Antigen-induced tolerance by intrathymic modulation of self-recognizing inhibitory receptors

CD1d-restricted invariant natural killer T cell (iNKT cells) have a limited T cell receptor (TCR) repertoire and share characteristics common to T cells and natural killer cells. While intrathymic selection facilitates the production of T cells carrying self major histocompatibility complex-restricted TCRs, natural killer cells carry an appropriate repertoire of self major histocompatibility complex-recognizing receptors to avoid self-reactivity. Here we show that chronic exposure to specific glycolipid antigen resulted in iNKT cell disappearance and thymus-dependent repopulation of iNKT cells with increased expression of inhibitory Ly-49 molecules that resulted in impaired responsiveness. Thymic selection of peripheral Ly-49-expressing iNKT cell repertoire inhibited cytokine production and other functions in vivo. These observations emphasize the acquisition of self-recognizing inhibitory receptors on NKT cells as a previously unknown mechanism of thymic tolerance after chronic antigen exposure.     

Simple,rapid, and accurate determination of deletion mutations by automated dna sequencing of heteroduplex fragments of the adenomatous polyposis coli (APC) gene generated by PCR amplification

Germline mutations in patients with familial adenomatous polyposis were analyzed by polymerase chain reaction (PCR) amplification of the adenomatous polyposis coli gene. PCR products from heterozygous patients for deletions of this gene formed four distinct bands on polyacrylamide gel electrophoresis. The four fragments were subsequently purified and both strands of each fragment were directly sequenced, using an automated DNA sequencer and the same primers as those for PCR amplification. It was found that the two slower migrating fragments were “bulge” heteroduplexes, while the other two were homoduplexes made up of two wild-type strands and two deletion-mutant strands, respectively. The sites of deletions in the adenomatous polyposis coli gene could be exactly determined in four of the five patients. In an attempt to identify deletion-carriers of familial adenomatous polyposis at the presymptomatic stage, a family study was also carried out, and two children were found to have the same mutations as those of their affected parents. The direct sequencing of heteroduplex fragments generated during PCR amplification is a potentially useful method for detecting mutations of not only the adenomatous polyposis coli gene but also many other genes of genetic diseases. © 1993 Wiley-Liss, Inc.     

Numerical aberrations of chromosome 9 in bladder cancer. A possible prognostic marker for early tumor recurrence

Bilharzial bladder cancer is one of the most common types of malignancy in both men and women in several developing countries including Egypt. It has several unique clinical, epidemiological, and histological characteristics, suggesting that it is an entity distinct from bladder cancer seen in Western countries. Genetic alterations in bilharzial-related bladder cancer have been studied infrequently, especially in the advanced stages of disease, that is, T3 and T4 classifications. The objective of this study was to extend establishing the baseline cytogenetic profile of this type of malignancy to early T1 and T2 classifications. For this purpose, fluorescence in situ hybridization was applied to interphase nuclei of frozen-stored samples with biotinylated repetitive DNA probes specific for all chromosomes to detect numerical chromosome changes in 35 patients presenting with relatively early-stage pT1 and pT2 disease. Eleven cases had squamous cell carcinoma (SCC) and 24 had transitional cell carcinoma. Six of 24 transitional cell carcinomas had diploid chromosome counts with all the probes. Numerical chromosome aberrations were detected in 18 cases (75%). In 12 cases, a loss of chromosome 9 was observed. In three cases, an additional loss of chromosome 17 was detected. One case demonstrated a loss of chromosome 10, whereas another two cases showed a gain of chromosome 7, next to a loss of chromosome 9. Loss of chromosome Y was observed in nine of the 27 male cases studied (33.3%), in which only one case showed an abnormality whereas four cases were detected next to loss of chromosome 9, and one case showed gain of chromosome 7. Five cases showed loss of chromosome 19 whereas gain of chromosome 4 was detected in two cases. Two of 11 samples of SCC had normal diploid chromosome counts with all the probes used. In four of 11 cases (36.4%) underrepresentation of chromosome 9, compared with the other chromosomes, was detected. An additional loss of chromosome 17 and gain of chromosome 7, next to loss of chromosome 9, was detected in three cases. One case showed loss of chromosome 17 as the only numerical aberration. Loss of the Y chromosome was detected in three cases of which one case had gain of chromosome 7 and one case had loss of chromosome 19. No correlation was found between any of the clinicopathologic parameters examined in this study and the presence or absence of any numerical chromosomal aberrations except for the significant association between schistosomal history and loss of Y chromosome (P=0.007).     

Abscess-forming granulomatous lymphadenitis: Histological typing of suppurative granulomas and clinicopathological findings with special reference to cat scratch disease

In order to clarify the histological and immunohistochemical characteristics of suppurative granuloma in abscess-forming granulomatous lymphadenitis (AGL), and the relation between AGL and cat scratch disease (CSD), 36 cases of AGL were studied. The combined results showed that there were two types of suppurative granulomas. The suppurative granulomas histologically revealed small lymphocytes of predominantly T cell phenotype distributed among the epithelioid histiocytes bordering central necrotic areas in the suppurative granulomas. These suppurative granulomas could be further subdivided into two groups, mainly those with and without the intermingling of large transformed cells of B-cell phenotypes: Type B granuloma with large transformed B cells and Type A without large transformed B cells. Both types of granulomas were observed in a varying degree in most cases. According to the predominant type of granulomas, 36 patients with AGL were further classified into two groups: Group I of Type A dominance and Group II of Type B dominance. Warthin-Starry (WS) silver stain positive bacteria, which are said to be a causative agent of CSD, were present in about 50% of both groups. No Brown-Hopps' Gram-positive bacteria, fungus, toxoplasma, Chlamydia or Bacillus Calmette-Guerin antigen were found in any case. Clinically, there was no significant difference between these two groups. On the other hand, the detection of WS-positive bacteria seemed to have some relationship with the duration of disease and the history of exposure to cats, and 70% of AGL cases occurred in autumn without a single concurrent epidemic.     

Endomyometriosis arising in the uterosacral ligament: a case report including a literature review and immunohistochemical analysis

We report a case of endomyometriosis arising in the left uterosacral ligament of a 29-year-old woman. The central cavity of the uterine-like mass was lined by pseudo-stratified columnar epithelium and endometrial stroma. The wall of the cyst consisted of bundles of smooth muscle cells. Immunohistochemical analysis demonstrated both alpha-estrogen receptor and progesterone receptor immunoreactivities in the epithelial, stromal and smooth muscle cells. A relatively high proliferating activity was also demonstrated in these cells by Ki-67 immunostaining. These findings suggest that the mass was hormone dependent and had a relatively rapid evolution.     

Catalase, a Specific Antigen in the Feces of Human Subjects Infected with Helicobacter pylori

Recently, we reported the production of three new monoclonal antibodies with high specificity for a Helicobacter pylori antigen suitable for diagnosis of H. pylori infection. The aim of the present study was to identify the antigen recognized by these monoclonal antibodies concerning both H. pylori and the feces of human subjects infected with H. pylori. The cellular antigen was purified from an H. pylori cell extract by immunoaffinity column chromatography with the monoclonal antibody as a ligand. The amino-terminal amino acid sequences (eight residues) of the purified antigen and H. pylori catalase were the same. The molecular weights of native and subunit, specific catalase activity, and UV and visible spectra of the purified antigen were in good agreement with those of H. pylori catalase. The human fecal antigens were purified from two fecal samples of two H. pylori-positive subjects by ammonium sulfate precipitation, CM-Sephadex C₅₀ chromatography, and the same immunoaffinity chromatography used for the H. pylori cellular antigen. The fecal antigens had catalase activity. The amino-terminal amino acid sequences (five residues) of the human fecal antigen and H. pylori catalase were the same. The monoclonal antibodies reacted with the native cellular antigen, but did not react with the denatured antigen, human catalase, and bovine catalase. The results show that the target antigen of the monoclonal antibodies is native H. pylori catalase and that the monoclonal antibodies are able to specifically detect the antigen, which exists in an intact form, retaining the catalase activity in human feces.     

Frequent co-localization of Cox-2 and laminin-5 gamma2 chain at the invasive front of early-stage lung adenocarcinomas

Laminin-5 is an extracellular matrix protein that plays a key role in cell migration and tumor invasion. Cox-2 is an induced isoform of cyclooxygenases that plays an important role in carcinogenesis, suppression of apoptosis, angiogenesis, and metastasis of colon cancer. We report frequent co-expression of cox-2 and laminin-5 at the invasive front of early-stage lung adenocarcinomas. We investigated the expression of cox-2 and laminin-5 immunohistochemically in 102 cases of small-sized lung adenocarcinoma (maximum dimension, 2 cm or less). Cox-2 and laminin-5 were expressed in 97 (95.1%) and 82 (80.4%) cases, respectively. Both were preferentially localized in cancer cells at the cancer-stroma interface, although cox-2 tended to show a diffuse staining pattern in some cases. A comparison of their staining patterns revealed a striking similarity in their distribution in 24 cases, and a partial overlap between their localization in another 20 cases. Moreover, an overall correlation was found between the expression levels of cox-2 and laminin-5 (P = 0.018). To gain insight into the mechanisms that regulate the expression of these proteins, we additionally studied their expression in 58 cases of stage I lung adenocarcinoma, in which p53 status was determined by immunohistochemistry, polymerase chain reaction-single strand conformation polymorphism analysis, and direct sequencing. The results showed that tumors with mutant p53 tended to express more cox-2 than those with wild-type p53 (P = 0.080). Also, tumors that overexpressed p53 had higher levels of cox-2 and laminin-5 than those without p53 overexpression (P = 0.032 and 0.047, respectively). Further immunohistochemical analysis showed that tumors that overexpressed both epidermal growth factor receptor (EGFR) and erbB-2 had higher levels of cox-2 and laminin-5 than those without concomitant overexpression of these proteins (P = 0.014 and P = 0.018, respectively). To see whether EGFR signaling is involved in cox-2 and laminin-5 expression, we further conducted in vitro analyses using six lung adenocarcinoma cell lines (A549, HLC-1, ABC-1, LC-2/ad, VMRC-LCD, and L27). Western blot analyses showed that cox-2 mRNA levels, and to a lesser extent laminin-5 gamma2 mRNA levels, correlated with the expression levels of erbB-2 and the phosphorylated form of MAPK/ERK-1/2 protein. The addition of transforming growth factor-alpha increased both cox-2 and laminin-5 gamma2 mRNA levels in A549, ABC-1, and L27 with different kinetics; the induction of cox-2 occurred earlier than that of laminin-5 gamma2. Finally, the migration of ABC-1 cells was inhibited by MAP kinase kinase inhibitor PD98059 and a selective cox-2 inhibitor NS-398. In contrast, the migration of A549 cells was inhibited by PD98059, but much less effectively by NS-398. These results suggest that co-stimulatory mechanisms may exist that increase the expression of cox-2 and laminin-5 at the invasive front of lung adenocarcinomas and that EGFR signaling could be one of the mechanisms. Further investigations are warranted concerning the role of cox-2 and laminin-5 in cancer cell invasion and the significance of p53 and EGFR signaling in the regulation of cox-2 and laminin-5 expression.     

Human cytomegalovirus infection in foci of Langerhans cell histiocytosis

 Langerhans cell histiocytosis (LCH) has been thought to be a disorder of immune regulation, and increasingly, evidence showing that the tissue damage in LCH involves lymphokines and pro-inflammatory cytokines is reported. We detected human cytomegalovirus (HCMV)-DNA in LCH cells in the foci of LCH lesions by immunohistochemistry, in situ hybridization and PCR. HCMV was detected in the nuclei and/or cytoplasm of LCH cells in 9 of 27 LCH cases by immunostaining. HCMV was probably an early antigen. In situ hybridization revealed signals for HCMV-DNA only in the nuclei of LCH cells in 10 of the 27 LCH cases. PCR analysis was performed in 20 of the LCH cases, and HCMV-DNA was detected in 7 of these. All 7 positive cases were also positive for HCMV by ISH and IHC. These findings suggested that early phase infection or reactivation of HCMV occurred in the LCH lesions. HCMV infection may be accompanied by impaired cytokine production. Our study also suggested a relationship between HCMV infection and expression of TNFα. In tissues affected by LCH, dermatopathic lymphadenopathy or malignant fibrous histiocytoma and in normal tissues no signals for Epstein-Barr virus-RNA were detected. These findings suggest that in some cases LCH is associated with HCMV infection. Received: 24 November 1998 / Accepted: 24 April 1998     

Microinvasive ductal carcinoma (T1mic) of the breast. The clinicopathological profile and immunohistochemical features of 28 cases

Microinvasive ductal carcinoma of the breast, namely ductal carcinoma in situ with microinvasion (T1mic) as defined by the American Joint Committee on Cancer (AJCC) Staging Manual, is a rare disease, although it is increasing because of widespread use of mammography. The aim of the present study was to describe the clinicopathological and immunohistochemical features of this entity. Twenty-eight patients who were diagnosed as T1mic from January 1997 to August 2002 were studied by using 3-5 mm-thick serial sections with hematoxylin-eosin staining. Immunohistochemical staining for the estrogen receptor (ER), progesterone receptor (PR), p53, Ki-67, and HER-2 were performed. All 28 patients were female, with a mean age of 48.8 years. Twenty-six patients (93%) revealed mammographic abnormalities on routine examination. All foci of the invasions were measured using an ocular micrometer. Invasive foci consisted of isolated cells or cell clusters, or appeared as a tongue-like projection of tumor through the basement membrane of the duct of ductal carcinoma in situ (DCIS). The mean number of invasive foci was 3, and the mean size was 0.6 mm. We found that high nuclear grade and predominant comedo subtype of DCIS components were 57.1% and 46.4%, respectively. Twenty-four cases (86%) demonstrated necrosis of DCIS components. Microinvasion was often associated with periductal stromal reaction (71.5%) and/or a lymphocytic infiltration (78.6%). All patients, excluding two, received axillary resection (the mean number of lymph nodes examined per case was 12), and none had lymph node metastasis. The positive expression of ER and PR strongly related to low grade nuclei and non-comedo subtype; however, the positive expression of HER-2 and P53 related to high grade nuclei and comedo subtype (P<0.01). Ki-67 expression was significantly higher in the high grade nuclei group than in the low grade group (P<0.01). Our study suggested that high nuclear grade and comedo DCIS were more aggressive and more common with microinvasion, and that microinvasion is more likely to be multifocal.