Contribution of the lateral lemniscus to the control of swallowing in decerebrate cats.

Lateral lemniscus, a relay nucleus of auditory sensation, is involved in the control of phonatory movements such as human speech and vocalization of animals. The present study was designed to test whether neurons in the lateral lemniscus contributed to the control of swallowing, one of non-phonic oro-pharyngolaryngeal movements. In acutely decerebrated cats (n=15), swallowing was induced by electrical stimulation (20-80µA at 10Hz for 20s with rectangular pulses of 0.2ms duration) delivered to the superior laryngeal nerve (SLN). Repetitive electrical stimulation (30-50µA at 50Hz for 10-20s) applied to the dorsal nucleus of the lateral lemniscus (LLD) increased the number and reduced the latency to the onset of the SLN-induced swallowing. On the other hand, stimulation of the ventral nucleus of the lateral lemniscus and the paralemniscal area, corresponding to the ventrolateral part of the parabrachial nucleus and the Kölliker-Fuse nucleus, often suppressed the SLN-induced swallowing. Microinjection of NMDA (0.1-0.15µl, 5.0-10mM) into the LLD through a stereotaxically placed glass micropipette facilitated the SLN-induced swallowing, i.e., the number was increased and the latency of swallowing was reduced. We also injected muscimol (a gamma amino-butyric acid (GABA)A receptor agonist), bicuculline (a GABAA receptor antagonist) and baclofen (a GABAB receptor agonist) into the LLD (0.1-0.15µl and 5.0mM for each substance). It was observed that an injection of muscimol suppressed the SLN-induced swallowing. However, an injection of bicuculline facilitated the swallowing. An injection of baclofen did not alter the swallowing. These results suggest the presence of functional topography in the lateral lemniscus and the paralemniscal area in relation to the control of swallowing. The facilitatory LLD-effects on swallowing are modulated by glutamatergic and GABAergic receptors on neurons in the LLD.

EGFR inhibitors augment antitumour helper T-cell responses of HER family-specific immunotherapy.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a major cause of cancer-related morbidity and mortality worldwide. Epidermal growth factor receptor (EGFR)-targeted therapy is an attractive strategy alternative to conventional cancer treatments for HNSCC, but its efficacy remains controversial. T-cell-based immunotherapy has been proposed as a novel therapeutic approach to improve the clinical outcome for HNSCC. In this study, we report human epidermal receptor (HER) family epitopes that induced CD4 T-cell responses to HNSCC. The results provide support for a novel strategy to treat HNSCC by combining EGFR-targeted therapy with T-cell-based immunotherapy. METHODS: We evaluated the capacity of predicted CD4 T-cell peptide epitopes from EGFR to induce antitumour immune responses in vitro. In addition, EGFR inhibitors were evaluated for their ability to augment tumour MHC class II expression in HNSCC cell lines and subsequently increase T-cell recognition. RESULTS: Among several predicted peptide epitopes, EGFR875-889 elicited CD4 T-cell responses that were restricted by HLA-DR4, DR15, or DR53 molecules, indicating that the peptide functions as a promiscuous T-cell epitope. The peptide-reactive T cells responded to autologous dendritic cells loaded with EGFR-expressing tumour cell lysates, indicating that these epitopes are naturally processed. In addition, the CD4 T cells were capable of directly recognising and killing HNSCC cells expressing EGFR and the appropriate HLA class II molecule. T cells reactive with the EGFR875-889 epitope could be detected in the blood of HNSCC patients. EGFR875-889-reactive CD4 T cells were also able to recognise several peptide analogues derived from homologous regions of EGFR family members, HER-2, HER-3 and c-MET. Finally, we examined the effects of EGFR tyrosine kinase inhibition or EGFR-blocking antibodies on CD4 T-cell tumour reactivity. Treatment of tumour cells with the EGFR inhibitors enhanced tumour recognition by EGFR875-889-reactive T cells presumably due to the upregulation of HLA-DR expression in the HNSCC cells. CONCLUSION: We identified novel CD4 T-cell EGFR epitopes and amongst these, EGFR875-889 functions as a promiscuous helper T-cell epitope that can elicit effective antitumour T-cell responses against tumours expressing HER family members and c-MET. These observations should facilitate the translation of T-cell-based immunotherapy into the clinic for the treatment of HNSCC and provide a rational basis for EGFR inhibition, immune-targeted combination therapy.

Tumour suppressive microRNA-874 regulates novel cancer networks in maxillary sinus squamous cell carcinoma.

BACKGROUND: On the basis of the microRNA (miRNA) expression signature of maxillary sinus squamous cell carcinoma (MSSCC), we found that miR-874 was significantly reduced in cancer cells. We focused on the functional significance of miR-874 in cancer cells and identification of miR-874-regulated novel cancer networks in MSSCC. METHODS: We used PCR-based methods to investigate the downregulated miRNAs in clinical specimens of MSSCC. Our signature analyses identified 23 miRNAs that were significantly reduced in cancer cells, such as miR-874, miR-133a, miR-375, miR-204, and miR-1. We focused on miR-874 as the most downregulated novel miRNA in our analysis. RESULTS: We found potential tumour suppressive functions such as inhibition of cancer cell proliferation and invasion. A molecular target search of miR-874 revealed that PPP1CA was directly regulated by miR-874. Overexpression of PPP1CA was observed in MSSCC clinical specimens. Silencing of the PPP1CA gene significantly inhibited cancer cell proliferation and invasion. CONCLUSION: The downregulation of miR-874 was a frequent event in MSSCC, which suggests that miR-874 functions as a tumour suppressive miRNA, directly regulating PPP1CA that has a potential role of an oncogene. The identification of novel miR-874-regulated cancer pathways could provide new insights into potential molecular mechanisms of MSSCC oncogenesis.

Primary small cell carcinoma of lacrimal sac: case report and literature review.

OBJECTIVE: We present the first reported case of primary small cell carcinoma of the lacrimal sac. CASE REPORT: A 67-year-old Japanese woman was referred to our department with a two-month history of left medial canthal swelling, epiphora and occasional nasal bleeding. Nasal endoscopy revealed a readily bleeding tumour in the left inferior meatus. Computed tomography and magnetic resonance imaging scans demonstrated that the tumour was mainly located in the left lacrimal sac. Histopathological studies of a biopsy specimen revealed small cell carcinoma. The patient was treated with four cycles of chemotherapy consisting of cisplatin and etoposide, in combination with radiotherapy. There was no evidence of recurrence or metastasis for five years. CONCLUSION: Small cell carcinoma originating in the head and neck region has been reported to be highly aggressive and to have a poor prognosis. We report a case of primary small cell carcinoma of the lacrimal sac successfully treated with chemo-radiotherapy.

Up-regulation of CC chemokine receptor 6 on tonsillar T cells and its induction by in vitro stimulation with alpha-streptococci in patients with pustulosis palmaris et plantaris.

Pustulosis palmaris et plantaris (PPP) is a tonsil-related disease; tonsillectomy is somewhat effective in treating the condition. However, the aetiological association between the tonsils and PPP has not yet been elucidated fully. Recently, some chemokines and chemokine receptors, including CC chemokine receptor (CCR) 4, CCR6 and CX chemokine receptor (CXCR) 3, have been reported to play important roles in the development of psoriasis, a disease related closely to PPP. In this study, we found that CCR6 expression on both tonsillar and peripheral blood T cells was up-regulated more intensively in PPP patients than in non-PPP patients (P < 0.001 for both), but CCR4 and CXCR3 expressions were not. In vitro stimulation with alpha-streptococcal antigen enhanced CCR6 expression significantly on tonsillar T cells in PPP patients (P < 0.05), but this was not observed in non-PPP patients. The chemotactic response of tonsillar T cells to the CCR6 ligand CC chemokine ligand (CCL) 20 was significantly higher in PPP patients than in non-PPP patients (P < 0.05). The percentage of CCR6-positive peripheral blood T cells decreased after tonsillectomy in PPP patients (P < 0.01); this decrease correlated with an improvement of skin lesions (P < 0.05, r = -0.63). The numbers of CCR6-positive cells and the expression of CCL20 were increased significantly in pathological lesions compared with non-pathological lesions in PPP skin (P < 0.01, P < 0.05 respectively). These results suggest that a novel immune response to alpha-streptococci may enhance CCR6 expression on T cells in tonsils and that CCR6-positive T cells may move to peripheral blood circulation, resulting in recruitment to target skin lesions expressing CCL20 in PPP patients. This may be one of the key roles in pathogenesis of the tonsil-related disease PPP.

Selective expansion of T cell receptor (TCR) V beta 6 in tonsillar and peripheral blood T cells and its induction by in vitro stimulation with Haemophilus parainfluenzae in patients with IgA nephropathy.

IgA nephropathy (IgAN), the most common form of primary glomerulonephritis, is recognized as a disease that often becomes worse during acute tonsillitis. Although many reports have shown that tonsillectomy is an effective treatment for IgAN patients, the immunological evidence has not yet been investigated fully. In this study, we compared the expression of T cell receptor (TCR) V beta families in tonsillar T cells of IgAN patients to those of non-IgAN patients. The reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric analyses showed that the TCR V beta 6 was used more frequently in tonsillar T cells of IgAN patients than in those of non-IgAN patients (P < 0.01 each). Similarly, the proportions of TCR V beta 6-positive cells in peripheral blood T cells were significantly higher in IgAN patients than in non-IgAN patients (P < 0.05). After tonsillectomy, the proportions decreased in IgAN patients (P < 0.05), but did not in non-IgAN patients. Furthermore, in vitro stimulation with Haemophilus parainfluenzae antigen, which is reported to deposit in the glomerular mesangium of IgAN, enhanced expression of TCR V beta 6 in tonsillar T cells from both IgAN and non-IgAN patients. These results suggest that TCR V beta 6-positive tonsillar T cells might be activated by H. parainfluenzae, move into the kidney through blood circulation and induce glomerulonephritis.

Development and characterization of human constitutive proteasome and immunoproteasome subunit-specific monoclonal antibodies.

Delta (Y), MB1 (X), and Z are the three catalytic beta-subunits located in the inner rings of the constitutive proteasome, an intracellular multicatalytic complex responsible for the generation of peptides presented by human leukocyte antigen (HLA) class I antigens to T cells. When cells are incubated with interferon-gamma, delta (Y), MB1 (X), and Z are replaced by LMP2, LMP7, and LMP10, respectively, leading to the expression of immunoproteasome which generates peptides with increased affinity for HLA class I antigens. The characterization of the expression of constitutive proteasome and immunoproteasome subunits in cells, normal tissues, and malignant lesions has been hampered by the lack or limited availability of constitutive proteasome and immunoproteasome subunit-specific monoclonal antibodies (mAbs), which are suitable for immunohistochemical staining. To overcome this limitation, we generated human delta (Y), MB1 (X), Z, LMP2, LMP7, and LMP10-specific mAb-secreting hybridomas from BALB/c mice immunized with peptides and recombinant fusion proteins. The mAbs SY-5, SJJ-3, NB-1, SY-1, HB-2, and TO-7 were shown to be specific for delta (Y), MB1 (X), Z, LMP2, LMP7, and LMP10, respectively, as they react specifically with the corresponding molecules when tested with a human B lymphoid LG2 cell lysate in Western blotting and with the peptide derived from each molecule in enzyme-linked immunosorbent assay. The reactivity of the six mAbs with the corresponding intracellular antigens resulted in intracellular staining when the mAbs were tested with microwave-treated and saponin-permeabilized cells in indirect immunofluorescence and with formalin-fixed, paraffin-embedded tissue sections in immunohistochemical reactions. These results suggest that the constitutive proteasome and immunoproteasome subunit-specific mAbs we have developed are useful probes to characterize the expression of proteasome subunits in normal tissues and in pathological lesions.

Endoplasmic reticulum chaperone-specific monoclonal antibodies for flow cytometry and immunohistochemical staining.

Endoplasmic reticulum (ER) chaperones of the antigen processing machinery play a crucial role in HLA class I antigen complex assembly and antigen presentation. The characterization of the expression of these chaperones in normal tissues and malignant lesions has been hampered by the lack or limited availability of ER chaperone-specific monoclonal antibodies (mAb) that are suitable for immunohistochemical staining. To overcome this limitation, we have generated human calnexin, ERp57, calreticulin and tapasin-specific mAb-secreting hybridomas from BALB/c mice immunized with peptides and recombinant proteins. The mAb TO-5, TO-2, TO-11 and TO-3 were shown to be specific for calnexin, ERp57, calreticulin and tapasin, respectively, as they react specifically with the corresponding immunizing peptides in ELISA and with the corresponding proteins when tested with human lymphoid cell lysates in Western blotting. Furthermore, the reactivity of the four mAb with the corresponding intracellular antigens yielded intracellular staining when the mAb were tested with formalin-fixed, microwave-treated and saponin-permeabilized cells in indirect immunofluorescence and with formalin-fixed, paraffin-embedded tissue sections in the immunoperoxidase reaction. These results suggest that the ER chaperone-specific mAb we have developed are useful probes for characterizing the expression of ER chaperones of the antigen processing machinery in normal and pathological cells. This information will contribute to defining the effects of abnormalities in their expression on HLA class I antigen expression and function and on the interactions of target cells with the host's immune system.

Treatment outcome of maxillary sinus squamous cell carcinoma.

BACKGROUND: Optimal treatment policies of maxillary sinus carcinoma remain to be defined. METHODS: Seventy-four patients with squamous cell carcinoma of maxillary sinus were treated at Department of Otolaryngology, Asahikawa Medical College between 1983 and 1997. The T classification according to the 1997 International Union Against Cancer was as follows: 9 with T2, 35 with T3, and 30 with T4. Eight patients had lymph node metastasis with N1 at diagnosis. Of 62 patients who started multimodality therapy that comprised preoperative radiochemotherapy including local irradiation with total dose of 50 grays along with concomitant intramaxillary arterial infusion of 5-fluorouracil with total dose of 5000 mg followed by total or partial maxillectomy, 59 received the complete therapy. Eleven patients had to be treated with radiotherapy alone, and 1 patient received postoperative radiotherapy. The median follow-up time for surviving patients was 117 months. RESULTS: The 5-year overall survival, disease free survival, and local control (LC) rates for all patients were 58.5%, 63.7%, and 73.6%, respectively. The patients who underwent multimodality therapy showed significantly better 5-year overall survival, disease free survival, and LC rates as compared with those who underwent radiotherapy alone (68.5% vs. 9.1%; 73.2% vs. 18.2%; 84.0% vs. 18.2%; P < 0.0001 each). Multivariate analysis revealed that T classification and treatment modality are independent predictors for disease free survival. CONCLUSIONS: The authors' treatment method, which did not include any complicated techniques, produced higher survival and LC rates because of high effectiveness of multimodality therapy. They concluded that their multimodality therapy could offer a better chance for cure from maxillary sinus carcinoma at many institutions.

Outcome of acute otitis media and its relation to clinical features and nasopharyngeal colonization at the time of diagnosis.

Children (n = 115; age range 1-9 years) with a diagnosis of acute otitis media (AOM) were eligible for this study and were enrolled within 24 h of the onset of symptoms/signs. A nasopharyngeal culture was obtained at the initial visit. Children were treated with a single oral antibiotic for 7 days. Changes in symptoms/signs and tympanic membrane features assessed by a scoring system were monitored for 1 month and related to the nasopharyngeal pathogen recovered on Day 1. More than 80% of children had no symptoms/signs by Day 3, and 60% of cases had normal tympanic membranes by Day 29. Children without any symptoms/signs on Day 3 had significantly lower symptom/sign scores on Day 1 (p = 0.005). Seventy-nine percent of cases carried middle ear pathogens in the nasopharynx at diagnosis: Streptococcus pneumoniae (48%); Haemophilus influenzae (24%); and Moraxella catarrhalis (16%). Children with S. pneumoniae showed significantly higher tympanic membrane scores than children with no pathogen at Days 8, 15 and 29 (p < 0.01 for each comparison). Multivariate regression analysis revealed that lower tympanic membrane score on Day 1, the absence of S. pneumoniae in the nasopharynx and treatment with amoxillin were independent factors for rapid normalization of the tympanic membrane. These data suggest that the clinical course of AOM may be predicted, in part, at the time of diagnosis by means of careful evaluation of symptoms/signs and the tympanic membrane as well as knowledge of pathogens harbored in the nasopharynx.

[Efficacy of tonsillectomy on psoriasis and tonsil histology].

Treatment outcomes of tonsillectomy were studied in 7 Japanese patients with psoriasis--3 males and 4 females aged 9 to 46 years (median: 23 years)--followed up 2 to 9 years after tonsillectomy. All skin lesions disappeared in 3 patients, 80% of those in 2, and no change in the remaining 2 during follow-up. Of 5 in whom skin lesions improved, 4 were females and had a history of tonsillitis making skin lesions worse. In quantitative immunohistologic analysis on tonsillar tissues by CD20 and anti-ssDNA antibodies, areas of T cell-nodules were significantly expanded, but those of the B-lymphoid follicles were smaller, and the number of apoptotic cells increased in tonsils from patients with psoriasis and PPP compared to those with recurrent tonsillitis. The area of T cell-nodules and the number of apoptosis cells were significantly larger in tonsils from 4 patients with complete recovery after tonsillectomy compared to the remaining 9 without complete recovery. This suggests that histologic evaluation may be helpful in estimating the effectiveness of tonsillectomy.

[Prognostic factors in non-Hodgkin's lymphoma of Waldeyer's ring and the lymph nodes of the neck].

Prognostic factors and treatment outcome of 74 patients with non-Hodgkin's lymphoma of Waldeyer's ring (male 34, female 16, median age 63 years) and cervical lymph nodes (male 17, female 7, median age 62 years) were analyzed retrospectively. The prognostic factors analyzed were: age, B-symptoms, serum LDH levels, and Ann Arbor stage. We also analysed the TNM classification (1987) of Waldeyer's ring lymphoma (previously referred as nasopharyngeal and oropharyngeal cancer) as a new prognostic factor. The 5-year-survival rate of the patients with Waldeyer's ring non-Hodgkin's lymphoma was 67%, compared with 34% for cervical lymph node lymphoma. In univariate analyses, unfavorable outcome was associated with age (age > 60 years), B-symptoms, elevated serum LDH level, and stage. Multivariate analysis showed that age and elevated serum LDH level were significant independent risk factors for death. The 5-year-survival rate according to TNM classification of Waldeyer's ring lymphoma was: T1 (4 cases) 75%, T2 (23 cases) 83%, T3 (10 cases) 56%, T4 (4 cases) 0%; according to N stage, N0 (15 cases) 91%, N1 (7 cases) 73%, N2 (11 cases) 55%, N3 (5 cases), M1 (3 cases) 0%, and according to TNM stage, stage I (one case) stage II (9 cases) 100%, stage III (12 cases) 80%, stage IV (19 cases) 34%. These results suggest that the N classification of Waldeyer's ring non-Hodgkin's lymphoma may be a new prognostic factor.

Low frequency of HLA-A*0201 allele in patients with Epstein-Barr virus-positive nasal lymphomas with polymorphic reticulosis morphology.

Lymphoproliferative diseases of the nasal cavity and paranasal sinuses occur frequently in Asian countries and are histologically categorized as monomorphic ordinary lymphoma and polymorphic reticulosis (PR) with apparent inflammatory cell infiltration. The large atypical cells in PR show natural-killer cell nature and frequently contain Epstein-Barr virus (EBV) DNA. Among the EBV genes involved in latent infection, those encoding EBV latent membrane proteins are frequently expressed in PR. Several cytotoxic T-lymphocyte (CTL) defined epitopes have been mapped to latent membrane proteins restricted with HLA-A2, -A11 or -A24 antigens. Thus, the HLA-A allele may affect the development of PR. To examine this possibility, HLA-A alleles of 25 patients with EBV(+) PR were determined with low-resolution polymerase chain reaction-based typing using HLA-A locus sequence-specific primer combinations. The frequency of HLA-A alleles including HLA-A2 and -A24 antigens in PR patients was lower than that in the normal Japanese population, but the difference was not significant. Since HLA-A2-restricted CTL responses are well delineated at the A2-subtype level, the A2-subtype of PR cases with HLA-A2 antigen was further determined by high-resolution genetic typing. The frequency of HLA-A*0201 in PR was significantly lower than in the normal population (p=0.0314). The HLA-A*0201-restricted CTL responses may thus function in vivo to suppress the development of overt lymphoma.

Cellular immune response of adenoidal and tonsillar lymphocytes to the P6 outer membrane protein of non-typeable Haemophilus influenzae and its relation to otitis media.

Cellular immune responses to the P6 outer membrane protein of non-typeable Haemophilus influenzae (NTHi) were determined in vitro by measuring immunoglobulin (Ig) secreting cells and lymphocyte proliferation in adenoidal and tonsillar lymphocytes from 19 children. Preliminary tests showed that P6 did not stimulate naive cells such as cord blood lymphocytes, but did stimulate sensitized cells in adenoids and tonsils. Cellular proliferation was significantly higher in adenoidal lymphocytes than in tonsillar lymphocytes (median: quadratile of stimulation index = 3.7:2.3-5.5 vs. 1.2:1.0-2.1, p < 0.02). A comparison between children with or without otitis media revealed that proliferative responses to P6 of adenoidal lymphocytes from children with otitis media were significantly decreased (2.0:1.8-3.6 vs. 3.7:2.3-5.5, p < 0.04). P6-specific antibody secreting cells were identified in a total of 14 adenoids and the number of cells secreting IgA was decreased in the otitis media group compared to controls (median: quadratile/10(6) cells = 435:359-499 vs. 755:593-1870, p < 0.05). Cultivation with P6 stimulated IgA secretion in children without otitis media, while no response was seen in children with otitis media (median: quadratile/10(6) cells = 1323:915-2410 vs. 2240:1900-2830, p < 0.02). These preliminary data demonstrate that lymphocytes from adenoids and tonsils recognize P6 as a specific antigen and that the adenoid is the more reactive of the two organs. Impaired P6-specific cellular immune responses of adenoids in children with otitis media may explain the recurrent nature of otitis media due to NTHi in the otitis prone population.

Increased interleukin-6, interferon-gamma and tumour necrosis factor-alpha production by tonsillar mononuclear cells stimulated with alpha-streptococci in patients with pustulosis palmaris et plantaris.

In vitro cytokine production by tonsillar mononuclear cells under culture conditions with or without lyophilized streptococcal antigens was measured by enzyme-linked immunosorbent assay (ELISA). Under culture conditions without any stimulus, a certain amount of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, and interleukin (IL)-6, was detected in the supernatant culture fluids from all 29 donors tested and just detectable levels of IL-1alpha and IL-2 were identified in 5 of 12 donors tested; but IL-4 and IL-5 were not detected in any supernatants from 21 donors tested. In 16 patients with pustulosis palmaris et plantaris (PPP), all three alpha-streptococcal antigens from Streptococcus, (S.) sanguis, S. salivarius and S. mitis induced production of IL-6. IFN-gamma and TNF-alpha by tonsillar mononuclear cells at a significant level. However, no significant induction of such cytokines by alpha-streptococcal stimulus was seen in 13 donors without PPP. No significant difference in cytokine production by tonsillar mononuclear cells was seen between the PPP and non-PPP subjects, when stimulated with S. pyogenes, Staphylococcus aureus Cowan I and pokeweed mitogen. In the experiments using tonsillar cell subsets, the cytokine inductions by alpha-streptococcal stimulus were found in monocyte-depleted cells as well as in purified T-cells. but not in B-cells. These data suggest that IL-6, IFN-gamma and TNF-alpha may play an important role in immunological reactions in the tonsils and that a hyper-immune response to some of alpha-streptococcal antigens through increased production of such cytokines by tonsillar mononuclear cells, to which T-cells mainly contribute, may play a key role in the pathogenesis of PPP.

Immunophenotypic and genotypic characterization of nasal lymphoma with polymorphic reticulosis morphology.

Nasal lymphoma with polymorphic reticulosis (PR) morphology is now categorized as T/natural killer (T/NK) cell lymphoma. In this study, immunophenotypes and genotypes of proliferating cells in 21 cases with PR were examined. The patients included 13 men and 8 women ranging in age from 20 to 74 (median 37) years. All patients presented with lesions in the upper respiratory tract, mostly in the nasal cavity. Histological specimens obtained from the primary lesions (19 cases) and metastatic cervical lymph nodes (2 cases) were used for analyses. Histologically, polymorphous proliferation was found in 20 cases, and these were thus diagnosed as PR. A monomorphous pattern was found in the remaining last case. Immunohistochemical analysis revealed that the proliferating cells were CD56 (123C3)+ and/or CD16 (2H7)+, TIA-1+ and frequently stained CD3 epsilon+. Tumor cells were frequently stained positively with monoclonal antibodies (mAbs) for T lymphocytes, but were negative for T-cell receptor (TCR) beta and delta chain expression. In situ hybridization analysis using an Epstein-Barr virus-encoded early RNA 1 (EBER-1) probe revealed positive signals in 13 of the 15 cases examined. Southern blotting analysis for clonality of the Epstein-Barr virus (EBV) genome in 12 positive cases confirmed the presence of monoclonal proliferation in 7 cases. The pattern of TCR gamma chain gene rearrangement was examined by PCR analysis of DNA from tumor tissues by the denaturing gradient gel electrophoresis method. The results demonstrated no clonal rearrangement in any of the 21 cases examined, including 7 cases with proven clonal proliferation of EBV-infected cells, indicating the absence of T-cell clones. Our findings strongly suggested that nasal T-cell lymphoma is in fact a NK cell lymphoma.

[Analysis of serum antibodies to alpha-streptococci in patients with tonsil-related pustulosis palmaris et plantaris].

To determine the systemic immune response to alpha-streptococci (Str. sanguis, Str. salivarius and Str. mitis) and beta streptococcus (Str. pyogenes T12) in patients with tonsil related pustulosis palmaris et plantaris (PPP), we measured serum antibody levels to whole cell body antigens of Streptococcus (Str.) sanguis, Str. salivarius, Str. mitis or Str. pyogenes by enzyme-linked immunosorbent assay (ELISA). The serum IgG antibody levels to alpha-streptococci, Str. sanguis and Str. salivarius were significantly higher in patients with PPP (n = 44) than in patients with recurrent tonsillitis (RT: n = 25) and in healthy adults (n = 17). Serum antibody IgG level to Str. pyogenes was not different among the 3 groups. The IgA antibody levels against any Streptococcus strain were not different among the 3 groups. The IgM antibody levels to Str. pyogenes were significantly higher in patients with RT than in patients with PPP. In western blot analysis, the serum IgG antibodies against 25-27 kDa protein from whole cell body of Str. sanguis, Str. salivarius and Str. mitis were found more frequently in patients with PPP than in healthy adults. However, the western blot profile in Str. pyogenes was not different between PPP and healthy adults. No significant difference was seen in the western blot profile of IgM or IgM antibodies to any streptococcal whole cell bodies. These data suggest that systemic hyper immune response to alpha-streptococci may be present in patients with tonsil-related PPP and the 25-27 kDa protein of the organism may be the target for this immunologic abnormality.

[A study of cytokine in palatine tonsil--flow cytometric analysis of cytokine production in tonsillar mononuclear cells].

Using two-color flow cytometry, we measured intracellular expression of interleukin-1 alpha (IL-1 alpha), IL-2, IL-4, IL-6, IL-8, interferon gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in tonsillar mononuclear cells freshly isolated and stimulated by phorbol myristate acetate (PMA) and ionomycin. In freshly isolated tonsillar mononuclear cells, IL-1 alpha was produced in 0.39% of CD3 cells, 0.48% of CD4 cells, 0.66% of CD19 and 11.2% of CD14 cells; TNF-alpha was found in 5.4% of CD14 cells. After 8-hour culture without any mitogens, IL-4, IL-8, TNF-alpha and IL-1 alpha were detected in 2.1%, 0.8%, 0.55%, and 0.42% of tonsillar mononuclear cells, respectively. Flow cytometric detection of intracellular cytokines in tonsillar mononuclear cells stimulated with PMA and ionomycin revealed that CD3 cells produced IL-1 alpha, IL-2, IL-4, IL-8, IFN-gamma and TNF-alpha, and CD19 cells produced IL-1 alpha, IL-6, IL-8 and TFN-alpha. In CD3 cells, the number of cells producing IL-2 and TNF-alpha were significantly higher than those expressing other cytokines; and the number of cells producing IFN-gamma and IL-8 were significantly higher than those expressing IL-4 and IL-1 alpha. In CD19 cells, the number of cells producing IL-6 and TNF-alpha were significantly higher than those of IL-8 and IL-1 alpha; and the number of cells producing IL-8 was significantly higher than that of IL-1 alpha. There was no difference in the number of CD3 and CD19 cells producing any cytokine between the adult recurrent tonsillitis group and adult obstructive sleep apnea syndrome group. However, the number of CD3 cells producing IL-2 or TNF-alpha and CD19 cells producing IL-1 alpha, IL-6 or TNF-alpha were significantly lower in children than that of adults (p < 0.05). These findings indicate that the cytokine production in tonsillar mononuclear cells is heterogenous according to the subset and activation and that flow cytometric analysis of intracellular cytokines is a useful means to investigate the pathophysiological role of cytokines in the tonsils.