <20%. Response to therapies needs to be ascertained in randomised controlled trials.

Persistent Tachypnea of Infancy. Usual and Aberrant.

RATIONALE: Persistent tachypnea of infancy (PTI) is a specific clinical entity of undefined etiology comprising the two diseases neuroendocrine cell hyperplasia of infancy (NEHI) and pulmonary interstitial glycogenosis. The outcome of typical NEHI is favorable. The outcome may be different for patients without a typical NEHI presentation, and thus a lung biopsy to differentiate the diseases is indicated. OBJECTIVES: To determine whether infants with the characteristic clinical presentation and computed tomographic (CT) imaging of NEHI (referred to as "usual PTI") have long-term outcome and biopsy findings similar to those of infants with an aberrant presentation and/or with additional localized minor CT findings (referred to as "aberrant PTI"). METHODS: In a retrospective cohort study, 89 infants with PTI were diagnosed on the basis of clinical symptoms and, if available, CT scans and lung biopsies. Long-term outcome in childhood was measured on the basis of current status. MEASUREMENTS AND MAIN RESULTS: Infants with usual PTI had the same respiratory and overall outcomes during follow-up of up to 12 years (mean, 3.8 yr) as infants who had some additional localized minor findings (aberrant PTI) visualized on CT images. Both usual and aberrant PTI had a relatively favorable prognosis, with 50% of the subjects fully recovered by age 2.6 years. None of the infants died during the study period. This was independent of the presence or absence of histological examination. CONCLUSIONS: PTI can be diagnosed on the basis of typical history taking, clinical findings, and a high-quality CT scan. Further diagnostic measures, including lung biopsies, may be limited to rare, complicated cases, reducing the need for an invasive and potentially harmful procedure.

Genotype alone does not predict the clinical course of SFTPC deficiency in paediatric patients.

Patients with interstitial lung disease due to surfactant protein C (SFTPC) mutations are rare and not well characterised. We report on all subjects collected over a 15-year period in the kids-lung register with interstitial lung disease and a proven SFTPC mutation. We analysed clinical courses, interventions and outcomes, as well as histopathological and radiological interrelations. 17 patients (seven male) were followed over a median of 3 years (range 0.3-19). All patients were heterozygous carriers of autosomal dominant SFTPC mutations. Three mutations (p.L101P, p.E191 K and p.E191*) have not been described before in the context of surfactant protein C deficiency. Patients with alterations in the BRICHOS domain of the protein (amino acids 94-197) presented earlier. At follow-up, one patient was healthy (2 years), six patients were "sick-better" (2.8 years, range 0.8-19), seven patients were "sick-same" (6.5 years, 1.3-15.8) and three patients were "sick-worse" (0.3 years, 0.3-16.9). Radiological findings changed from ground-glass to increasing signs of fibrosis and cyst formation with increasing age. Empiric treatments had variable effects, also in patients with the same genotype. Prospective studies with randomised interventions are urgently needed and can best be performed in the framework of international registers.

Case Report: A rare case of bilateral middle ear tophaceous gout.

Introduction: Hypacusia can be caused by various etiologies; however, hearing loss attributed to gouty tophi remains a rare occurrence. This case report presents, for the first time, a bilateral gouty tophi causing hearing impairment. Case presentation: This report describes a case study involving an 83-year-old Caucasian female patient who presented symptoms of hypacusia, pruritus, and a sensation of pressure in her right ear. A computed tomography scan revealed the presence of non-homogeneous calcified structures in both ears. Following a comprehensive assessment that included pure-tone audiometry and a thorough evaluation of the patient's clinical complaints, a tympanoplasty procedure was initially performed on the right ear. Pathological analysis revealed the presence of gouty tophi. After surgical removal of the tophus, a notable improvement in the patient's hearing threshold was observed. Four months later, a similar surgical intervention was performed on the contralateral ear, achieving a similar positive outcome. The substantial postoperative decrease of bone conduction indicates an inner ear affection by the gout tophi. Conclusion: Gout tophus in both ears is a very rare but possible cause of hypacusia, even in the absence of a pre-existing diagnosis of systemic gout disease. We report a case of gout tophi in both ears as a rare cause of hearing loss.

Human papillomavirus-associated head and neck squamous cell carcinoma cells lose viability during triggered myocyte lineage differentiation.

Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease, and death rates have remained at approximately 50% for decades. New tumor-targeting strategies are desperately needed, and a previous report indicated the triggered differentiation of HPV-negative HNSCC cells to confer therapeutic benefits. Using patient-derived tumor cells, we created a similar HNSCC differentiation model of HPV+ tumor cells from two patients. We observed a loss of malignant characteristics in differentiating cell culture conditions, including irregularly enlarged cell morphology, cell cycle arrest with downregulation of Ki67, and reduced cell viability. RNA-Seq showed myocyte-like differentiation with upregulation of markers of myofibril assembly. Immunofluorescence staining of differentiated and undifferentiated primary HPV+ HNSCC cells confirmed an upregulation of these markers and the formation of parallel actin fibers reminiscent of myoblast-lineage cells. Moreover, immunofluorescence of HPV+ tumor tissue revealed areas of cells co-expressing the identified markers of myofibril assembly, HPV surrogate marker p16, and stress-associated basal keratinocyte marker KRT17, indicating that the observed myocyte-like in vitro differentiation occurs in human tissue. We are the first to report that carcinoma cells can undergo a triggered myocyte-like differentiation, and our study suggests that the targeted differentiation of HPV+ HNSCCs might be therapeutically valuable.

Long-term follow-up and treatment of congenital alveolar proteinosis.

BACKGROUND: Clinical presentation, diagnosis, management and outcome of molecularly defined congenital pulmonary alveolar proteinosis (PAP) due to mutations in the GM-CSF receptor are not well known. CASE PRESENTATION: A 2 1/2 years old girl was diagnosed as having alveolar proteinosis. Whole lung lavages were performed with a new catheter balloon technique, feasible in small sized airways. Because of some interstitial inflammation in the lung biopsy and to further improve the condition, empirical therapy with systemic steroids and azathioprin, and inhaled and subcutaneous GMCSF, were used. Based on clinical measures, total protein and lipid recovered by whole lung lavages, all these treatments were without benefit. Conversely, severe respiratory viral infections and an invasive aspergillosis with aspergilloma formation occurred. Recently the novel homozygous stop mutation p.Ser25X of the GMCSF receptor alpha chain was identified in the patient. This mutation leads to a lack of functional GMCSF receptor and a reduced response to GMCSF stimulation of CD11b expression of mononuclear cells of the patient. Subsequently a very intense treatment with monthly lavages was initiated, resulting for the first time in complete resolution of partial respiratory insufficiency and a significant improvement of the overall somato-psychosocial condition of the child. CONCLUSIONS: The long term management from early childhood into young adolescence of severe alveolar proteinosis due to GMCSF receptor deficiency requires a dedicated specialized team to perform technically demanding whole lung lavages and cope with complications.

Lymphocytic interstitial pneumonia and follicular bronchiolitis in children: A registry-based case series.

OBJECTIVES: Pediatric lymphocytic interstitial pneumonia (LIP) and follicular bronchiolitis (FB) are poorly characterized lymphoproliferative disorders. We present and quantify demographics, radiological and histopathologic patterns, treatments and their responses, and outcomes in non-HIV-infected children with LIP and FB. METHODS: This structured registry-based study included a retrospective chart review, blinded analysis of imaging studies and lung biopsies, genetic testing, and evaluation of treatments and outcomes. RESULTS: Of the 13 patients (eight females) studied, eight had FB, four had combined LIP/FB, and one had isolated LIP; diagnoses were highly concordant between the pathologists. Most patients became symptomatic during the first 2 years of life, with a mean lag time to diagnosis of 4 years. The most common symptoms were coughing and respiratory infections (11 out of 13 each), dyspnea (10 out of 13), and wheezing (eight out of 13). Autoantibodies were found in eight out of 13 patients. In three patients, disease-causing mutations in the COPA gene were identified. CT revealed hilar lymphadenopathy (five out of 12), ground-glass opacity (eight out of 12), consolidation (five out of 12), and cysts (four out of 13). Systemic steroids as intravenous pulses (11 out of 13) or oral intake (10 out of 13) were the main treatments and showed high response rates of 100% and 90%, respectively. Within the mean observation period of 68 months, all children had chronic courses, eight out of 13 had severe diseases, two died, and one worsened. CONCLUSIONS: Children with LIP/FB have chronic diseases that occurred in early childhood and were commonly associated with immune dysregulation as well as high morbidity and mortality. Early diagnosis and treatment may be crucial to improve the outcome.

Hermansky-Pudlak syndrome type 2 manifests with fibrosing lung disease early in childhood.

BACKGROUND: Hermansky-Pudlak syndrome (HPS), a hereditary multisystem disorder with oculocutaneous albinism, may be caused by mutations in one of at least 10 separate genes. The HPS-2 subtype is distinguished by the presence of neutropenia and knowledge of its pulmonary phenotype in children is scarce. METHODS: Six children with genetically proven HPS-2 presented to the chILD-EU register between 2009 and 2017; the data were collected systematically and imaging studies were scored blinded. RESULTS: Pulmonary symptoms including dyspnea, coughing, need for oxygen, and clubbing started 3.3 years before the diagnosis was made at the mean age of 8.83 years (range 2-15). All children had recurrent pulmonary infections, 3 had a spontaneous pneumothorax, and 4 developed scoliosis. The frequency of pulmonary complaints increased over time. The leading radiographic pattern was ground-glass opacities with a rapid increase in reticular pattern and traction bronchiectasis between initial and follow-up Computer tomography (CT) in all subjects. Honeycombing and cysts were newly detectable in 3 patients. Half of the patients received a lung biopsy for diagnosis; histological patterns were cellular non-specific interstitial pneumonia, usual interstitial pneumonia-like, and desquamative interstitial pneumonia. CONCLUSIONS: HPS-2 is characterized by a rapidly fibrosing lung disease during early childhood. Effective treatments are required.

Tools to explore ABCA3 mutations causing interstitial lung disease.

BACKGROUND: Interstitial lung diseases (ILD) comprise disorders of mostly unknown cause. Among the few molecularly defined entities, mutations in the gene encoding the ATP-binding cassette (ABC), subfamily A, member 3 (ABCA3) lipid transporter represent the main cause of inherited surfactant dysfunction disorders, a subgroup of ILD. Whereas many cases are reported, specific methods to functionally define such mutations are rarely presented. MATERIALS AND METHODS: In this study, we exemplarily utilized a set of molecular tools to characterize the mutation K1388N, which had been identified in a patient suffering from ILD with lethal outcome. We also aimed to correlate in vitro and ex vivo findings. RESULTS: We found that presence of the K1388N mutation did not affect protein expression, but resulted in an altered protein processing and a functional impairment of ABCA3. This was demonstrated by decreased dipalmitoyl-phosphatidylcholine (PC 32:0) content and malformed lamellar bodies in cells transfected with the K1388N variant as compared to controls. CONCLUSIONS: Here we present a set of tools useful for categorizing different ABCA3 mutations according to their impact upon ABCA3 activity. Knowledge of the molecular defects and close correlation of in vitro and ex vivo data will allow us to define groups of mutations that can be targeted by small molecule correctors for restoring impaired ABCA3 transporter in the future. Pediatr Pulmonol. 2016;51:1284-1294. © 2016 Wiley Periodicals, Inc.

Decrease of the surface fraction of surfactant proteins containing clara cells and type II pneumocytes in a rat asthma model.

In asthma surfactant proteins (SP) might differ in distribution and composition and thus play a role in pathophysiology of this disease. Therefore, the well-established animal model of ovalbumin sensitized and challenged rats were used to study the distribution of surfactant proteins in Clara cells and type II pneumocytes. Serial sections of paraffin embedded lung tissue were sequentially immunostained by the avidin-biotin-peroxidase complex (ABC) technique. Antisera against SP-A, SP-B and Clara cell specific protein (CC10) were used. We determined stereologically' the surface fraction of immunolabelled cells and semiquantitatively the percentage of test fields containing labelled alveolar macrophages. In allergen sensitized and provocated rat lungs: (1) the surface fraction of SP-A and SP-B positive Clara cells was significantly reduced, (2) the surface fraction of Clara cells stained with CC10 was coincided with controls, (3) the surface fraction of SP-A and not of SP-B possitive type II pneumocytes decreased significantly, (4) a significantly higher percentage of test fields with SP-A labelled alveolar macrophages was evaluated. Thus, in this animal model of asthma the inflammatory process after allergen challenge is accompanied by alterations in the distribution patterns of SP in Clara cells and type II pneumocytes.

Categorizing diffuse parenchymal lung disease in children.

BACKGROUND: Aim of this study was to verify a systematic and practical categorization system that allows dynamic classification of pediatric DPLD irrespective of completeness of patient data. METHODS: The study was based on 2322 children submitted to the kids-lung-register between 1997 and 2012. Of these children 791 were assigned to 12 DPLD categories, more than 2/3 belonged to categories manifesting primarily in infancy. The work-flow of the pediatric DPLD categorization system included (i) the generation of a final working diagnosis, decision on the presence or absence of (ii) DPLD and (iii) a systemic or lung only condition, and (iv) the allocation to a category and subcategory. The validity and inter-observer dependency of this workflow was re-tested using a systematic sample of 100 cases. RESULTS: Two blinded raters allocated more than 80% of the re-categorized cases identically. Non-identical allocation was due to lack of appreciation of all available details, insufficient knowledge of the classification rules by the raters, incomplete patient data, and shortcomings of the classification system itself. CONCLUSIONS: This study provides a suitable workflow and hand-on rules for the categorization of pediatric DPLD. Potential pitfalls were identified and a foundation was laid for the development of consensus-based, international categorization guidelines.

HNPCC: six new pathogenic mutations.

BACKGROUND: Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disease with a high risk for colorectal and endometrial cancer caused by germline mutations in DNA mismatch-repair genes (MMR). HNPCC accounts for approximately 2 to 5% of all colorectal cancers. Here we present 6 novel mutations in the DNA mismatch-repair genes MLH1, MSH2 and MSH6. METHODS: Patients with clinical diagnosis of HNPCC were counselled. Tumor specimen were analysed for microsatellite instability and immunohistochemistry for MLH1, MSH2 and MSH6 protein was performed. If one of these proteins was not detectable in the tumor mutation analysis of the corresponding gene was carried out. RESULTS: We identified 6 frameshift mutations (2 in MLH1, 3 in MSH2, 1 in MSH6) resulting in a premature stop: two mutations in MLH1 (c.2198_2199insAACA [p.N733fsX745], c.2076_2077delTG [p.G693fsX702]), three mutations in MSH2 (c.810_811delGT [p.C271fsX282], c.763_766delAGTGinsTT [p.F255fsX282], c.873_876delGACT [p.L292fsX298]) and one mutation in MSH6 (c.1421_1422dupTG [p.C475fsX480]). All six tumors tested for microsatellite instability showed high levels of microsatellite instability (MSI-H). CONCLUSIONS: HNPCC in families with MSH6 germline mutations may show an age of onset that is comparable to this of patients with MLH1 and MSH2 mutations.

Collagenous colitis: implications for the role of vascular endothelial growth factor in repair mechanisms.

OBJECTIVES: Collagenous colitis is a chronic inflammatory bowel disease with a band-like subepithelial deposition of immature extracellular matrix. Because the extracellular matrix deposition is potentially reversible, an imbalance between fibrogenesis and fibrolysis with reduced matrix degradation has been suspected. Vascular endothelial growth factor plays a central role in extracellular matrix degradation. Therefore, we investigated the expression of vascular endothelial growth factor in the colonic mucosa of patients with collagenous colitis before and after long-term treatment with oral budesonide. METHOD: A quantitative immunohistochemical method was used to measure the amount of immunoreactive vascular endothelial growth factor, tenascin and leucocyte common antigen within the epithelium and the lamina propria of colonic biopsies by area morphometry. RESULTS: Strong immunostaining for vascular endothelial growth factor within the epithelium and the lamina propria, and for tenascin, was seen in patients with collagenous colitis compared with normal controls. The enhanced immunostaining for vascular endothelial growth factor within the lamina propria was accompanied by the accumulation of leucocytes, detected by staining for leucocyte common antigen. After long-term treatment with oral budesonide, the amount of immunostaining for leucocyte-derived vascular endothelial growth factor within the lamina propria decreased significantly to normal levels. In contrast, staining for vascular endothelial growth factor within the epithelium remained significantly increased. CONCLUSIONS: Our data suggest an important role of vascular endothelial growth factor in counteracting the local imbalance of fibrogenesis and fibrolysis, leading to an accumulation of immature subepithelial matrix in collagenous colitis.

Immunohistochemical localization of vascular endothelial growth factor in colonic mucosa of patients with inflammatory bowel disease.

BACKGROUND/AIMS: Significantly enhanced serum levels of VEGF (vascular endothelial growth factor) were found in patients with inflammatory bowel disease. Peripheral blood mononuclear cells have been identified as one of the origins of the circulating VEGF. The present investigation examines the localization of VEGF at the site of inflammation in colonic mucosa of patients with Crohn's disease and ulcerative colitis. METHODOLOGY: Immunohistochemical localization of VEGF and immunostaining for leukocytes were performed in colonic mucosal biopsies of 41 patients with Crohn's disease, 26 patients with ulcerative colitis and normal mucosal specimens of 5 patients with irritable bowel syndrome. Measurement of immunohistochemical staining for VEGF and for leukocytes within the epithelium and the lamina propria was performed separately by area morphometry using a computerized cell analysis system. RESULTS: In both patients with Crohn's disease and ulcerative colitis immunohistochemical staining for VEGF within the lamina propria of inflamed colonic mucosa was significantly higher compared with noninflamed mucosa (Crohn's disease: 4.26% vs. 0.07%, P < 0.001; ulcerative colitis: 3.68% vs. 0.32%, P = 0.001). There was a significant correlation between immunostaining for leukocytes and VEGF within the lamina propria in both patients with Crohn's disease (r = 0.73, P < 0.05)) and ulcerative colitis (r = 0.67, P < 0.05). In Crohn's disease immunostaining for VEGF within the epithelium was significantly higher in inflamed mucosa compared with noninflamed mucosa (9.85% vs. 0.63%, P < 0.001). In contrast, strong immunostaining for VEGF has been observed in the epithelium of noninflamed mucosa (7.60%, P < 0.003), as well as in inflamed mucosa of patients with active ulcerative colitis (9.68%, P < 0.002) compared with noninflamed mucosa of patients with inactive ulcerative colitis (1.39%). CONCLUSIONS: The present data indicate, that the increased VEGF expression within the epithelium and the interstitial accumulation of VEGF-producing leukocytes in inflamed mucosa may play an important role in the inflammatory mechanisms of Crohn's disease and ulcerative colitis.