bluuebunny/crossref_metadata_2025 huggingface dataset:
Pleomorphic carcinoma of the breast: clinicopathological analysis of 26 cases of an unusual high‐grade phenotype of ductal carcinoma
Authors: Silver S A , Tavassoli F A
Published: March 2003

Aims

Pleomorphic carcinoma is a poorly described entity whose phenotype is not well recognized as within the morphological spectrum of breast carcinoma. The purpose of this report is to describe the clinicopathological features of this tumour, and to promote its recognition as an unusual high‐grade morphological variant of mammary ductal carcinoma.

Methods and results

Histological slides of breast carcinomas (N = 64) coded between 1978 and 1995 as having pleomorphic or anaplastic features were reviewed. Pleomorphic carcinoma (N = 26) was diagnosed when ≥ 50% of the tumour manifested a pleomorphic cell population (> sixfold variation in nuclear size). Tumours of lobular origin were excluded. All neoplasms occurred in women with a mean age of 53 years. Patients underwent biopsy and/or mastectomy (n = 24) or lumpectomy (n = 2). The tumours' mean size was 54 mm. All were high‐grade neoplasms. The pleomorphic cell population comprised 50–100% of the tumour; 31% had a prominent spindled morphology. Fifty‐eight per cent of the tumours were initially misclassified by referring pathologists as sarcomas or carcinomas, possibly metastatic. Adjacent DCIS or a transition to classic ductal carcinoma was present in 73%. Five (19%) patients were stage I and three (12%) had stage IV disease. Axillary dissections yielded ≥ 3 (mean 7.2) positive lymph nodes in 52%. Most (68%) tumours were aneuploid; a high S‐phase (> 10%) was present in 63%. All neoplasms were ER negative and all but one were PR negative. p53 expression was present in 71%; none expressed bcl‐2. c‐erbB‐2 was detected in four (19%) node‐positive and in 0 (0%) node‐negative cases (P = 0.01). Of 16 patients with follow‐up, 6 (38%) were disease‐free (mean, 74 months), four (25%) alive with disease (mean, 33 months) and six (38%) dead of disease at a mean of 22 months.

Conclusions

Pleomorphic carcinoma is a prognostically unfavourable lesion and represents the extreme end of the morphological spectrum of grade III infiltrating ductal carcinoma.
Diagnostic gold standard for soft tissue tumours: morphology or molecular genetics?
Authors: Pfeifer J D , Hill D A , O'sullivan M J , Dehner L P
Published: March 2003

The recognition of recurrent genetic alterations in specific tumour types has provided the basis for the reclassification of certain soft tissue neoplasms, and molecular analysis of patient material has the potential to provide both diagnostic and prognostic information. In this review, we evaluate the role of molecular genetic testing as the prospective ‘gold standard’ for sarcoma diagnosis. Molecular genetic testing, as with every new method, promises to improve accuracy and to be more sensitive and less subjective, claims that have been made previously by histochemistry, electron microscopy and immunohistochemistry. Technical limitations in molecular assays, as well as more general specificity issues, decrease the clinical usefulness of molecular pathological testing significantly and suggest that, at present, molecular evaluation is best considered an ancillary technique that neither supersedes other ancillary techniques nor eclipses traditional pathological examination.
Extremely high Langerhans cell infiltration contributes to the favourable prognosis of HPV‐infected squamous cell carcinoma and adenocarcinoma of the lung
Authors: Miyagi J , Kinjo T , Tsuhako K , Higa M , Iwamasa T , Kamada Y , Hirayasu T
Published: March 2003

Aims: The infiltration of Langerhans cells in adenocarcinomas and squamous cell carcinomas of the lung was examined in relation to prognostic implications and human papillomavirus (HPV) infection.

Methods and results: Samples from 62 adenocarcinoma and 59 squamous cell carcinoma patients in 1995–97, the prognosis of which had been followed up, were used. The Langerhans cells were demonstrated immunohistochemically using anti S100a and CD1 antibodies. Human papillomavirus (HPV) infection was examined by polymerase chain reaction (PCR) and nonisotopic in‐situ hybridization (NISH) methods. Statistical analysis was carried out using the Kaplan–Meier method (Wilcoxon analysis) and multiple regression analysis. HPV infection was demonstrated in 12 cases (19.4%) of adenocarcinoma. The HPV‐infected adenocarcinomas had abundant faintly eosinophilic cytoplasm, and were immunohistochemically positive for the surfactant apoprotein A. In the 59 cases of squamous cell carcinomas 19 were of the well differentiated form, and 29 and 11 were moderately and poorly differentiated cases, respectively. HPV was detected in 29 cases (49.2%) (13 well and 16 moderately differentiated cases). In all HPV‐infected adenocarcinoma and squamous cell carcinoma cases, extremely large numbers of Langerhans cells (more than 100 per high‐power field) were demonstrated in the tumour nests. In contrast, in the non‐HPV‐infected adenocarcinomas and squamous cell carcinomas, only a few (less than about 10 per high‐power field) Langerhans cells were observed. The squamous cell carcinoma cases with high Langerhans cell infiltration, which were also infected with HPV, showed a significantly good prognosis (P = 0.007). The adenocarcinoma cases with high Langerhans cell infiltration tended to have a better prognosis than the cases with low Langerhans cell infiltration, but the difference was not statistically significant. The low number of highly infiltrated cases was insufficient for an adequate statistical analysis. Furthermore, there was no significant correlation between either Langerhans cell infiltration and smoking, or HPV infection and smoking, in either squamous cell carcinoma or adenocarcinoma cases.

Conclusions: It was considered that the extremely high Langerhans cell infiltration in the tumours was caused by HPV infection. The extremely large number of Langerhans cells in the tumours contributes to the favourable prognosis for HPV‐infected lung cancer.
Use of calretinin in the differential diagnosis of unicystic ameloblastomas
Authors: Coleman H , Altini M , Ali H , Doglioni C , Favia G , Maiorano E
Published: March 2003

Aims: Calretinin, a 29‐kDa calcium‐binding protein is expressed widely in normal human tissues and tumours including both unicystic and solid and multicystic ameloblastomas. The histological distinction between unicystic ameloblastomas and certain non‐neoplastic odontogenic cysts can be problematic. The objective of this study was to determine whether calretinin was expressed in the lining epithelium of odontogenic keratocysts, residual and dentigerous cysts and to determine whether this calcium‐binding protein could be used to distinguish these cysts from the unicystic ameloblastoma.

Methods and results: The lining epithelium in 22 cases of odontogenic keratocyst, 26 cases of residual cyst and 20 cases of dentigerous cyst were examined for the expression of calretinin. No positive epithelial staining was observed in any of these cystic lesions. In comparison, however, 81.5% of cases of unicystic ameloblastoma showed a coarse dark brown staining of the more superficial epithelial cell layers. Scattered positive stromal and epithelial cells were present; these were interpreted as mast cells.

Conclusions: Calretinin appears to be a specific immunohistochemical marker for neoplastic ameloblastic epithelium and we suggest that it may be an important diagnostic aid in the differential diagnosis of cystic odontogenic lesions and ameloblastic tumours.
Cytokeratin 14 expression in epithelial neoplasms: a survey of 435 cases with emphasis on its value in differentiating squamous cell carcinomas from other epithelial tumours
Authors: Chu P G , Lyda M H , Weiss L M
Published: March 2003

Cytokeratin 14 expression in epithelial neoplasms: a survey of 435 cases with emphasis on its value in differentiating squamous cell carcinomas from other epithelial tumours

Aims: The tissue distribution of cytokeratin 14 (CK14) in epithelial neoplasms is not well defined. We have evaluated 435 cases of epithelial neoplasm of various origins with cytokeratin 14 monoclonal antibody with special attention to possible use in differential diagnosis.

Methods and results: Immunohistochemistry (ABC–HRP method) was performed for detection of CK14. We found that the expression of cytokeratin 14 was generally restricted to: (i) the majority of cases of squamous cell carcinoma regardless of origin (67/74) and degree of differentiation; (ii) neoplasms with focal squamous differentiation, including endometrial, and ovarian adenocarcinoma, malignant mesothelioma and transitional cell carcinoma; (iii) thymoma (8/8); (iv) myoepithelial components of salivary gland pleomorphic adenoma (3/4); and (v) oncocytic neoplasms, including thyroid Hurthle cell adenoma (1/1) and salivary gland Warthin’s tumour (2/2).

Conclusion: CK14 protein is a useful marker in differential diagnosis of squamous cell carcinomas.
A new method of margin evaluation in breast conservation surgery using an adjustable mould during fixation
Authors: Ichihara S , Suzuki H , Kasami M , Aoyama H , Sato Y , Oiwa M , Kurokawa K , Endo T
Published: March 2003

A new method of margin evaluation in breast conservation surgery using an adjustable mould during fixation

Aims: We have developed a new method of breast resection margin assessment in quadrantectomy using an adjustable mould to prevent the three‐dimensional specimen from distorting during fixation.

Methods and results: The new method has been applied to 10 consecutive quadrantectomies (six invasive duct carcinomas, four duct carcinoma in situ with or without microinvasion). The precise configuration of the fixed specimen enabled pathologists to examine the side slices, the 5 mm thick slices cut parallel to the flat lateral margins of the specimen, permitting the separation of margin evaluation from tumour characterization. Eight cases with negative margins by our method would also be negative by assessment of inked margins since the margin widths were estimated to be from 5 to 30 mm (average 16.3 mm); two cases with positive margins would also be positive by inked margins.

Conclusions: Our new method was as reliable as the inked margins employing sequential slicing of the entire tissue, although it reduced the number of blocks by more than half in invasive carcinomas. A further advantage of this method is that the accuracy in margin evaluation is not influenced by the extent of tumour sampling. In addition, our system can pinpoint the positive margins facilitating re‐excision to obtain tumour‐free margins.
Vascular smooth muscle cells and pericytes express MMP‐1, MMP‐9, TIMP‐1 and type I procollagen in inflammatory bowel disease
Authors: Arihiro S , Ohtani H , Hiwatashi N , Torii A , Sorsa T , Nagura H
Published: March 2003

Vascular smooth muscle cells and pericytes express MMP‐1, MMP‐9, TIMP‐1 and type I procollagen in inflammatory bowel disease

Aims: Matrix metalloproteinases (MMPs) are involved in tissue remodelling, which is one of the important aspects of inflammatory disease. To assess the balance between the matrix degradation and production, we analysed the in situ expression of MMP‐1, ‐3, ‐8 and ‐9, tissue inhibitor of metalloproteinases (TIMP)‐1 and ‐2, and type I procollagen (PC‐I) in inflammatory bowel disease.

Methods and results: Immunohistochemistry using frozen sections was performed in 17 patients with ulcerative colitis (UC) and 16 with Crohn’s disease (CD). In both UC and CD, MMPs and TIMPs were expressed by inflammatory cells as well as by fibroblastic cells most prominently in actively inflamed areas in ulcer bases, but sparsely in intact inflamed mucosa in both UC and CD. In UC, inflamed mucosa with erosions expressed these substances focally. Fibroblasts also expressed PC‐I. We identified that vascular smooth muscle cells of venules in ulcer bases expressed MMP‐1 and ‐9, TIMP‐1 and PC‐I. These venules also expressed E‐selectin, a cell adhesion molecule to facilitate the leucocyte extravasation, and vascular endothelial growth factor (VEGF) receptor 2, consistent with their property of newly formed vessels.

Conclusions: Our results suggest that MMPs are involved in the tissue remodelling, angiogenesis and promotion of leucocyte extravasation in the actively inflamed area in the ulcer base in both UC and CD. MMP‐1 expression in the mucosa may be related to the initial step of ulceration in UC. Therapeutic manipulation of extracellular matrix turnover would be an effective therapy to alleviate active inflammation and accelerate ulcer healing.
Over‐diagnosis of hydatidiform mole in early tubal ectopic pregnancy
Authors: Burton J L , Lidbury E A , Gillespie A M , Tidy J A , Smith O , Lawry J , Hancock B W , Wells M
Published: March 2003

Over‐diagnosis of hydatidiform mole in early tubal ectopic pregnancy

Aims: Tubal ectopic hydatidiform moles are rare lesions, and only 40 cases have been reported in the world literature. We investigated the apparently high incidence of tubal ectopic hydatidiform moles in women referred for treatment to a Supraregional Trophoblastic Tumour Screening and Treatment Centre between 1986 and 1996.

Methods and results: Of 4261 women referred during the study period, 25 (0.6%) had a suspected tubal ectopic hydatidiform mole and paraffin‐embedded tissue was available in 20 (80%) of these. Each case was reviewed by two pathologists and DNA flow cytometric analysis was undertaken when the histological diagnosis was initially deemed equivocal or suggestive of hydatidiform mole. On review, 17 cases (85%) showed no evidence of hydatidiform mole (circumferential trophoblastic proliferation, hydrops, scalloped villi, and stromal karyorrhexis). Of these, 11 cases (65%) showed features of early placentation and six (35%) showed hydropic abortion. DNA flow cytometry was performed in 14 (82%) of these cases and revealed a diploid population in each case. Three cases of molar pregnancy (15%) were identified. Each of these cases had the histological features of an early complete hydatidiform mole. Sufficient tissue was available for DNA flow cytometric analysis in two of these cases and confirmed the presence of diploidy in each.

Conclusions: Our results show that tubal ectopic hydatidiform mole is a rare entity and demonstrate that it is over‐diagnosed. Polar trophoblastic proliferation and hydropic villi are features of early placentation and of hydropic abortion. Sheets of extravillous trophoblast may be particularly prominent in tubal ectopic gestation. In the absence of circumferential trophoblastic proliferation combined with hydropic change a diagnosis of gestational trophoblastic disease should be avoided.
Distribution of alpha glutathione S‐transferase in ovarian neoplasms: an immunohistochemical study
Authors: Tiltman A J , Ali H
Published: March 2003

Distribution of alpha glutathione S‐transferase in ovarian neoplasms: an immunohistochemical study

Aims: Alpha glutathione S‐transferase (α‐GST) has been shown to be an immunohistochemical marker for δ4–5 isomerase, an enzyme active in steroidogenesis. The purpose of this study was to document the distribution of α‐GST in ovarian neoplasms in order to evaluate its usefulness as a diagnostic tool.

Methods and results: A total of 92 tumours (25 sex cord/stromal, 53 epithelial and 14 germ cell) were subjected to immunohistochemistry using a commercially available polyclonal antibody to α‐GST. The avidin–biotin complex was used as a detection system. Positive staining was found in luteinized stromal cells of all tumour types (58/92). This included the Leydig cells of Sertoli–Leydig cell tumours (7/7) and was particularly prominent in the stromal cells of both benign and malignant mucinous tumours (24/25). Granulosa and Sertoli cells showed weak or no intracytoplasmic staining, which is expected because they do not normally produce androstenedione. They did show some intranuclear staining. Malignant mucinous (12/25) and occasional other epithelial tumours showed focal intracytoplasmic positive staining. Yolk sac tumours showed focal positivity (7/8).

Conclusions: Intracytoplasmic staining of stromal cells is considered to indicate steroidogenesis and intranuclear staining the intracytoplasmic transport function of α‐GST. The intracytoplasmic staining of mucinous carcinomas might represent an up‐regulation of some detoxification function. The findings suggest that antibody to α‐GST has some value in the investigation of ovarian pathology and could readily be included in any panel of antibodies used to investigate ovarian neoplasms of uncertain histogenesis.
c‐erbB‐2 and c‐Met expression relates to cholangiocarcinogenesis and progression of intrahepatic cholangiocarcinoma
Authors: Aishima S‐I , Taguchi K‐I , Sugimachi K , Shimada M , Sugimachi K , Tsuneyoshi M
Published: March 2003

c‐erbB‐2 and c‐Met expression relates to cholangiocarcinogenesis and progression of intrahepatic cholangiocarcinoma

Aims: The c‐erbB‐2 and c‐Met proto‐oncogenes are important for tumour invasiveness and metastasis in many types of malignant tumours. Previous studies have indicated that these proteins are associated with carcinogenesis in intrahepatic cholangiocarcinoma. In this study, we examined c‐erbB‐2 and c‐Met expression by immunohistochemistry in hepatolithiasis, intrahepatic cholangiocarcinoma and metastatic lymph node, in order to clarify whether these proteins play a role in carcinogenesis and tumour metastasis in intrahepatic cholangiocarcinoma.

Methods and results: In hepatolithiasis, the staining for c‐erbB‐2 was positive in 14 of the 23 (61%) cases, while staining for c‐Met was positive in eight of the 23 (35%) cases. In intrahepatic cholangiocarcinoma, staining for c‐erbB‐2 was positive in 45 of the 81 (55%) cases, while staining for c‐Met was positive in 28 (35%) cases. The positivity of c‐Met staining in intrahepatic cholangiocarcinoma was significantly higher in the differentiated type of cholangiocarcinoma than in the undifferentiated type. In addition, c‐Met‐positive staining had an inverted correlation with tumour size, the presence of perineural invasion and the presence of lymph node metastasis. c‐Met staining had a significantly higher positivity in cases at an early stage of intrahepatic cholangiocarcinoma. In contrast, the positivity of c‐erbB‐2 staining in intrahepatic cholangiocarcinoma was significantly higher in cases with lymph node metastasis than in cases without. In metastatic lymph nodes, the staining for c‐erbB‐2 was positive in 20 of the 25 (80%) cases, while staining for c‐Met was positive in six of the 25 (24%) cases. There was no difference in survival between c‐erbB‐2‐positive and negative patients. However, the patients with c‐Met‐positive tumours had a significantly longer survival than those with c‐Met‐negative tumours in the medium survival term. The multivariate analysis showed the presence of lymph node metastasis, lymphatic permeation and histological differentiation to be independent prognostic factors.

Conclusion: These results indicate that increased c‐Met expression participates in cholangiocarcinogenesis and in the early developmental stages of intrahepatic cholangiocarcinoma, while increased c‐erbB‐2 expression contributes to the development of cholangiocarcinogenesis into an advanced stage associated with tumour metastasis.
Respiratory failure due to micronodular type II pneumocyte hyperplasia
Authors: Cancellieri A , Poletti V , Corrin B
Published: March 2003

Aims:  To report the first case of respiratory failure due to micronodular type II pneumocyte hyperplasia.

Methods and results:  Biopsy, explant and autopsy material from a 16‐year‐old girl, a smoker, with no personal or family history of tuberous sclerosis, who died following lung transplantation necessitated by progressive respiratory failure, was evaluated histologically. Micronodular pneumocyte hyperplasia was identified histologically as the cause of the respiratory failure. Foci of hyperplastic type II pneumocytes measuring up to 4 mm were widely scattered through the lungs. The hyperplastic cells had abundant eosinophilic cytoplasm, vesicular nuclei and prominent nucleoli. Immunohistochemistry showed that they stained for cytokeratins AE1, AE3 and CAM 5.2 and for epithelial membrane antigen (EMA), but not for carcinoembryonic antigen (CEA), S100, smooth muscle actin, CD68 and HMB‐45.

Conclusions:  Although micronodular type II pneumocyte hyperplasia is usually of no clinical significance, in our patient the process was so florid as to cause respiratory failure, which was severe enough to necessitate lung transplantation.
Endocervical adenocarcinoma and its variants: their morphology and differential diagnosis
Authors: Young R H , Clement P B
Published: March 2003

Adenocarcinoma of the uterine cervix and its variants account for a much greater number of cases in routine practice of histopathology than they did several decades ago. The varied morphology of these tumours results in diverse problems in differential diagnosis. The overall area of glandular pathology of the cervix, of which invasive adenocarcinoma is only one subset, is further complicated by the fact that there are many benign glandular proliferations of the cervix that can potentially be misinterpreted as adenocarcinoma. In this review the histopathology of endocervical adenocarcinoma and its variants is presented with the emphasis on evaluation of routinely stained sections, still the bedrock of routine practice, relatively little aid being provided by immunohistochemistry or other new techniques, contrary to what is sometimes implied in the literature. Description of the appearance of each subtype of adenocarcinoma or variant thereof is followed by a section on their differential diagnosis. Eighty percent of endocervical carcinomas are of the so‐called usual type being characterized by cells with eosinophilic cytoplasm and generally brisk mitotic activity. It is sometimes stated that endocervical adenocarcinomas are mucinous but the usual form just noted often has little or no mucin. Pure or almost pure mucinous adenocarcinoma do occur, however, and have an important subtype, the so‐called adenoma malignum (minimal deviation adenocarcinoma). Although treacherous because of its bland cytological features and sometimes deceptive pattern, a cone biopsy or hysterectomy specimen showing this neoplasm typically has easily recognizable features that indicate the presence of an infiltrative adenocarcinoma. An important variant of usual endocervical adenocarcinoma is the well differentiated villoglandular papillary adenocarcinoma, a designation that should be reserved for tumours with grade 1 cytologic features as usual endocervical adenocarcinoma, which is typically grade 2, may have papillae. In our opinion all other variants of pure adenocarcinoma, including endometrioid, are rare and include in addition to the latter clear cell, serous and mesonephric neoplasms. Tumours witha glandular and nonglandular component are also reviewed: adenosquamous carcinoma, glassy cell carcinoma, adenoid basal carcinoma, ‘adenoid cystic’ carcinoma and adenocarcinoma admixed with a neuroendocrine tumour.
ALK protein expression in rhabdomyosarcomas
Authors: Pillay K , Govender D , Chetty R
Published: March 2003

Aims:  The ALK p80 chimeric protein is thought to be up‐regulated as a result of the t(2;5) as classically seen in anaplastic large cell lymphoma. However, rhabdomyosarcomas (in particular, the alveolar subtype) have also been noted to show expression of this protein. This study set out to examine ALK expression in a large number of rhabdomyosarcomas.

Methods and results:  Eighty‐three cases of rhabdomyosarcomas and 16 cases of malignant mixed müllerian tumours with a rhabdomyosarcomatous component were retrieved from the archives of the Department of Anatomical Pathology for the period 1983–2001. The sections were stained with polyclonal ALK antibody. There were 52 male and 30 female patients. In one case, the gender of the patient was not indicated. The ages ranged from 1 week to 77 years. The most common site was the head and neck region, followed by the pelvis and extremities. Thirty‐one cases were of the alveolar subtype while 40 cases were embryonal. There were four mixed embryonal/alveolar, six pleomorphic and two unclassifiable rhabdomyosarcomas. Fourteen of the 31 (45%) alveolar rhabdomyosarcomas stained positively for the ALK protein, while only six of the 40 embryonal (15%) cases showed positivity. One case each of the mixed embryonal/alveolar, pleomorphic and unclassified cases was also immunopositive. The rhabdomyosarcomatous component in the malignant mixed müllerian tumours was positive in four of the 16 cases.

Conclusion:  We conclude that a proportion of alveolar rhabdomyosarcomas (in particular) exhibit ALK protein expression. However, ALK expression is not restricted to this subtype. An extension of this study is to determine if this over‐expression is as a result of the t(2;5) translocation.
Malignant myopericytoma: expanding the spectrum of tumours with myopericytic differentiation
Authors: McMenamin M E , Fletcher C D M
Published: March 2003

Aims:  The spectrum of tumours showing myopericytic differentiation is increasingly being defined and includes lesions such as myofibroma and infantile haemangiopericytoma. Here we seek to describe for the first time and clinicopathologically characterize examples of malignant myopericytoma.

Methods and results:  Five cases of malignant myopericytoma were identified in the authors' consultation files. Immunostains were performed and clinical information was obtained. Tumours arose in three females and two males (median age 67 years, range 19–81 years) on the neck, arm, thigh and foot. One patient presented with disseminated metastases. One patient had a prior history of multiple benign myopericytomas in the same location. Four patients developed metastases and three died within 1 year. Tumours were composed of highly mitotic myoid‐appearing ovoid‐to‐spindle cells showing at least focally striking perivascular orientation resembling that seen in benign myopericytoma; three cases were focally fascicular and three showed thin‐walled branching vessels. All tumours showed at least focally prominent positivity for smooth muscle actin. One case showed dot‐like desmin positivity.

Conclusions:  In reporting examples of malignant myopericytoma, we further characterize and broaden the morphological spectrum of myopericytic neoplasms. Available data indicate that malignant myopericytomas are associated with aggressive clinical behaviour.
Nitric oxide‐mediated cytotoxic effects of alveolar macrophages on transformed lung epithelial cells are independent of the β2 integrin‐mediated intercellular adhesion
Authors: Hirano
Published: March 2003

It is known that murine macrophages produce nitric oxide (NO) when stimulated with lipopolysaccharide (LPS) or interferon‐γ (IFN‐γ), and NO mediates the tumoricidal activity of activated macrophages. The present study was designed to investigate whether the intercellular adhesion is necessary for activated rat alveolar macrophages to exert the full cytotoxic effects. Rat alveolar macrophages produced NO dose dependently in response to either LPS or IFN‐γ, and caused DNA fragmentation in rat type II pneumocytes transformed with SV40 (SV40T2). Chemically produced NO also caused the DNA fragmentation and viability loss in SV40T2, and both of them were inhibited by a NO radical scavenger. The cytotoxicity of activated macrophages was reduced by NG‐monomethyl‐l‐arginine, a competitive nitric synthase inhibitor, and neither superoxide dismutase nor catalase modulated the cytotoxicity. Although alveolar macrophages stimulated with either LPS or IFN‐γ caused DNA fragmentation of SV40T2, only LPS increased the intercellular adherence between macrophages and SV40T2. The intercellular adhesion was reduced by both anti‐CD18 and anti‐CD11a. However, those antibodies did not affect the cytotoxicity of LPS‐stimulated macrophages. These results clearly indicate that NO‐mediated cytotoxicity is caused predominantly by diffusion of NO, and the β2 integrin‐mediated intercellular adhesion does not play an important role, if any, in activated macrophage‐mediated cytotoxic effects on SV40T2.
Antigen direction of specific T‐cell clones into gingival tissues
Authors: Kawai  , Shimauchi  , Eastcott  , Smith  , Taubman
Published: March 2003

This study was performed to investigate T‐cell traffic to periodontal tissues during infection with a periodontal pathogen Actinobacillus actinomycetemcomitans (Aa). Rowett rat T‐cell clones, A3 (CD4+ CD8−, αβTCR+, NKRP‐1−, specific to Aa) and G2 (CD4− CD8−, αβTCR+, NKRP‐1+, which reacts to Aa, Gram‐negative and ‐positive bacteria), both expressed the same prominent adhesion molecules (LFA‐1, VLA‐4) to the same extent. Binding of both T‐cell clones to rat endothelial cells in vitro was blocked by antibody to VLA‐4. Rowett rats were infected with Aa and infused with Aa‐stimulated, isogenic T‐clone lymphocytes that had been labelled in vitro with 125IUdR. Radioactivity associated with recovery of clone A3, but not G2, was significantly elevated in the gingivae of infected rats, suggesting migration to infected animals’ gingival tissues. Migration of radioactive Aa‐specific A3 clone cells traced by autoradiography reached a maximum at 24 hr (1·2% of total lymphocytes as radiolabelled cells in infected gingiva versus 0·6% in non‐infected), indicating an apparent antigen‐directed retention in infected rats’ gingival tissues. The G2 clone was not retained in the gingival tissues (0·20% of total lymphocytes as radiolabelled cells in infected gingiva versus 0·26% in non‐infected). However, the possibility of A3 retention directed by inflammation or tissue‐selective homing could not be excluded. In further experiments, other adoptively transferred T‐clone lymphocytes [clones G23 (Th1) and F13 (Th2)] with specificity for the 29 000 MW outer membrane protein of Aa with the same prominent adhesion molecules could be recovered from rat gingivae previously challenged with this antigen. However, transferred T‐clone lymphocytes [clone G26 (Th1)] with specificity for a different Aa antigen were not recovered. Therefore, the dynamics of cell entry into periodontal lesions vary for activated T lymphocytes with different antigenic specificities, indicating the significance of antigen in lymphocyte traffic to periodontal tissues.
Cytokine regulation on the synthesis of nitric oxide in vivo by chronically infected human polymorphonuclear leucocytes
Authors: TAKEICHI  , SAITO  , OKAMOTO  , TSURUMACHI  , SAITO
Published: March 2003

To determine if nitric oxide (NO) is produced by chronically infected human polymorphonuclear leucocytes (PMNs) in vivo, inflamed exudates (periapical exudates: PE) collected from periapical periodontitis patients were examined. Cell‐free supernatants and cells were separated by centrifugation. Significant levels of nitrite concentrations were observed in the supernatants. The production of inducible NO synthase (iNOS) in highly purified PMNs derived from PEs was then immunocytochemically determined using rabbit anti‐human iNOS antiserum. In vitro, human peripheral blood PMNs (PB‐PMNs) isolated from patients were cultured with a combination of Esherichia coli–lipopolysaccharide (LPS), recombinant human interferon‐γ (rhIFN‐γ) and/or interleukin‐1β (rhIL‐1β). The stimulated PB‐PMNs showed steady‐state levels of nitrite. The stimulation of LPS, rhIFN‐γ and rhIL‐1β showed more NO induction than that of LPS with either IFN‐γ or IL‐1β, suggesting the synergistic effects of cytokines. Cryostat sections of surgically removed periapical tissues were also immunohistochemically examined for iNOS, IFN‐γ and IL‐1β. Two‐colour immunohistochemistry revealed the interaction of iNOS‐producing PMNs and IFN‐γ‐ or IL‐1β‐producing mononuclear cells. On the basis of these data, we concluded that with the stimulation of inflammatory cytokines derived from mononuclear cells, PMNs can spontaneously produce NO at the site of chronic infection. The present studies are consistent with a hypothesis suggesting that PMNs could be regulated and delicately balanced to produce NO by mononuclear cell‐derived cytokines in vivo. NO‐producing cells may play a pivotal role in chronic inflammation.
Intranasal immunization with polymer‐grafted microparticles activates the nasal‐associated lymphoid tissue and draining lymph nodes
Authors: HERITAGE  , BROOK  , UNDERDOWN  , McDERMOTT
Published: March 2003

Waldeyer’s ring is located at the juncture of the respiratory and alimentary tracts, where it is bombarded by inhaled and ingested antigens. However, knowledge of its exact function or consequences of its removal is incomplete. Recently, the murine nasal‐associated lymphoid tissue (NALT) has been reported to have functional similarities to Waldeyer’s ring and, thus, might be a suitable model to examine the function of oronasopharyngeal lymphoid tissues. To explore the capability of NALT to incite local mucosal and systemic immunity, we immunized mice intranasally (i.n.) with 3‐(triethoxysilyl)‐propyl‐terminated polydimethylsiloxane (TS‐PDMS)‐grafted microparticles (MP), an inoculant previously shown to induce robust systemic and mucosal humoral immunity following intragastric (i.g.) administration. We demonstrated that i.n. immunization with low doses of microentrapped, but not soluble, human serum albumin (HSA) evoked robust circulating IgG responses (P<0·05). Additionally, NALT cells isolated from MP‐treated mice proliferated in vitro when restimulated with HSA (P<0·05), suggesting that i.n. immunization with HSA‐containing MP incited specific immunity via NALT cell activation. Coinciding with these observations, after i.n. MP administration HSA‐specific spot‐forming cells (SFC) were observed in NALT, and later posterior cervical lymph nodes (pCLN) and spleen (SPL), suggesting that the observed MP‐induced specific systemic antibody responses emanated from the NALT. We also showed that i.n. immunization with HSA‐containing TS‐PDMS‐grafted MP stimulated interleukin‐4 (IL‐4)‐secreting lymphocytes in the NALT. This cytokine microenvironment was probably responsible for driving the IgG1 sera response observed after i.n. MP administration, via the migration of NALT‐derived IgG1‐committed B cells. Interestingly, unlike i.g. MP administration, i.n. immunization with HSA‐containing MP did not evoke detectable specific IgA in any lymphoid tissue examined, or in nasal secretions, probably reflecting differences between NALT and other mucosae‐associated lymphoid tissues (MALT).
Rat mucosal mast cells: the cultured bone marrow‐derived mast cell is biochemically and functionally analogous to its counterpart in vivo
Authors: MACDONALD  , PICK  , BISSONNETTE  , BEFUS
Published: March 2003

Mast cells (MC) are biochemically and functionally heterogeneous and the mixture of MC phenotypes varies according to anatomical location. Intestinal mucosal MC (IMMC) have been used to study the mucosal MC subset in the rat, but they are difficult to isolate in sufficient numbers and with consistent purity and viability. Bone marrow‐derived MC (BMMC), with an apparent mucosal MC phenotype, can be cultured in large numbers and with high purity from normal rat bone marrow using supernatants from mesenteric lymph node cells of rats infected with the nematode, Nippostrongylus brasiliensis. We have compared serine proteinase content, tumour necrosis factor‐α (TNF‐α) storage and secretion, and TNF‐α‐dependent cytotoxicity of IMMC and BMMC to assess the appropriateness of BMMC as in vitro models of mucosal MC. Two‐dimensional gel electrophoretic analysis revealed that the overall protein constituents of BMMC and IMMC were highly homologous. Immunoblotting confirmed that both MC types expressed the MMC‐associated enzyme, rat mast cell proteinase‐2 (RMCP‐2), but not RMCP‐1, mast cell proteinase‐5 (MCP‐5) or carboxypeptidase A (CPA), which characterize the connective tissue MC in the rat and which were detected in a representative of this subset, namely, the peritoneal MC (PMC). BMMC demonstrated levels of TNF‐α‐dependent cytotoxicity that were equivalent to those of IMMC. Like IMMC, BMMC contained little stored TNF‐α, in comparison with PMC, but both MC types generated substantial amounts of TNF‐α 6 hr following IgE‐mediated activation. Pretreatment of PMC with recombinant rat interferon‐γ (IFN‐γ) for 20 hr inhibited anti‐immuoglobulin E (anti‐IgE)‐mediated release of the granule‐associated enzyme, β‐hexosaminidase, whereas identically treated BMMC were unresponsive to this cytokine. Similar results have previously been reported for IMMC. Rat BMMC, unlike their more immature and less phenotypically committed counterparts in the mouse, appear therefore to be more appropriate models for studies on the mucosal MC.
Inhibition of cytotoxic alloreactivity by human allogeneic mononuclear cells: evidence for veto function of CD2+cells
Authors: RADDATZ  , DEIWICK  , SATO  , SCHLITT
Published: March 2003

In animal models of organ transplantation, infusion of donor‐derived leucocytes or bone marrow cells can support tolerance induction. To date, little is known about the suppressive effects of human allogeneic mononuclear cells on alloreactivity in the human system. To study this, mixed leucocyte cultures (MLC) were incubated in the presence and absence of viable allogeneic mononuclear cells (MNC) (modulator cells) of stimulator/donor origin, and the cytotoxic and proliferative potential of the resulting effector cells was determined. The experiments showed that: viable allogeneic MNC from bone marrow and from lymph nodes and peripheral blood (PBMC) were able to suppress allospecific cytotoxicity by an average of 60%; that allospecific as well as non‐specific inhibitory effects could be observed with unseparated PBMC; that CD2+PMNC showed predominantly allospecific inhibition of cytotoxicity with little effect on proliferation whereas CD2−PBMC showed non‐specific inhibitory effects (both for cytotoxicity and proliferation), which could be eliminated by indomethacin; that addition of interleukin‐2 (IL‐2) up to 50 U/ml to the MLC could not reverse the inhibitory effect; and that selective removal of CD8+cells from the CD2+modulator population diminished the specific inhibitory effect only partially. These findings demonstrate that viable human MNC from different compartments can have a marked suppressive effect on alloreactivityin vitro. For peripheral blood mononuclear cells (PBMC) the data suggest that various mechanisms can contribute to allosuppression, including specific suppressive veto effects by CD2+cells. Such inhibitory effects might be applicablein vivofor down‐regulating allospecific cytotoxicity and to facilitate the acceptance of allografts.
Changes in cellular response to mycobacterial antigens and cytokine production patterns in leprosy patients during multiple drug therapy
Authors: Trao V. T. , Huong P. L. T. , Thuan A. T. , Anh D. D. , Trach D. D. , Rook G. A. W. , Wright E. P.
Published: March 2003

Changes inMycobacterium leprae‐induced lymphoproliferative responses and mediator release by leprosy patients’ lymphocytes were followed during multiple drug therapy (MDT). At the time of diagnosis, multibacillary (MB) patients who did not develop reactions responded to both sonicatedM. lepraeand synthetic disaccharide coupled to bovine serum albumin (ND‐BSA) antigens, but those who would later develop reactions did not respond, even in the presence of added cytokines. The paucibacillary (PB) group initially had high responses to sonicatedM. lepraebut no response to ND‐BSA, even in the presence of added cytokines. In the first year of treatment, the supernatants of PB patients’ cell cultures contained factors that enhanced the phytohaemagglutinin (PHA) response of normal cells. In contrast, those MB patients who did not develop reactions at a later stage produced culture supernatants that were inhibitory. Interestingly, the MB patients who later developed reactions during treatment, and did not initially respond toM. leprae, produced supernatants containing enhancing factors, like those of the PB group. Later on in the treatment, all patients had the same patterns: when response toM. lepraedecreased from its highest level, inhibitory factors were produced. Further studies revealed that the supernatants which inhibited the PHA response of normal cells contained the active form of transforming growth factor‐β1(TGF‐β1), whatever the disease type or treatment status of the donor. These TGF‐β1levels correlated directly with the degree of inhibition. Similarly, supernatants that neither inhibited nor enhanced PHA responses contained the highest levels of interleukin‐10 (IL‐10), while those from treated patients that enhanced contained the lowest levels of interleukin‐4 (IL‐4) and interferon‐γ (IFN‐γ). These cytokine correlations transcended the conventional disease classification, and imply that all patients pass through a sequence of patterns of immune response during treatment. These treatment‐induced changes may explain occasional reports of response patterns at variance with the ‘immunological spectrum’ of leprosy.

BB, borderlineBL, borderline lepromatousBT, borderline tuberculoidIFN‐γ, interferon gammaIL‐4, interleukin‐4IL‐10, interleukin‐10MB, multibacillaryPB, paucibacillaryRT–PCR, reverse transcriptase–polymerase chain reactionLL, polar lepromatousTGF‐β1, transforming growth factor‐β1TT, tuberculoid.
Evidence for epitope‐specific thymus‐independent response against a repeat sequence in a protein antigen
Authors: KUMAR  , ZHENG
Published: March 2003

We have studied the imm_unogenicity of a recombinant heat‐shock protein‐related stress protein of 70 000 MW (Pfhsp) of the human malaria parasite Plasmodium falciparum in H‐2 congenic and non‐congenic strains of inbred and outbred mice. Most mice of different genetic backgrounds produced antibodies to Pfhsp, indicating a lack of any apparent genetic restriction of imm_une response. A peptide corresponding to the GGMP repeat sequence in the C‐terminal region of Pfhsp was recognized by more than 75% of sera from imm_unized mice. The GGMP repeat epitope‐specific antibodies were largely of the IgM isotype, especially in all seven inbred strains of mice tested. The lack of significant boosting of the imm_une response, a predominantly IgM isotype of antibodies and generation of antibody responses in athymic nude mice suggest a thymus‐independent response against the GGMP repeat epitope in the Pfhsp molecule.
Staphylococcal enterotoxin B‐induced T‐cell anergy is mediated by regulatory T cells
Authors: WANG Z.‐Q. , ORLIKOWSKY T. , DUDHANE A. , TREJO V. , DANNECKER G. E. , PERNIS B. , HOFFMANN M. K.
Published: March 2003

Naive T cells mount a vigorous proliferative response to superantigen (SAg) stimulation in vivo. The proliferative response is followed by a partial deletion of responder T cells. Part of the deletion process has recently been attributed to the action of regulatory cytotoxic T cells that recognize major histocompatibility complex (MHC) class I‐associated antigen receptor determinants on the target cell surface. Responder T cells that survived the SAg response were found to be incapable of generating a secondary proliferative response to a SAg challenge. We show here that this ‘anergy’ is enforced by CD8‐positive regulatory suppressive T cells. These regulatory cells inhibit cell division of preactivated T cells but not the SAg response of naive T cells. Regulatory T cells are not generated in the presence of cyclosporin A and, once activated, become inactivated or deleted when restimulated in the presence of this immunosuppressive drug.
The effect of renin–angiotensin system inhibitors on pro‐ and anti‐inflammatory cytokine production
Authors: PEETERS A. C. T. M. , NETEA M. G. , KULLBERG B. J. , THIEN T. , VAN DER MEER J. W. M.
Published: March 2003

The balance between pro‐ and anti‐inflammatory cytokines has been implicated in the pathogenesis of infectious and auto‐immune diseases, and its modulation has been proposed as a potential therapeutic target. The results reported in the present study show that modulators of the renin–angiotensin system, such as the angiotensin‐converting enzyme (ACE)‐inhibitor captopril and the angiotensin II receptor type I antagonist valsartan, have potent inhibitory effects on the lipopolysaccharide (LPS)‐stimulated production of pro‐inflammatory cytokines tumour necrosis factor (TNF) and interleukin‐1 (IL‐1) in vitro. The anti‐inflammatory cytokine IL‐1Ra is increased by captopril, whereas IL‐6 production is decreased by valsartan. These effects are exerted mainly at high concentrations of the drugs. Administration of one dose of captopril or valsartan in therapeutic dosages to patients with essential hypertension did not influence LPS‐stimulated production of cytokines by whole blood. In conclusion, despite inhibitory effects on pro‐inflammatory cytokine production in vitro, it is unlikely that captopril or valsartan could be used in anticytokine therapeutic strategies in vivo.
Interferon‐γ‐activated primary enterocytes inhibit Toxoplasma gondii replication: a role for intracellular iron
Authors: DIMIER  , BOUT
Published: March 2003

Toxoplasma gondii is an obligate intracellular parasite that infects a wide variety of nucleated cells in its numerous intermediate hosts including man. The oral route is the natural portal of entry of T. gondii. Ingested organisms are released from cysts or oocysts within the gastrointestinal tract and initially invade the intestinal epithelium. We show that T. gondii invades and proliferates in cultured primary rat enterocytes, obtained with an original procedure. Activation of the enterocytes with rat recombinant interferon‐γ (IFN‐γ) inhibits T. gondii replication, the inhibition being dose dependent. Neither nitrogen and oxygen derivatives nor tryptophan starvation appear to be involved in the inhibition of parasite replication by IFN‐γ. Experiments using Fe2+ salt, carrier and chelator indicate that intracellular T. gondii replication is iron dependent, suggesting that IFN‐γ‐treated enterocytes inhibit T. gondii replication by limiting the availability of intracellular iron to the parasite. Our data show that enterocytes probably play a major role on mucosal surfaces as a first line of defence against this coccidia, and possibly other pathogens, through an immune mechanism. The results suggest that limiting the availability of iron could represent a broad antimicrobial mechanism through which the activated enterocytes exert control over intracellular pathogens.
Anti‐DNA IgA autoantibodies are spontaneously generated in mouse Peyer’s patches
Authors: SHIMODA  , INOUE  , AMETANI  , FUJIWARA J. , TSUJI N. M. , KURISAKI J.‐I. , AZUMA N. , KANNO
Published: March 2003

IgA antibodies in the mucosal immune system are produced specifically to environmental antigens such as virus and bacteria, and possibly to some food components, which will provide a potential luminal antigen, DNA. To study the immune response to DNA in the gut, we established B‐cell hybridomas producing IgA monoclonal antibodies (mAb) from Peyer’s patches (PP) of non‐immunized, non‐autoimmune, specific pathogen‐free BALB/c mice, and examined their specificity by enzyme‐linked immunosorbent assay (ELISA). Three mAb out of 18 bound strongly to self, bacterial and synthetic DNA, with Kd of about 10−7 m. One of the three mAb also reacted with the histone component and another reacted with some mouse food component. The VH genes of these three mAb have not previously been reported to have anti‐DNA specificity, and carry putative somatically mutated sites favouring DNA binding in CDR. The features resemble those of anti‐DNA antibodies found in human and murine models of systemic lupus erythmatosus (SLE), and are indicative of an antigen‐driven selection process. Our findings suggest that even in normal healthy animals, anti‐DNA antibodies of IgA isotype can be produced in certain peripheral environments such as in PP by spontaneous antigenic stimulation.