Differential expression of genes encoding tight junction proteins in colorectal cancer: frequent dysregulation of claudin-1, -8 and -12.

BACKGROUND AND AIMS: As integral membrane proteins, claudins form tight junctions together with occludin. Several claudins were shown to be up-regulated in various cancer types. We performed an expression analysis of genes encoding tight junction proteins to display differential gene expression on RNA and protein level and to identify and validate potential targets for colorectal cancer (CRC) therapy. PATIENTS AND METHODS: Amplified and biotinylated cRNA from 30 microdissected CRC specimen and corresponding normal tissues was hybridized to Affymetrix U133set GeneChips. Quantification of differential protein expression of claudin-1, -8 and -12 between normal and corresponding tumour tissues was performed by Western blot analyses. Paraffin-embedded CRC tissue samples, colon cancer cell lines and normal tissue microarray were analysed for protein expression of claudin-1 by immunohistochemistry (IHC). RESULTS: Claudin-1 (CLDN1) and -12 (CLDN12) are frequently overexpressed in CRC, whereas claudin-8 (CLDN8) shows down-regulation in tumour tissue on RNA level. Quantification of proteins confirmed the overexpression of claudin-1 in tumour tissues, whereas changes of claudin-8 and -12 were not significantly detectable on protein level. IHC confirmed the markedly elevated expression level of claudin-1 in the majority of CRC, showing membranous and intracellular vesicular staining. CONCLUSIONS: Differential expression of genes encoding claudins in CRC suggests that these tight junction proteins may be associated to and involved in tumorigenesis. CLDN1 is frequently up-regulated in large proportion of CRC and may represent potential target molecule for blocking studies in CRC.

Immunohistochemical expression of extracellular matrix proteins and adhesion molecules in pancreatic carcinoma.

BACKGROUND/AIMS: Carcinoma invasion and metastasis in general involve multiple steps including dynamic changes in the composition and structure of extracellular matrix proteins and cell surface receptors. In the present study, the usually highly invasive carcinoma of the pancreas was investigated regarding the expression of various extracellular matrix proteins and their corresponding integrin receptors, as well as E-cadherin. METHODOLOGY: Phenotypic expression of various markers was investigated immunohistochemically in frozen sections of 16 pancreatic carcinomas and normal pancreatic tissue. RESULTS: An irregular and discontinuous deposition of type IV collagen and laminin in the basement membrane was found in cancer tissue and a pronounced desmoplastic reaction with deposition of type I, type III, and type IV collagen in the tumor stroma. In contrast, the noninvolved pancreas showed an intact basement membrane and a sparse stroma. The collagen type IV and laminin receptors alpha 2, alpha 3, and beta 1 integrin subunits were expressed on pancreatic cancer cells but not the alpha 6 integrin subunit normally present on epithelial cells, suggesting anchorage independence of the carcinoma cells. An increased capacity for cancer cell motility was suggested by the abundant expression of the "antiadhesive" extracellular matrix proteins, tenascin and vitronectin close to the cancer cells, and the expression of cell surface receptors such as alpha v (vitronectin-binding). Expression of the alpha 4 integrin subunit was also increased on cancer cells. CONCLUSIONS: The distribution of extracellular matrix proteins and the cell surface immune phenotype differed in pancreatic carcinoma as compared to normal pancreatic tissue. The present findings substantiate the notion that disseminated growth of highly malignant carcinomas of the pancreas reflects an invasive interaction of the tumor cells with extracellular matrix proteins of a well-established stroma. Similar findings were observed regardless of tumor histology and patient survival time.

Proliferation and apoptosis in the evolution of endemic and acquired immunodeficiency syndrome-related Kaposi's sarcoma.

Kaposi's sarcoma (KS) is a multifocal lesion that occurs predominantly in the skin, most frequently in people infected with HIV-1, and that evolves through early stages (patch and plaque) to a tumor-like late stage (nodular). Both, endemic African (EKS) and AIDS-associated (AKS) KS expressed human herpesvirus 8 (HHV-8) as shown by PCR. By immunohistochemistry the expression of cellular Bcl-2 and c-myc was confined in early stages of both EKS and AKS to relatively few endothelial cells (EC) whereas in nodular KS most of spindle cells (SC) strongly expressed both genes. CD40 was usually strongly expressed in SC at all KS stages as well as in EC of non-involved tissue whereas CD40L (CD154) was not demonstrable. Fas (CD95) was moderately to weakly expressed by SC whereas p53 and Waf-1 were found in less than 5% of the SC. In both AKS and EKS at nodular stage almost no apoptotic SC were detected. In most AKS and EKS low levels of cell proliferation were seen but AKS showed consistently higher values compared to EKS. All clinical types and stages of KS showed a diploid cellular DNA content by flow cytometric analysis of microselected lesions. Thus, we conclude that KS during evolution represents diploid, probably reactive, cell proliferation, which progressively increases the expression of strong cellular and also viral (HHV-8) antiapoptotic factors.

The apoptotic v-cyclin-CDK6 complex phosphorylates and inactivates Bcl-2.

v-cyclin encoded by Kaposi's sarcoma herpesvirus/human herpesvirus 8 (KSHV or HHV8) associates with cellular cyclin-dependent kinase 6 (CDK6) to form a kinase complex that promotes cell-cycle progression, but can also induce apoptosis in cells with high levels of CDK6. Here we show that whereas HHV8-encoded v-Bcl-2 protects against this apoptosis, cellular Bcl-2 has lost its anti-apoptotic potential as a result of an inactivating phosphorylation in its unstructured loop region. Moreover, we identify Bcl-2 as a new substrate for v-cyclin-CDK6 in vitro, and show that it is present in a complex with CDK6 in cell lysates. A Bcl-2 mutant with a S70A S87A double substitution in the loop region is not phosphorylated and provides resistance to apoptosis, indicating that inactivation of Bcl-2 by v-cyclin-CDK6 may be required for the observed apoptosis. Furthermore, the identification of phosphorylated Bcl-2 in HHV8-positive Kaposi's sarcoma indicates that HHV8-mediated interference with host apoptotic signalling pathways may encourage the development of Kaposi's sarcoma.

Abolished angiogenicity and tumorigenicity of Burkitt lymphoma by interleukin-10.

Because of its immunosuppressive properties, interleukin-10 (IL-10) is thought to play an important role in a number of human disease states, including inflammation, autoimmunity, and transplant rejection. In this study, we demonstrate that introduction of human or viral IL-10 genes into Burkitt's lymphoma cells markedly reduced their ability to grow as subcutaneous (sc) tumors in SCID mice. In vivo assays for angiogenesis revealed an inhibition of the angiogenic capacity of the IL-10-transfected lines. Recombinant human IL-10 abolished and viral IL-10 reduced vascular endothelial growth factor (VEGF)-165-induced neovascularization. Furthermore, IL-10 blocked the VEGF- and fibroblast growth factor (FGF)-2-induced proliferation of microvascular endothelial cells in vitro. The current observations suggest a direct role for IL-10 in the prevention of angiogenesis in human lymphoid malignancies.

Sequence analysis and variation of EBNA-1 in Epstein-Barr virus-related herpesvirus of cynomolgus monkey.

OBJECTIVES: The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is an important protein for immortalization and tumorigenesis of infected cells. EBNA-1 gene variants may play a role in tumorigenesis. We determined the nucleotide and amino acid (aa) sequences of EBNA-1 in EBV-related herpesviruses from cynomolgus monkeys (cynomolgus-EBV) which induced malignant lymphomas in its natural host and in rabbits, and compared them with sequences of EBV and other lymphocryptoviruses (LCVs). METHODS: Polymerase chain reaction and direct sequencing methods were performed using extracted DNA from cynomolgus-EBV-infected cell lines. RESULTS: The amino acid sequences of cynomolgus-EBV EBNA-1 from two cell lines (Si-IIA: 588 aa; Ts-B6: 619 aa) which are antigenically cross-reactive to human EBV EBNA-1 showed homology with human EBV (Si-IIA: 53%; Ts-B6: 58%) and other LCVs from baboons (54 and 52%) and rhesus monkeys (60 and 58%), especially in the C-terminal unique domain. Homology of the EBNA-1 sequence between Si-IIA and Ts-B6 was 92%. The sequence difference between EBV and the related LCVs was manifested mainly in the length of the internal repeat 3-corresponding region, which contains serine in the glycine/alanine repeat region of nonhuman LCVs. CONCLUSION: Sequence variation of cynomolgus-EBV EBNA-1 from different cell lines was observed. However, their sequences show a relatively high homology with human EBV and share the common features of EBNA-1 of EBV and other LCVs.

Combined analysis of DNA ploidy, proliferation, and apoptosis in paraffin-embedded cell material by flow cytometry.

A flow cytometric assay was developed for correlated measurement of DNA content and apoptotic DNA strand breaks in cell nuclei of formalin-fixed, paraffin-embedded tissues. The assay allows a combined analysis of cell ploidy, proliferation, and apoptosis in sections of fixed paraffin-embedded archival or fresh tissue/cell specimens. It is based on (a) proteolytic release of cell nuclei from deparaffinized and rehydrated 90-microm thick sections of the fixed embedded specimen, (b) the inactivation of the protease, (c) FITC-labeling of DNA strand breaks by the terminal deoxynucleotidyl transferase (TdT)-mediated FITC-dUTP nick end-labeling (TUNEL) reaction, and (d) DNA staining with 4'6-diamidino-2-phenyleindole. The fluorescence was recorded with a double-beam flow cytometer equipped with a mercury arc lamp and an argon ion laser. Cytograms obtained with this assay correlated closely with those produced using nonembedded material from the same specimen. Furthermore, a significant correlation was found between flow cytometric analysis of apoptosis in cell nuclei released from paraffin blocks and conventional evaluation of TUNEL on (corresponding) sections (p < 0.001). Since necrotic cells can stain positively by TUNEL, the possibility to microscopically select nonnecrotic tumor regions for flow cytometric analysis is an important advantage of the assay.

Anti-CD3 induced thymocyte apoptosis in vivo require the antibody Fc domain.

We have earlier found that explanted thymic epithelial cells (TEC) can produce glucocorticoid (GC) activity in vitro and that the GC receptor (GR) antagonist RU486 partially inhibit thymic apoptosis induced by the anti-CD3 monoclonal antibodies (MoAb) 2C11, both in vivo and in new-born thymic organ cultures. To explain the inhibitory effect of RU486 in this system we have now investigated the importance of the 2C11 Fc as this MoAb bind with high affinity to cellular FcR. We have found both that whole 2C11 MoAb can bind to explanted TEC in vitro and that F(ab)'2 fragments from this MoAb loose this ability, in addition with the capacity to induce thymic apoptosis in vivo. We interpret our results to indicate that the injected 2C11 MoAb may establish a close contact between GC producing, FcR positive TEC cells and CD3 positive thymocytes and thereby subject the later to high paracrine GC concentrations and subsequent induction of apoptosis.

Protective DNA immunization against Chlamydia pneumoniae.

We have investigated the efficacy of the DNA vaccination using the heat shock protein 60 (HSP-60) gene of C. pneumoniae, for protection of mice against infection with the bacteria. C57Bl/6 mice had a 5-20-fold reduction of C. pneumoniae numbers in lungs when immunized intranasally (i.n.) with plasmids (p) encoding pHSP-60. The reduction of the bacterial load coincided with a decreased severity of disease. No specific antibodies were detected after protective i. n. immunization. In contrast, mice immunized intradermally (i.d.) were not protected against challenge with C. pneumoniae, although specific humoral Immunoglobulin (Ig)G responses were generated. Co-inoculation i.n. of pHSP-60 with pIL-12 but not with pGM-CSF further increased protection of mice against infection with C. pneumoniae. Lungs from pHSP-60 i.n. immunized and infected mice showed higher levels of interferon (IFN)-gamma mRNA, and spleen cells from these mice co-cultured with r-HSP-60 released higher levels of IFN-gamma and displayed higher proliferative responses than nonimmunized and infected controls. pHSP-60 immunized IFN-gamma receptor (R)-/- mice were not protected against infection with C. pneumoniae. Likewise, i.n. administration of pIFN-gamma alone induced significant protection. DNA vaccine-induced protection was CD4+ and CD8+ T-cell dependent, as shown by DNA-vaccination of MHC class II-/-, CD4-/-, CD8-/- and CD4-/-CD8-/-mice. Interestingly, DNA vaccine induced CD4+ T cells, in the absence of CD8+ T cells, were involved in worsening the outcome of infection. This worsening was linked with a shift towards a Th2 cytokine pattern.

Topoisomerase II-mediated alterations of K562 drug resistant sublines.

In order to further elucidate the roles of DNA topoisomerase II (topo II) subtypes, alpha and beta, as drug targets in chemotherapy, we have determined the enzyme levels in K562 cells selected for resistance to mitoxantrone (K562/Mxn), daunorubicin (K562/Dnr) and idarubicin (K562/Ida 20 and K562/Ida 60), as well as topo II-DNA complex formation, DNA damage and cytotoxicity, induced by topo II interactive agents, for example etoposide, teniposide, mitoxantrone and amsacrine. As compared to the parental cells, topo IIalpha/beta protein levels in K562/Mxn, K562/Dnr, K562/Ida 20 and 60 lines, measured with Western blot, were 17/67%, 85/88, 24/31% and 10/7% respectively. DNA damage, determined by DNA unwinding technique, induced by teniposide and amsacrine correlated with both topo IIalpha/beta protein levels (r2 = 0.8/0.9, P = 0.03/0.01 and r2 = 0.8/0.9, P = 0.04/0.01, respectively). Topo II-DNA complex formation induced by all studied drugs correlated with topo IIbeta protein levels (r2-range 0.8-0.9, P-range 0.01-0.04), while the correlation with topo IIalpha was weaker. Topo IIalpha/beta protein levels tended to show an inverse correlation with the cytotoxicity of etoposide (r2 = -0.9/-0.7, P = 0.01/0.06). The overall topo II-DNA complex formation correlated with drug-induced DNA damage (r2 = 0.9, P = 0.0001), whilst not with the cytotoxicity. Our findings indicate that both topo II isozymes are the targets of the antitumor agents studied, and of potential clinical relevance for prediction of treatment efficacy. They could play a role in tailored chemotherapy.

Kaposi's sarcoma-associated herpesvirus-encoded v-cyclin triggers apoptosis in cells with high levels of cyclin-dependent kinase 6.

Kaposi's sarcoma-associated herpesvirus (KSHV) has a key etiological role in development of Kaposi's sarcoma (KS). v-Cyclin is a KSHV-encoded homologue to D-type cyclins that associates with cellular cyclin-dependent kinase 6 (CDK6). v-Cyclin promotes S-phase entry of quiescent cells and has been suggested to execute functions of both D- and E-type cyclins. In this study, expression of v-cyclin in cells with elevated levels of CDK6 led to apoptotic cell death after the cells entered S phase. The cell death required the kinase activity of CDK6 because cells expressing a kinase-deficient form of CDK6 did not undergo apoptosis upon v-cyclin expression. Studies on the mechanisms involved in this caspase-3-mediated apoptosis indicated that it was independent of cellular p53 or pRb status, and it was not suppressed by Bcl-2. In contrast, the KSHV-encoded v-Bcl-2 efficiently suppressed v-cyclin-/CDK6-induced apoptosis, demonstrating a marked difference in the antiapoptotic properties of c-Bcl-2 and v-Bcl-2. In KS lesions, high CDK6 expression was confined to a subset of cells, some of which displayed signs of apoptosis. These results suggest that v-cyclin may exert both growth-promoting and apoptotic functions in KS, depending on factors regulating CDK6 and v-Bcl-2 levels.

Expression of K13/v-FLIP gene of human herpesvirus 8 and apoptosis in Kaposi's sarcoma spindle cells.

BACKGROUND: Human herpesvirus 8 (HHV8) infection is associated with all forms of Kaposi's sarcoma (KS). The HHV8 genome locus ORFK13-72-73 (ORF = open reading frame) encodes proteins that may be important in HHV8-mediated pathogenesis, i.e., the latency-associated nuclear antigen (encoded by ORF73), viral-cyc-D (v-cyc-D), a viral homologue of cellular cyclin D (encoded by ORF72), and viral-FLIP (v-FLIP), a homologue of the cellular FLICE (Fas-associated death domain-like interleukin 1 beta-converting enzyme) inhibitory protein (encoded by ORFK13; is an inhibitor of apoptosis [programmed cell death]). Through differential splicing events, this locus expresses individual RNA transcripts that encode all three proteins (tricistronic transcripts) or just two of them (v-FLIP and v-cyc-D; bicistronic transcripts). We examined expression of these transcripts in KS tissues. METHODS: We collected tissues from patients with KS of different stages. By use of an optimized in situ hybridization procedure, we examined different ORFK13-72-73 locus transcripts in HHV8-infected cells in skin lesions and in one adjacent lymph node. Apoptosis in KS lesions was determined by use of an in situ assay. RESULTS AND CONCLUSIONS: Our results indicate the following: 1) Transcripts from the ORFK13-72-73 locus appear to be spliced differentially in latently infected KS cells in skin lesions and in HHV8-infected cells in lymph nodes; specifically, ORFK13-ORF72 bicistronic transcripts were expressed abundantly in KS cells, whereas ORFK13-ORF72-ORF73 tricistronic transcripts were detected only in lymph node cells. 2) Sequences encoding the antiapoptotic protein v-FLIP are expressed at very low levels in early KS lesions, but expression increases dramatically in late-stage lesions. 3) The increase in expression of v-FLIP-encoding transcripts is associated with a reduction in apoptosis in KS lesions. IMPLICATIONS: These data suggest that functional v-FLIP is produced in vivo and that antiapoptotic mechanisms may be involved in the rapid growth of KS lesions, where only a few cells undergoing mitosis are generally observed.

Antibacterial components in bronchoalveolar lavage fluid from healthy individuals and sarcoidosis patients.

Antibacterial peptides and proteins are an integral part of the epithelial defense barrier that provides immediate protection against bacterial invasion. In humans, alpha-defensins are mainly bactericidal effectors in circulating granulocytes, beta-defensin-1 is synthesized in epithelial cells, and LL-37 is produced in granulocytes but is also induced in skin epithelia during inflammation. To investigate the importance of these defense effectors in disease, we analyzed bronchoalveolar lavage fluid (BALF) for bactericidal activity. Antibacterial activity was found in BALF material from healthy individuals and sarcoidosis patients, with enhanced activity in BALF from the patients. The activity was present as several antibacterial components, of which we have so far characterized LL-37, lysozyme, alpha-defensins, and antileukoprotease. In addition, the antibacterial peptide LL-37 was located in alveolar macrophages, bronchial epithelial cells, and bronchial glands, suggesting that it has a defensive role in airway mucosa. In conclusion, the airway epithelium is protected by a complex antibacterial defense system. This is activated in sarcoidosis, and may explain why these patients seldom develop severe respiratory tract infections.

Lymphoid disorders associated with HHV-8/KSHV infection: facts and contentions.

Following the demonstration in 1994, that Kaposi's sarcoma (KS) was associated with a novel virus (KSHV or HHV-8) belonging to the lymphotropic herpes family, this virus was also found in certain lymphoid neoplasias of immunodeficient (HIV+) and immune competent hosts. The association of HHV-8/KSHV infection is now well established with primary effusion lymphoma (PEL) or body cavity based lymphoma (BCBL) and multicentric Castleman's disease (MCD) of the plasma cell type. A possible pathogenic role of HHV-8/KSHV in other lymphoid tumours including primary central nervous system lymphoma (PCNSL) and multiple myeloma (MM) as well as some atypical lymphoproliferations and sarcoidosis has also been suggested, but this is at present a controversial matter, or not confirmed. Several HHV-8/KSHV genes, including potential oncogenes, genes homologous to various cellular genes and growth factors have been incriminated in the pathogenesis of KS and PEL/BCBL, but a common pathogenic mechanism for the clearly diverse proliferations represented by PEL, MCD and KS is at present not evident.

Fetal porcine islet-like cell clusters transplanted to cynomolgus monkeys: an immunohistochemical study.

BACKGROUND: The mechanism(s) involved in acute cellular xenograft rejection have hitherto been generated in vitro or in different experimental models, with pig tissue being transplanted to rodents. There is an urgent need to validate these results in a clinically more relevant combination of species. METHODS: Fetal porcine islet-like cell clusters (ICC) were transplanted under the kidney capsule in cynomolgus monkeys, either untreated or given immunosuppression with cyclosporine (CsA; 10 mg/kg body weight, intramuscularly) and 15-deoxyspergualin (DSG; 5 mg/kg body weight, intramuscularly). ICC xenografts were examined at 1, 3, 6, or 10-12 days after transplantation, using immunohistochemical techniques. Serum levels of xenoreactive antibodies were measured with ELISA. RESULTS: No deposits of IgM, IgG, Clq, or C3 were detected within the ICC xenograft in any of the monkeys. Likewise, no significant increase in the levels of xenoreactive antibodies were found after transplantation. In untreated animals, a few N-Elastase-positive cells (neutrophil granulocytes) were seen in the xenograft at day 1. A few mononuclear cells were present in the adjacent renal parenchyma, but they did not infiltrate the xenograft. At this time (day 1), early signs of necrosis were observed in the central parts of the graft. On day 3, the graft had a large, central necrotic area that contained polymorphonuclear cells; the remaining parts of the xenograft showed severe infiltration with CD8+ T cells. Occasional CD68+ cells (macrophages) were seen on days 1 and 3. On day 6, large numbers of macrophages were found infiltrating the entire graft. A few CD20+ B cells, accumulated as small clusters, were also found. Only a few natural killer cells (CD56+) were detected. The CsA/DSG-treated monkeys showed markedly fewer CD2+/CD8+ T cells on day 6 than the untreated monkeys, and the ICC graft was clearly better preserved. However, the number of CD8+ and CD68+ cells had increased considerably at 12 days after transplantation and diffusely infiltrated the whole ICC xenograft. CONCLUSION: Porcine ICC transplanted under the kidney capsule in cynomolgus monkeys were rejected by an acute cell-mediated rejection progressing during the first 6 days after transplantation. The process was not dependent on host Ig or C3 binding to the graft. Although the rejection of porcine ICC was significantly delayed in CsA/DSG-treated monkeys, the ICC xenografts were almost completely destroyed 12 days after transplantation.

Proliferation and apoptosis-related gene expression in experimental acquired immunodeficiency syndrome-related simian lymphoma.

Lymphomas in 10 cynomolgus monkeys infected with a simian immunodeficiency virus (SIVsm) were studied with regard to proliferative activity and apoptosis-related gene expression. All were diffuse large-cell lymphomas, showed mono or oligoclonality and a 9/10 diploid cellular DNA content. Expression of a simian homologue to Epstein-Barr virus (HVMF-1) was shown in nine cases. The lymphomas showed moderate to high proliferative activity by Ki67 immunostaining and DNA flow cytometry, and a low number of apoptotic cells detected by TdT-mediated dUTP nick-end labeling (TUNEL). Immunohistochemistry showed abundant tumor infiltrating TIA-1(+) cytotoxic lymphocytes (CTL) and macrophages. Bcl-2, Mcl-1, and also Bax and Bak, but not p53 were demonstrable in the tumor cells by immunostaining. Our findings suggest a causal relationship between HVMF-1 infection and a low apoptotic index of the lymphomas due to the expression of Bcl-2. The apparent inefficient function of tumor-infiltrating CTL could be due to inactivation of CTL and/or resistance of the lymphoma cells to CTL effects. The tumors showed immunoreactivity for CD18, CD29, and CD49d, but not for CD11a, mimicking the phenotype of human Epstein-Barr virus (EBV)-related lymphomas. In summary, our observations indicate a high similarity between this simian model of acquired immunodeficiency syndrome (AIDS)-related lymphomas (ARL) and human ARL and other immunosuppression-related lymphomas.

Cytotoxic intraepithelial lymphocytes in colorectal polyps and carcinomas.

The frequency of intraepithelial lymphocytes (IEL) was investigated in 255 colorectal lesions: in 18 metaplastic polyps, in 159 tubular, serrated and villous adenomas, in 28 incipient adenocarcinomas and in 50 overt adenocarcinomas. > or = 21 IELs were found in 13.6% (12/88) of the adenomas with high grade dysplasia (HGD) but in none of the 18 metaplastic polyps, the 71 adenomas with low grade dysplasia (LGD), or the 50 overt adenocarcinomas. CD3-T, MHCII and TIA1 cell antigens were positive in IELs from tubular adenomas, villous adenomas, incipient carcinomas and overt adenocarcinomas. In the normal colorectal mucosa and in metaplastic polyps, CD3-T was positive whereas MHCII and TIA 1 were not expressed. On the other hand, Fas cell antigen was expressed in the normal mucosa and in metaplastic polyps but it was downregulated in neoplastic lesions. The upregulation of CD3-T, MHC class II molecules, and TIA 1 in IELs of neoplastic colorectal lesions suggests that they are activated and cytotoxic; however, they undergo apoptosis due to the downregulation of the FAS molecules.

Measles virus antigen in macrophage/microglial cells and astrocytes of subacute sclerosing panencephalitis.

In two patients with subacute sclerosing panencephalitis (SSPE) of 10 and 25 months duration we demonstrated by immunohistochemistry the presence of measles-virus nucleocapsid antigen (MVNA) in CD68+ cells and astrocytes of brain tissues. In both cases, CD68+ hematogenous monocyte/ macrophages and perivascular microglial cells (Mphi) were found infiltrating the brain parenchyma, and often partially or completely invested by perivascular reactive astrocytes expressing glial fibrillary acidic protein (GFAP). Mphi with cytoplasmic MVNA were often seen in the Virchow-Robin spaces and in close association with perivascular astrocytes, which often also contained MVNA+ intracytoplasmic inclusions. Reactive astrocytosis was more severe in the patient with long-standing illness, and a correspondingly elevated number of strongly GFAP+ MVNA+ or MVNA- perivascular binucleated astrocytes was observed. An uptake of MVNA+ cell debris by reactive astrocytes was evident in areas of white matter displaying extensive demyelination and necrosis. Taken together, these observations seem to indicate that the brain infiltration by Mphi carrying measles virus could represent one pathway of virus entry and dissemination in the central nervous system. Virus transfer to perivascular astrocytes via cell-to-cell contacts with infected macrophages is also suggested.

Trypanosoma cruzi infection in tumor necrosis factor receptor p55-deficient mice.

Tumor necrosis factor receptor p55 (TNFRp55) mediates host resistance to several pathogens by allowing microbicidal activities of phagocytes. In the studies reported here, TNFRp55-/- mice infected with the intracellular parasite Trypanosoma cruzi showed clearly higher parasitemia and cumulative mortality than wild-type (WT) controls did. However, gamma interferon (IFN-gamma)-activated macrophages from TNFRp55-/- mice produced control levels of nitric oxide and killed the parasite efficiently in vitro. Trypanocidal mechanisms of nonphagocytic cells (myocardial fibroblasts) from both TNFRp55-/- and WT mice were also activated by IFN-gamma in a dose-dependent way. However, IFN-gamma-activated TNFRp55-/- nonphagocytes showed less effective killing of T. cruzi than WT control nonphagocytes, even when interleukin 1beta (IL-1beta) was added as a costimulator. In vivo, T. cruzi-infected TNFRp55-/- mice and WT mice released similar levels of NO and showed similar levels of IFN-gamma mRNA and inducible nitric oxide synthase mRNA in their tissues. Instead, increased susceptibility to T. cruzi of TNFRp55-/- mice was associated with reduced levels of parasite-specific immunoglobulin G (IgG) (but not IgM) antibodies during infection, which is probably linked to abnormal B-cell differentiation in secondary lymphoid tissues of the mutant mice. Surprisingly, T. cruzi-infected TNFRp55-/- mice showed increased inflammatory and necrotic lesions in several tissues, especially in skeletal muscles, indicating that TNFRp55 plays an important role in controlling the inflammatory process. Accordingly, levels of Mn2+ superoxide dismutase mRNA, a TNF-induced enzyme which protects the cell from the toxic effects of superoxide, were lower in mutant than in WT infected mice.

Positive and negative thymic selection in T cell receptor-transgenic mice correlate with Nur77 mRNA expression.

The orphan nuclear receptor Nur77 has been implicated in thymic negative selection. We studied the effect of two T cell receptor (TCR) transgenes on positive selection and Nur77 mRNA expression in thymus. DO11.10 mice, expressing a transgenic TCR specific for an ovalbumin (OVA) 323-339 peptide presented by I-Ad, were found to have an enlarged thymus with a reduced apoptotic activity, measured by flow cytometry, reduced mitochondrial membrane potential and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) techniques. In contrast, in F5 mice expressing a transgenic TCR recognizing the influenza virus nucleoprotein (NP) 366-374 peptide restricted by Db, this positive selection effect was much less pronounced. Positive thymic selection in DO11.10 TCR+ mice correlated with a reduced level of Nur77 mRNA expression shown by Northern blot. F5 mice expressed levels close to those expressed by the wild type. Both transgenic mouse strains responded with extensive cortical apoptosis, and with up-regulation of Nur77 mRNA, to injection of cognate peptides. As 9-cis-Retinoic acid (9-cis-RA) inhibits Nur77-dependent apoptosis in T cell hybridomas in vitro, mice were pretreated with the drug to investigate a similar effect in vivo. However, the drug itself, at saturating concentrations, caused extensive apoptosis in immature CD4+/CD8+ thymocytes. The result demonstrates a correlation between Nur77 expression and thymic apoptotic activity, both during positive and negative selection events.