Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings

High-level microsatellite instability (AISI-H) is demonstrated in 10 to 15% of sporadic colorectal cancers and in most cancers presenting In the inherited condition hereditary nonpolyposis colorectal cancer (HNPCC). Distinction between these categories of MSI-H cancer is of clinical importance and the aim of this study was to assess clinical, pathological, and molecular features that might he discriminatory. One hundred and twelve MSI-H colorectal cancers from families fulfilling the Bethesda criteria were compared with 57 sporadic MSI-H colorectal cancers. HNPCC cancers presented at a lower age (P < 0.001) with no sporadic MSI-H cancer being diagnosed before the age of 57 years. MSI was less extensive in HNPCC cancers with 72% microsatellite markers showing band shifts compared with 87% in sporadic tumors (P < 0.001). Absent immunostaining for hMSH2 was only found in HNPCC tumors. Methylation of bMLH1 was observed in 87% of sporadic cancers but also in 55% of HNPCC tumors that showed loss of expression of hMLH1 (P = 0.02). HNPCC cancers were more frequently characterized by aberrant beta -catenin immunostaining as evidenced by nuclear positivity (P < 0.001). Aberrant p53 immunostaining was infrequent in both groups. There were no differences with respect to 5q loss of heterozygosity or codon 12 K-ras mutation, which were infrequent in both groups. Sporadic MSI-H cancers were more frequently heterogeneous (P < 0.001), poorly differentiated (P = 0.02), mucinous (P = 0.02), and proximally located (P = 0.04) than RNPCC tumors. In sporadic MSI-H cancers, contiguous adenomas were likely to be serrated whereas traditional adenomas were dominant in HNPCC. Lymphocytic infiltration was more pronounced in HNPCC but the results did not reach statistical significance. Overall, HNPCC cancers were more like common colorectal cancer in terms of morphology and expression of beta -catenin whereas sporadic MSI-H cancers displayed features consistent with a different morphogenesis. No individual feature was discriminatory for all RN-PCC cancers. However, a model based on four features was able to classify 94.5% of tumors as sporadic or HNPCC. The finding of multiple differences between sporadic and familial MSI-H colorectal cancer with respect to both genotype and phenotype is consistent with tumorigenesis through parallel evolutionary pathways and emphasizes the importance of studying the two groups separately.

Methylation of O-6-methylguanine DNA methyltransferase characterizes a subset of colorectal cancer with low-level DNA microsatellite instability

The significance of low-level DNA microsatellite instability (MSI-L) is not well understood. K-ras mutation is associated with MSI-L colorectal cancer and with the silencing of the DNA repair gene O-6-methylguanine DNA methyltransferase (MGMT) by methylation of its promoter region. MGMT methylation was studied in sporadic colorectal cancers stratified as DNA microsatellite instability-high (n = 23), MSI-L (n = 44), and microsatellite-stable (n = 23). Methylation-specific PCR was used to detect MGMT-promoter hypermethylation in 3 of 23 (13%) microsatellite instability-high, in 28 of 44 (64%) MSI-L, and in 6 of 23 (26%) microsatellite-stable cancers (P = 0.0001). K-ras was mutated in 20 of 29 (69%) methylated MSI-L cancers and in 2 of 15 (13%) unmethylated MSI-L cancers (P = 0.001), indicating a relationship between MGMT-methylation and mutation of K-ras. Loss of nuclear expression of MGMT was demonstrated immunohistochemically in 23 of 31 (74%) cancers with methylated MGMT and in 10 of 49 (20%) cancers with nonmethylated MGMT (P < 0.0001). Loss of expression of MGMT was also demonstrated in 9 of 31 serrated polyps. Silencing of MGMT may predispose to mutation by overwhelming the DNA mismatch repair system and occurs with greatest frequency in MSI-L colorectal cancers.

Proliferation, apoptosis, and survival in high-level microsatellite instability sporadic colorectal cancer

Sporadic colorectal cancer (CRC) characterized by high-level DNA microsatellite instability (MSI-H) has a favorable prognosis. The reason for this MSI-H survival advantage is not known. The aim of this study was to correlate proliferation, apoptosis, and prognosis in CRC stratified by MSI status. The proliferative index (PI) was measured by immunohistochemical staining with the Ki-67 antibody in a selected series of 100 sporadic colorectal cancers classified according to the level of MSI as 31 MSI-H, 29 MSI-Low (MSI-L), and 40 microsatellite stable (MISS). The Ki-67 index was significantly higher in MSI-H cancers (P < 0.0001) in which the PI was 90.1 1.2% (mean +/- SE) compared with 69.5 +/- 3.1 % and 69.5 +/- 2.3 % in MSI-L and MSS subgroups, respectively. There was a positive linear correlation between the apoptotic index (AI) and PI (r = 0.51; P < 0.001), with MSI-H cancers demonstrating an increased AI:PI ratio indicative of a lower index of cell production. A high PI showed a trend toward predicting improved survival within MSI-H cancers (P = 0.09) but did not predict survival in MSI-L or MSS cancers. The Al was not associated with survival in any MSI subgroup. In conclusion, this is the first study to show that sporadic MSI-H cancers are characterized by a higher AL:PI ratio and increased proliferative activity compared with MSI-L and MSS cancers, and that an elevated PI may confer a survival advantage within the MSI-H subset.

High frequency of low-level microsatellite instability in early colorectal cancer

Molecular events in early colorectal cancers (CRCs) have not been well elucidated because of the low incidence of early CRCs in clinical practice. Therefore, we studied 104 sporadic early CRCs with invasion limited to submucosa compared with 116 advanced CRCs. Loss of heterozygosity as well as microsatellite instability (MSI) status was examined. A significantly high frequency of low-level MSI (MSI-L) phenotype was detected in early CRCs (51.0%) compared with advanced CRCs (25.9%; P = 0.0001). In early and advanced CRCs, samples with MSI-L phenotype differed from microsatellite stable (MSS) phenotype with respect to loss of heterozygosity at 1p32 and 8p12-22. MSI-L is a frequent genetic event in early CRCs and may be a novel pathway in colorectal carcinogenesis distinct from both MSI-H and MSS.

Apoptosis and Expression of Bcl-2, Bcl-XL, and Bax in Renal Cell Carcinomas

There are at present disparate published results with regard to the relevance of the Bcl-2 gene family, levels of apoptosis, and cell proliferation in the development and progression of renal cell carcinoma (RCC). The present study v analyses the interrelationship between the expression of representatives of the anti-apoptotic (Bcl-2, Bcl-X-L) or pro-apoptotic (Bax) Bcl-2 proteins, incidence of apoptosis, and mitosis in a selected small group of 22 graded RCCs that had paired normal renal tissue, or non-neoplastic tissue in the renal biopsy specimen. The cases were chosen to determine the feasibility of measuring these parameters as potential surrogate markers of progression or treatment failure of the cancers. The results showed that in approximately 50% of the RCCs, where Bcl-2 and/or Bcl-X-L expression was high, apoptosis it-as not detected, and when expression of these proteins was low or not found, increased levels of apoptosis were seen. In most of the remaining 50% of samples, high levels of Bcl-X-L but not Bcl-2 were negatively correlated with low levels of apoptosis (Bcl-X-L: r = -0.437, P = 0.07 and Bcl-2: r = + 0.560, P = 0.02). For the same group of samples, high Bax expression was found in association with apoptosis (r = + 0.578, P = 0,02). A novel finding was an association between low expression of Bcl-2 an/or Bcl-X-L in normal tissue and the level of expression of these proteins in the RCCs, an intrinsic variation that may be an individual patient factor. The results indicate that, in RCCs with increased expression of Bcl-2 and/or Bcl-X-L, levels of apoptosis are minimal and these combined factors may assist in progression of the cancers and resistance to treatments.

Low Level Laser Effects On Pain to Palpation and Electromyographic Activity in TMD Patients: A Double-Blind, Randomized, Placebo-Controlled Study

The purpose of this study was to evaluate the effect of diode laser (GaAlAs - 780 nm) on pain to palpation and electromyographic (EMG) activity of the masseter and anterior temporalis muscles. The laser was applied on the temporalis and masseter muscles twice a week (four weeks). Forty-eight (48) patients with myofascial pain were randomly assigned between actual and placebo treatments and between the energetic doses of 25 J/cm(2) and 60 J/cm(2), and were evaluated using VAS before, immediately after the final application, and 30 days after the laser treatment. Surface electromyography was performed with maximum dental clenching before and after laser therapy. The results show there were no significant statistical differences in the EMG activity between the groups before and after laser treatment. With regard to the pain at palpation, although both groups presented a significant difference in the symptoms before and after the treatment, only the active doses showed statistically significant reductions in pain level in all the regions of the palpated muscles. However, there was no significant statistical difference between groups (experimental and placebo). In conclusion, low level laser did not promote any changes in EMG activity. The treatment did, however, lessen the pain symptoms in the experimental groups.

Low-level lead exposure and children

The adverse effects of environmental lead exposure on the mental development of young children are well established. There is no safe level of blood lead below which children are not affected. Recent research expands our understanding of the impact of lead exposure continuing into later childhood, as well as its effects on children's behaviour. However, social and other environmental factors also contribute to variance in measures of developmental and behavioural outcomes. Lead is associated with only modest effects on children's development, but is a potentially modifiable risk factor. As environmental exposure to lead declines for the whole population, continued specific attention is needed for children living in industrial areas.

A molecular comparison of T lymphocyte populations infiltrating the liver and circulating in the blood of patients with chronic hepatitis B: Evidence for antigen-driven selection of a public complementarity-determining region 3 (CDR3) motif

Despite a large number of T cells infiltrating the liver of patients with chronic hepatitis B, little is known about their complexity or specificity. To characterize the composition of these T cells involved with the pathogenesis of chronic hepatitis B (CHB), we have studied the clonality of V beta T cell receptor (TCR)-bearing populations in liver tissue by size spectratyping the complementarity-determining region (CDR3) lengths of TCR transcripts. We have also compared the CDR3 profiles of the lymphocytes infiltrating the liver with those circulating in the blood to see whether identical clonotypes may be detected that would indicate a virus-induced expansion in both compartments. Our studies show that in most of the patients examined, the T cell composition of liver infiltrating lymphocytes is highly restricted, with evidence of clonotypic expansions in 4 to 9 TCR V beta subfamilies. In contrast, the blood compartment contains an average of 1 to 3 expansions. This pattern is seen irrespective of the patient's viral load or degree of liver pathology. Although the TCR repertoire profiles between the 2 compartments are generally distinct, there is evidence of some T cell subsets being equally distributed between the blood and the liver. Finally, we provide evidence for a putative public binding motif within the CDR3 region with the sequence G-X-S, which may be involved with hepatitis B virus recognition.

Adoptive transfer of autologous Epstein-Barr virus-specific cytotoxic T cells for nasopharyngeal carcinoma

Tumor cells from NPC patients are regularly and latently infected with EBV. To examine whether the virus serves as target for immune intervention of the cancer, we determined levels of EBV-specific CTLp in peripheral blood from NPC patients, long-term survivors of the cancer and healthy subjects. CTLp levels of test subjects varied between 3-3,000/10(6) PBMCs. The plasma EBV burden increased when the CTLp level fell below 150, whereas the EBV burden of PBMCs was not correlated with CTLp level. Compared with healthy carriers, CTLp levels of patients were lower and varied over a wider range, between 3-1,500/10(6) PBMCs. The quantitative immune deficit was probably attributed to the tumor because, first, CTLp in survivors was restored to levels similar to those in healthy carriers after the tumor had been successfully treated. Second, the CTLp level changed as disease progressed, being lower in local disease, increased in locoregional disease and decreased again when the tumor metastasized. Based on these findings, 4 patients with advanced disease were infused with 5 x 10(7)-3 x 10(8) autologous EBV CTLs. The treatment was safe and unaccompanied by inflammatory or other complications, but whether it improved tumor control could not be discerned from the large tumor bulk. Nevertheless, the treatment regularly increased CTLp levels of patients, maintained it at higher levels for protracted periods and, in 3 patients, restored host surveillance of EBV replication, reducing the plasma EBV burden. Taken together, these results provided a rationale to further explore EBV as a target of immune intervention of NPC. (C) 2001 Wiley-Liss, Inc.

Quantitative and qualitative influences of tapasin on the class I peptide repertoire

Tapasin is critical for efficient loading and surface expression of most HLA class I molecules. The high level surface expression of HLA-B*2705 on tapasin-deficient 721.220 cells allowed the influence of this chaperone on peptide repertoire to be examined. Comparison of peptides bound to HLA-B*2705 expressed on tapasin-deficient and -proficient cells by mass spectrometry revealed an overall reduction in the recovery of B*2705-bound peptides isolated from tapasin-deficient cells despite similar yields of B27 heavy chain and beta (2)-microglobulin. This indicated that a proportion of suboptimal ligands were associated with B27, and they were lost during the purification process. Notwithstanding this failure to recover these suboptimal peptides, there was substantial overlap in the repertoire and biochemical properties of peptides recovered from B27 complexes derived from tapasin-positive and -negative cells. Although many peptides were preferentially or uniquely isolated from B*2705 in tapasin-positive cells, a number of species were preferentially recovered in the absence of tapasin, and some of these peptide ligands have been sequenced. In general, these ligands did not exhibit exceptional binding affinity, and we invoke an argument based on lumenal availability and affinity to explain their tapasin independence. The differential display of peptides in tapasin-negative and -positive cells was also apparent in the reactivity of peptide-sensitive alloreactive CTL raised against tapasin-positive and -negative targets, demonstrating the functional relevance of the biochemical observation of changes in peptide repertoire in the tapasin-deficient APC. Overall, the data reveal that tapasin quantitatively and qualitatively influences ligand selection by class I molecules.

A novel approach to antigen-specific deletion of CTL with minimal cellular activation using alpha 3 domain mutants of MHC class I/peptide complex

In this study, we have compared the effector functions and fate of a number of human CTL clones in vitro or ex vivo following contact with variant peptides presented either on the cell surface or in a soluble multimeric format. In the presence of CD8 coreceptor binding, there is a good correlation between TCR signaling, killing of the targets, and Fast-mediated CTL apoptosis. Blocking CD8 binding using (alpha3 domain mutants of MHC class I results in much reduced signaling and reduced killing of the targets. Surprisingly, however, Fast expression is induced to a similar degree on these CTLs, and apoptosis of CTL is unaffected. The ability to divorce these events may allow the deletion of antigen-specific and pathological CTL populations without the deleterious effects induced by full CTL activation.

Tolerance or immunity to a tumor antigen expressed in somatic cells can be determined by systemic proinflammatory signals at the time of first antigen exposure

Mice transgenic for the E7 tumor Ag of human papillomavirus type 16, driven from a keratin 14 promoter, express E7 in keratinocytes but not dendritic cells. Grafted E7-transgenic skin is not rejected by E7-immunized mice that reject E7-transduced transplantable tumors. Rejection of recently transplanted E7-transgenic skin grafts, but not of control nontransgenic grafts or of established E7-transgenic grafts, is induced by systemic administration of live or killed Listeria monocytogenes or of endotoxin. Graft recipients that reject an E7 graft reject a subsequent E7 graft more rapidly and without further L. monocytogenes exposure, whereas recipients of an E7 graft given without L. monocytogenes do not reject a second graft, even if given with L. monocytogenes. Thus, cross-presentation of E7 from keratinocytes to the adaptive immune system occurs with or without a proinflammatory stimulus, but proinflammatory stimuli at the time of first cross-presentation of Ag can determine the nature of the immune response to the Ag. Furthermore, immune effector mechanisms responsible for rejection of epithelium expressing a tumor Ag in keratinocytes are different from those that reject an E7-expressing transplantable tumor. These observations have implications for immunotherapy for epithelial cancers.

Individual and societal bases of national identity - A comparative multi-level analysis

In a previous study (Jones and Smith, 1999) we established that much the same core pattern of national identity characterizes many developed countries. Using the national identity module from the 1995 International Social Survey Programme, we identified two dimensions of national identity: an ascriptive dimension resembling the concept of ethnic identity described in the historical and theoretical literature, and a voluntarist dimension closer to the notion of civic identity. Some writers view these dimensions in terms of a historical sequence but we find that both constructs coexist in the minds of individual respondents in the nations we examine (we exclude Bulgaria and the Philippines from the present but not the earlier analysis). The dataset used for the multilevel analyses reported here consists of 28 589 respondents in the remaining 21 countries included in the national identity database for the 1995 round of surveys. The macrosociological literature on national identity does not offer well-defined predictions about what precise patterns of national identification we might expect to find among the masses of the developed countries. There are, however, recurring themes from which one can construct plausible hypotheses about how countries might differ according to their level of development, broadly conceived. Thus, we hypothesize that forces such as post-industrialism and globalization tend to favour the more open voluntaristic form of national identity over the more restrictive ascribed form. We develop different multi-level models in order to evaluate specific hypotheses pertaining to such issues, by simultaneously relating individual and societal characteristics to the relative strength of individual commitment to these different types of national identity.