Bacterial microbiome of the nose of healthy dogs and dogs with nasal disease.

The role of bacterial communities in canine nasal disease has not been studied so far using next generation sequencing methods. Sequencing of bacterial 16S rRNA genes has revealed that the canine upper respiratory tract harbors a diverse microbial community; however, changes in the composition of nasal bacterial communities in dogs with nasal disease have not been described so far. Aim of the study was to characterize the nasal microbiome of healthy dogs and compare it to that of dogs with histologically confirmed nasal neoplasia and chronic rhinitis. Nasal swabs were collected from healthy dogs (n = 23), dogs with malignant nasal neoplasia (n = 16), and dogs with chronic rhinitis (n = 8). Bacterial DNA was extracted and sequencing of the bacterial 16S rRNA gene was performed. Data were analyzed using Quantitative Insights Into Microbial Ecology (QIIME). A total of 376 Operational Taxonomic Units out of 26 bacterial phyla were detected. In healthy dogs, Moraxella spp. was the most common species, followed by Phyllobacterium spp., Cardiobacteriaceae, and Staphylococcus spp. While Moraxella spp. were significantly decreased in diseased compared to healthy dogs (p = 0.005), Pasteurellaceae were significantly increased (p = 0.001). Analysis of similarities used on the unweighted UniFrac distance metric (p = 0.027) was significantly different when nasal microbial communities of healthy dogs were compared to those of dogs with nasal disease. The study showed that the canine nasal cavity is inhabited by a highly species-rich bacterial community, and suggests significant differences between the nasal microbiome of healthy dogs and dogs with nasal disease.

Eosinophilic granuloma with Splendore-Hoeppli material caused by toxigenic Corynebacterium ulcerans in a heifer.

Raised lesions were present on the left nasal vestibule of a 20-month-old Japanese Brown heifer. The largest mass which caused partial nasal obstruction was removed surgically. Corynebacterium ulcerans was identified in the mass. 16S ribosomal RNA and RNA polymerase beta subunit genes were 100% and 98% identical to other C. ulcerans strains. Histologically, multiple foci of eosinophilic granuloma with Splendore-Hoeppli material were seen. Rod-shaped Gram-positive organisms were detected with metachromatic granules, producing diphtheria toxin with 5, 30 and 48 amino acid differences to another C. ulcerans strain, C. diphtheriae or C. pseudotuberculosis, respectively. The toxin is highly cytotoxic and may be responsible for the formation of abundant Splendore-Hoeppli material. The lesion was therefore judged to be an allergic reaction to bacterial antigens or diphtheria toxin.

Intracranial nasal natural killer/T-cell lymphoma: immunopathologically-confirmed case and review of literature.

Advances in immunophenotypic profiling now permit characterization of natural killer/T-cell (NK/T-cell) lymphoma as distinct from other extranodal T- and B-cell Non-Hodgkin's lymphomas. NK/T-cell lymphoma presents most commonly in the nasal cavity. Disease progression to the central nervous system (CNS) is a rare phenomenon. We present here, to our knowledge, the first immunophenotypically-confirmed case of direct extension of nasal NK/T-cell lymphoma to the brain. In addition, we review the literature with respect to NK/T-cell lymphoma metastasis to the CNS. The overall prevalence of NK/T-cell lymphoma CNS metastasis is less than 3%. Although rare, CNS invasion portends a poor prognosis, emphasizing the importance of early and accurate immunophenotype profiling and the need for novel, aggressive therapy.

Fungal DNA is present in tissue specimens of patients with chronic rhinosinusitis.

BACKGROUND: It has been postulated that fungal organisms might represent the immunologic target initiating and maintaining the disease process in patients with chronic rhinosinusitis (CRS). The presence of fungi in nasal mucus has been established by different groups, but so far it has not been shown how the immune system could even recognize such extramucosal--extracorporal--fungal targets. The aim of this study was to determine whether fungal DNA is present in tissue specimens taken from patients with polypoid CRS. METHODS: Twenty-seven surgical specimens were collected from patients suffering from CRS. Fifteen surgical specimens from healthy ethmoidal mucosa served as controls. A second set of controls consisted of five surgical specimens of acoustic neuroma, which were included to rule out contamination within the protocol. All paranasal tissue samples were treated and rinsed carefully with a solution of Dithiothreitol to digest any nasal mucus and ensure that only tissue was examined. A highly sensitive two-step polymerase chain reaction (PCR) was applied to detect fungal DNA, using one universal primer for unspecific detection of fungal DNA and a second primer pair specific for Alternaria. RESULTS: Fungal DNA was detected in all 27 CRS specimens equally with both PCR primers. Controls from healthy paranasal mucosa were positive using the panfungal primers in 10 of 15 cases but were all negative for Alternaria DNA. PCR was negative for fungal DNA in all five neuroma specimens. CONCLUSIONS: Fungal DNA can be detected within sinonasal tissue specimens of patients suffering from CRS. These findings need to be discussed with respect to the proposed hypothesis of the immune system recognizing extramucosal organisms and initiating an immune response in sensitized patients.

Minimally invasive treatment of the nasal inverted papilloma.

BACKGROUND: The purpose of this work is to evaluate our results in the treatment of the nasal inverted papillomas with an endoscopic approach using a retrospective case series. METHODS: Between 1993 and 2000 we treated 27 patients with nasal inverted papillomas. All patients underwent endoscopic nasal surgery under general anesthesia. None of the inverted papillomas extended outside of the paranasal sinuses. All tissue samples underwent polymerase chain reaction and hybridization in situ to detect genetic sequences of the human papilloma virus and Epstein Barr virus. RESULTS: The study population consisted of 16 men and 11 women with a median age of 52 years (range, 22-77 years). Ten patients (37%) had undergone a previous nasal surgery. The median follow-up was 5 years (range, 2-8 years). None of the patients presented with bilateral nasal involvement or a synchronous carcinoma. Seven patients underwent an additional surgical approach (two endoscopic approaches via a Caldwel-Luc approach, four sublabial approaches via a Caldwel-Luc approach, and one external ethmoidectomy). There were no surgical complications. Two patients (7%) had recurrent papilloma 4 and 6 years after surgery and again underwent endoscopic resection. The amplification both by polymerase chain reaction and hybridization in situ for human papilloma virus and Epstein Barr virus were negative in the specimens from all patients. CONCLUSIONS: According to the literature and our own experience, we believe that the initial surgical management of primary and recurrent inverted papillomas limited to the nasal cavity and paranasal sinuses should be endoscopic sinus surgery.

Detection of epstein-barr virus in nasal T-cell lymphoma.

The close relationship between Epstein-Barr virus (EBV) and nasal T-cell lymphoma (NTL) has frequently been reported. However, the status of the infection, either lytic or latent, is obscure. This study involved 16 patients with NTL. Phenotypes of lymphoma cells were examined by immunohistochemical staining using CD3, CD4, CD8, CD20 and CD45RO monoclonal antibodies. EBV-encoded small nuclear RNA (EBER)-1 and EBV NotI tandem repeat region were detected by reverse transcription, using a rapid (< or = 60 min) in situ hybridization technique. Tumor cells expressed at least one T-cell marker, such as CD3, CD4, CD8 and CD45RO. CD20 was not detected in any of the cases. EBER-1 was identified in all cases; no Notl tandem DNA repeat was demonstrated. All cases demonstrated a T-cell phenotype. These data suggest that NTL is associated with EBV infection in the latent phase.

MALT lymphoma of nasal mucosa treated with antibiotics.

Mucosa associated lymphoid tissue (MALT) lymphomas occur in sites other than the gastrointestinal tract and in the early stages respond to treatment with antibiotics. We report a rare case of nasal mucosal MALT lymphoma which responded to treatment with antibiotics and has since then remained in remission.

Detection of Epstein-Barr virus genome in sinonasal undifferentiated carcinoma by use of in situ hybridization.

Associations between Epstein-Barr virus and undifferentiated carcinomas of nasopharynx, parotid gland, and thymus have recently been reported. Epstein-Barr virus has also been associated with malignant lymphoma of the nose and paranasal sinuses. These findings raise the possibility that Epstein-Barr virus may additionally be linked to undifferentiated carcinoma of the nose and paranasal sinuses (SNUC), an uncommon but distinctive and highly aggressive neoplasm. Histologically, SNUC consists of small and medium cells, the precise characterization of which often requires immunocytochemical analysis. This study investigates the presence of DNA sequences of Epstein-Barr virus in biopsy specimens of 13 cases of SNUC that were defined immunocytochemically by use of previously reported criteria. In situ hybridization was used to detect Epstein-Barr virus genome in different cell types in routinely processed, paraffin-embedded tissues. Epstein-Barr virus-specific DNA sequences were detected in tumor cells of SNUC specimens from 5 of the 13 cases examined. No correlation was found between positive hybridization and primary tumor site, morphologic subtype, or disease course. Epstein-Barr virus DNA was detected in 38% (5 of 13) of the SNUC samples analyzed. This finding suggests that this virus may play a role in the pathogenesis of this rare neoplasm.

A majority of inverted sinonasal papillomas carries Epstein-Barr virus genomes.

BACKGROUND: The relationship between sinonasal inverted papilloma (IP) and various strains of human papilloma virus (HPV) has been examined previously. Yet there is little consensus regarding the incidence or role of HPV in IP. The possible role of Epstein-Barr virus (EBV), which, like HPV, is a DNA virus linked to human lymphoid and epithelial malignancies, was investigated. METHODS: The polymerase chain reaction (PCR) was used to detect EBV genomic sequences in surgical specimens of IP, in benign nasal polyps, and various control tissues. The IP specimens were similarly examined for the presence of HPV types 6, 11, 16, and 18. RESULTS: EBV DNA was found in 13 of 20 IP specimens (65%) and none of the 10 control tissues. Nine of the 20 specimens contained HPV DNA, and 5 of 20 specimens contained both EBV and HPV. CONCLUSIONS: These results imply a previously unsuspected role for Epstein-Barr virus in the pathogenesis of sinonasal inverted papilloma.

Epstein-Barr virus is localized in the tumour cells of nasal lymphomas of NK, T or B cell type.

Seven cases of nasal lymphoma were studied to identify the lineage of Epstein-Barr virus (EBV)+ cells using dual-labelling methods. Five cases were phenotypically and genotypically of natural killer cell (NK) type with germ-line configuration of T-cell receptor (TcR) beta-chain gene and immunoglobulin heavy-chain joining region (IgJH) gene, with one case each of T- and B-cell type showing rearranged TcR beta or IgJH and lambda-light chain genes respectively. EBV genome was clonal in all these cases except in the B-cell case where its clonality was undeterminable. Using in situ hybridization (ISH) for EBV-encoded small nuclear RNA 1 and 2 (EBER), signal was detected in 45% to 88% of nucleated cells in the tumours. Immunostaining for EBV latent membrane protein-I (LMP) also revealed numerous LMP+ cells in 3/5 NK-type cases and the T- and B-cell cases. Using ISH for EBER combined with immunostaining for CD markers and double immunohistochemistry for LMP and CD markers, the predominant lineage of the EBV+ cells was identified as: CD2+CD3-CD19-CD20- CD45R0 +/- CD56+CD68- in the NK-type cases, CD2+CD3 +/- CD19-CD20- CD45R0+CD56-CD68- in the T-cell case and CD20+CD45R0-CD68- in the B-cell case, in agreement with the genotype and phenotype of each tumour. These results show that, in EBV+ nasal lymphomas of NK, T- or B-cell lineage, EBV was consistently associated with the tumour-cell population and support the view that EBV serves a promoting role in the pathogenesis of different types of EBV+ nasal lymphoma.

Detection of Epstein-Barr viral RNA in malignant lymphomas of the upper aerodigestive tract.

Recent studies have suggested a probable etiologic association between Epstein-Barr virus (EBV) and nasal lymphomas, irrespective of geographic location. This study was performed to investigate the strength of association of EBV with non-Hodgkin's lymphomas of the upper aerodigestive tract, based on a large series of cases that have been thoroughly immunophenotyped on frozen tissues. A sensitive in situ hybridization technique was used to detect EBV encoded RNA (EBER) in paraffin sections. Among 30 cases of nasal/nasopharyngeal T-cell lymphoma, 25 (83.3%) were EBER-positive. In the positive cases, most of the neoplastic cells showed strong nuclear signals. Further analysis of this group of tumors showed that all 21 cases (100%) with a CD56+ CD3-phenotype were EBER positive, whereas four of nine cases (44.4%) with a CD56-negative immunophenotype were positive. Only one of 10 cases (10%) of nasal/nasopharyngeal B-cell lymphoma was EBER positive; the positive case was a diffuse mixed-cell lymphoma and could not be distinguished morphologically from the negative cases. Among the 21 cases of lymphoma of the tonsils and back of the tongue (20 B-lineage and one T-lineage), none was EBER positive. In the normal mucosa of the nose/nasopharynx or tonsil (20 cases studied), only very rare EBER-positive small lymphocytes were found in two cases. The almost exclusive detection of EBER in nasal/nasopharyngeal T-cell neoplasms among the lymphomas of the upper aerodigestive tract suggests that EBV probably plays an etiologic role in the pathogenesis of this group of tumors and is not simply a passenger virus, and neither is this merely a site-dependent phenomenon in view of the weak association with nasal/nasopharyngeal B-cell lymphoma.

Epstein-Barr virus in nasal T-cell lymphomas (polymorphic reticulosis/midline malignant reticulosis) in western China.

Polymorphic reticulosis (PR) or midline malignant reticulosis (MMR) is considered to be malignant, or at least pre-malignant T-cell proliferations of the nose or midline area. Recent reports of small series of nasal T-cell lymphomas have shown a strong association with Epstein-Barr virus (EBV). Furthermore, a peculiar phenotype is described, with expression of CD56 and not of CD3, suggesting a possible origin from natural killer (NK) cells. We have analysed a series of 38 cases of PR/MMR for the presence of EBV by in situ hybridization (ISH) of the EBV-encoded RNAs 1 and 2 (EBER). Twenty cases were tested for expression of EBV-encoded latent membrane protein 1 (LMP-1). Special attention was also paid to the expression of CD3 and the NK cell-related marker CD56. Thirty-two cases (84 per cent) showed positive EBER ISH. In 5 of 20 cases, LMP-1 expression was detected. In three cases, a few scattered cells were positive, and in two cases, LMP-1 was detected in clusters of atypical cells. Most of the neoplasms showed expression of CD3 (89 per cent) and in 27 cases (71 per cent), CD56 was detected. These results are consistent with an aetiopathogenetic role for EBV in most, but not all, cases of PR/MMR. Our findings are less supportive of a major role for LMP-1 in tumour genesis. CD3 expression in most of the cases of PR/MMR underlines the T-cell origin of these neoplasms, often with aberrant expression of CD56.

Epstein-Barr virus in patients with polymorphic reticulosis (lethal midline granuloma) from China and Japan.

BACKGROUND: Polymorphic reticulosis is one of several diseases constituting lethal midline granuloma (LMG). Previous immunohistochemical studies suggested a T-cell nature of proliferating cells; the term nasal T-cell lymphoma (NTL-LMG) has since been used widely. The authors' previous study in Asian countries showed the clustering of Mongolian patients with NTL-LMG, but the frequency varied with geographic area; it was much higher in Korea and southwest Japan (Okinawa) than in Shanghai and Honshu, Japan. Recently an etiologic role of Epstein-Barr virus (EBV) for the development of NTL-LMG has been postulated. METHODS: In this study, the presence of EBV and human T-cell lymphocytic leukemia virus type 1 (HTLV-1) genomes were examined in NTL-LMG patients from Southwest Japan (Okinawa, 10 patients), another Japanese district (Honshu, 21 patients), and Shanghai, China (5 patients). All of the tissues from different geographic sites were analyzed at one central location. RESULTS: Immunohistochemistry showed that proliferating large cells were positive for CD43 and/or CD45RO, identical with reported NTL-LMG cases. Polymerase chain reaction (PCR) revealed the presence of EBV genome in the NTL-LMG lesions, but the frequency varied according to the geographic area: 67% in Okinawa, 33% in Honshu, and 100% in Shanghai. In situ hybridization provided positive signals in the nuclei of proliferating cells. Expression of latent membrane protein in the proliferating cells of cases positive for EBV by PCR and in situ hybridization was confirmed. CONCLUSIONS: The results suggest that the EBV may play a role in the development of NTL-LMG. However, the variation of frequency of EBV genome in different geographic locations suggests that EBV infection may not be an indispensable condition for the disease.

[Epstein-Barr virus infection in midline malignant reticulosis].

An oligonucleotide probe directed against small RNAs encoded by EBV gene was used to detect EBV genome by in situ hybridization combined with immunophenotype analysis. The results showed that (1) in 18 of 19 cases (94.7%) atypical lymphoid cells (ALC) expressed T-cell differentiation antigen, 15 cases were CD3 positive, 9 cases were UCHL1 positive, 6 cases were both CD3 and UCHL1 positive, no ALC gave positive reaction to B-cell and histiocyte markers. (2) 15 of 19 cases gave positive reaction to EBER (small RNAs encoded by EBV gene) by in situ hybridization analysis and ALC were reactive to T-cell markers in all 15 cases but one. The results of this study suggest that EBV infection is also present in midline malignant reticulosis, which is characterized by active T-cell proliferation. The role and effect of EBV infection in pathogenesis of lymphoproliferative disorders was discussed.

Detection of Epstein-Barr viral RNA in sinonasal undifferentiated carcinoma from Western and Asian patients.

Undifferentiated carcinoma of the nasopharynx has a well-known association with Epstein-Barr virus (EBV), but only an inconsistent relationship has been identified in undifferentiated carcinomas occurring at other sites. We investigated 22 formalin-fixed, paraffin-embedded cases of sinonasal undifferentiated carcinomas (SNUCs) occurring in Western and Asian patients. A highly sensitive in situ hybridization method was performed using an antisense oligonucleotide probe to the EBER1 gene of EBV. We identified EBV RNA in seven of 11 SNUCs from Asian patients, but in none of the Western SNUC patients (0/11). The EBER1 signal was present in all or virtually all of the tumor cell nuclei in the seven EBV-RNA-positive Asian SNUCs. The latent membrane protein-1 (LMP1) of EBV was not identified in any of the five positive cases tested. Our results suggest that genetic predisposition or environmental/geographical cofactors play an important role in determining the strength of the association of SNUC with EBV.

Nasal T-cell lymphoma: a clinicopathologic entity associated with peculiar phenotype and with Epstein-Barr virus.

Recent evidence has shown that most nasal lymphomas (NL) are associated with a T-cell phenotype and are thus called nasal T-cell lymphomas (NTCL), but little information is available about the T-cell receptor (TCR) expression. The presence of Epstein-Barr virus (EBV) genome has been recently reported in NTCL in Oriental populations in which NL and EBV-associated tumors are more common and in occasional Occidental cases. This prompted us to investigate lymphoma biopsies from 7 non-Oriental patients with NTCL for the expression of natural killer (NK) and T-cell antigens, including TCR proteins, for the presence of EBV-encoded latent membrane protein (LMP) using immunohistochemistry and for the presence of EBV DNA and Epstein-Barr early region (EBER) RNA using in situ hybridization (ISH). Six cases displayed a CD3-, TCR alpha beta-, TCR gamma delta-, CD2+, CD7+, CD5-, CD4-, CD8-, CD56+ phenotype, suggesting that these tumors may be peripheral T-cell lymphomas (PTCL) with extensive loss of T-cell antigens and expression of the NK-cell (CD56) antigen or, alternatively, NK-cell neoplasias. The remaining case was a gamma delta PTCL, as shown by the CD3+, TCR gamma delta+ phenotype and the biallelic gamma and delta TCR gene rearrangements. Using ISH, EBER RNA transcripts were detected in tumor cells in all cases and EBV DNA was shown in the 6 tested cases. In all cases, tumor cells expressed LMP. These findings support the concept that NTCL constitute a distinct group of lymphomas that, in addition to their peculiar clinical features, exhibit an unusual TCR "silent" CD56+ or TCR gamma delta+ phenotype and harbor the EBV. In view of the LMP transforming potential, these data suggest that EBV may play a role in the pathogenesis of NTCL.

Nasal lymphomas in Peru. High incidence of T-cell immunophenotype and Epstein-Barr virus infection.

The incidence of non-Hodgkin's lymphoma of the nasal region is much higher in Peru than in the United States and is similar to the incidence of sinonasal lymphomas in Asian countries. To characterize these lymphomas, we evaluated the clinical, morphologic, and immunohistochemical features of 14 cases and also analyzed the cases for Epstein-Barr virus (EBV) RNA using a sensitive and specific in situ hybridization method. Morphologically, the cases consisted of nine large cell immunoblastic lymphomas, one diffuse mixed cell lymphoma, one diffuse small cleaved lymphoma, one small noncleaved lymphoma, and two cases unclassifiable in the Working Formulation. Eleven cases demonstrated evidence of T lineage, two were of B lineage and one of indeterminate immunophenotype. In 13 of the lymphoma cases including all of the T-cell lymphomas, EBV RNA was detected in a high percentage of cells. Double-labeling immunohistochemical and in situ hybridization studies identified CD43 positivity in the cells labeling for EBV RNA. Much smaller amounts of EBV RNA were detectable in six of eight control benign nasopharyngeal biopsy specimens, and two were completely negative. These findings are similar to the prevalence of EBV-positive T-cell lymphomas in Asian countries and differ from the findings of the more common EBV-negative B-cell nasal lymphomas in the United States. These findings suggest that EBV plays a role in the development of nasal T-cell lymphomas and that the incidence of EBV infection may explain the reported "East-West" difference in the incidence of nasal T-cell lymphomas.

Association of human papillomavirus with nasal neoplasia.

Since HPV-57b has been identified by two different techniques in benign, premalignant, and malignant lesions of the nasal cavity, but not in cases of chronic sinusitis, HPV-57 should be recognised as at least a co-factor in the aetiology of nasal neoplasia. Paraffin sections of 22 histologically confirmed nasal tumours were screened by in-situ hybridisation with riboprobes specific for HPV-57b. Virus was demonstrated in 6 of 7 fungiform papillomas, 6 of 8 inverted papillomas, 1 of 3 inverted papillomas with dysplasia, and 2 of 4 inverted papillomas with carcinoma. The presence of HPV-57b was confirmed with the polymerase chain reaction, which identified an additional 4 positive samples, bringing the total to 86% positive specimens. The results underscore the importance of HPVs in the aetiology of cancers at extragenital sites.