Caffeine and alcohol intakes have no association with risk of multiple sclerosis.

BACKGROUND: The association between alcohol and caffeine intakes and risk of multiple sclerosis (MS) is unclear; no prospective studies have examined this relationship. OBJECTIVE: We examined intakes of alcohol and caffeine in relation to risk of multiple sclerosis. METHODS: Intakes of alcohol and caffeine were examined in relation to the risk of MS in two large cohorts of women, the Nurses' Health Study (NHS; 92,275 women followed from 1980 to 2004) and Nurses' Health Study II (NHS II; 95,051 women followed from 1991 to 2005). Their diet was assessed at baseline and every four years thereafter. During the follow-up, 282 cases of MS were confirmed with onset of symptoms after baseline. Twenty-four cases were missing information on alcohol intake, leaving a total of 258 cases for the alcohol analyses. RESULTS: Neither total alcohol consumption, nor consumption of beer, wine, or liquor was related to MS risk. The multivariable-adjusted pooled relative risk (RR) found by comparing categories of alcohol intake to 0 gm/day was 1.07 (95% CI: 0.32-1.99) for 0.1-4.9 gm/day, 1.01 (0.32-1.99) for 5.0-14.9 gm/day, 1.21 (0.69-2.15) for 15.0-29.9 gm/day, and 0.80 (0.32-1.99) for 30+ gm/day; (p for trend=0.89). Caffeine intake was also not significantly associated with MS risk. The multivariable adjusted pooled RR comparing highest to lowest quintile of caffeine intake was 1.14; 95% CI: 0.79-1.66; p for trend=0.71. Consideration of caffeinated and decaffeinated coffee separately also yielded null results. CONCLUSION: These results do not support an association between alcohol and caffeine intakes and MS risk.

Anti-Epstein-Barr virus antibodies as serological markers of multiple sclerosis: a prospective study among United States military personnel.

BACKGROUND: Elevated Epstein-Barr virus (EBV) antibody titers are risk factors for multiple sclerosis (MS), but the strength and consistency of this association are not well characterized. OBJECTIVES: The objectives of this study were to determine whether this association is confounded by vitamin D or modified by gender or race, and the usefulness of EBV nuclear antigen (EBNA) antibodies as a marker for MS. METHODS: We conducted a prospective study among US military personnel. Antibody titers against EBV antigens were measured in serum samples from 222 individuals who developed MS and 444 age, sex, and race/ethnicity matched controls. Conditional logistic regression was used to estimate relative risks. RESULTS: MS risk increased with increasing titers of anti-EBNA complex (p < 10(-9)) and anti-EBNA-1 (p = 5.8 × 10(-9)) titers. MS risk was 36-fold higher among individuals with anti-EBNA complex IgG titers ≥320 than among those with titers <20 (95% confidence interval [CI] 9.6-136), and 8-fold higher among those with anti-EBNA-1 ≥320 than among those with anti-EBNA-1 <20 (95% CI 2.6-23). These associations were consistent across gender and race/ethnicity groups and independent from 25-hydroxyvitamin D levels. Areas under the receiver operating characteristic (ROC) curves were 0.67 for EBNA complex and 0.65 for EBNA-1. CONCLUSIONS: Serum titers of pre-onset anti-EBNA antibodies are strong, robust markers of MS risk and could be useful in an MS risk score.

EBNA1 and LMP1 variants in multiple sclerosis cases and controls.

BACKGROUND: Prior infection with Epstein-Barr virus (EBV) is an established risk factor for multiple sclerosis (MS). Some findings from observational studies, including possible epidemics and differences in prevalence, may be explained if different strains of EBV conferred different MS risk. METHODS: DNA was extracted from peripheral lymphocytes obtained from 66 MS cases and 66 age- and cohort-matched controls. Nested polymerase chain reaction (PCR) was performed to amplify the N- and C-terminus regions of EBNA1 and the hyper-variable region of the LMP1 gene. For EBNA1, we compared the presence of the prototype B95.8 vs variant sequence and the presence of multiple strains in MS cases and controls. For LMP1, we considered differences in the proportions of mutations between cases and controls. RESULTS: Comparing the proportion of mutant sequence between MS cases and controls in the EBNA1 N-terminal (0/28 vs 1/27) and C-terminal regions (3/40 vs 8/36) revealed no significant differences (P > 0.05). No individual variants in LMP1 were associated with risk of MS (all P > 0.05). Neither EBNA1 nor LMP1 variation was associated with anti-EBNA1 IgG antibody titers. CONCLUSIONS: These findings do not support a strong role for variation in EBNA1 N-terminus, EBNA1 C-terminus or LMP1 contributing to MS risk.

99th Dahlem conference on infection, inflammation and chronic inflammatory disorders: Epstein-Barr virus and multiple sclerosis: epidemiological evidence.

While the causes of multiple sclerosis (MS) are unknown, there is strong evidence that infection with Epstein-Barr virus (EBV) is an important factor. In this review, we discuss the epidemiological evidence and argue for a causal role of EBV in MS aetiology. One of the most striking and consistent observations is that MS is extremely rare among EBV-negative individuals. Further, the timing of EBV infection appears to be critical, with individuals who are infected during adolescence and young adulthood, when the infection is more likely to manifest as mononucleosis, having a two- to threefold greater risk of MS compared to individuals infected in early life. These observations challenge the hygiene hypothesis which states that being in a high hygiene environment in early life increases future risk of MS - if this general formulation were true, EBV-negative individuals would be expected to have an increased risk of MS. Additional support for the causal role of EBV comes from longitudinal, prospective studies which show remarkable consistency, in that antibodies against EBV are elevated prior to MS onset. However, while infection with EBV is consistent with many observations of MS epidemiology, there are some that remain unexplained, suggesting that other factors are also involved in determining risk.

The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product.

Deletions or mutations of the retinoblastoma gene, RB1, are common features of many tumors and tumor cell lines. Recently, the RB1 gene product, p105-RB, has been shown to form stable protein/protein complexes with the oncoproteins of two DNA tumor viruses, the adenovirus E1A proteins and the simian virus 40 (SV40) large T antigen. Neither of these viruses is thought to be associated with human cancer, but they can cause tumors in rodents. Binding between the RB anti-oncoprotein and the adenovirus or SV40 oncoprotein can be recapitulated in vitro with coimmunoprecipitation mixing assays. These assays have been used to demonstrate that the E7 oncoprotein of the human papilloma virus type-16 can form similar complexes with p105-RB. Human papilloma virus-16 is found associated with approximately 50 percent of cervical carcinomas. These results suggest that these three DNA viruses may utilize similar mechanisms in transformation and implicate RB binding as a possible step in human papilloma virus-associated carcinogenesis.

Plasma titers of antibodies against Epstein-Barr virus BZLF1 and risk of multiple sclerosis.

BACKGROUND/AIMS: Results of recently conducted prospective studies have demonstrated that the presence of high titers of anti-EBNA-1 or anti-EBNA complex IgG antibodies in healthy individuals is a strong risk factor for multiple sclerosis (MS). Antibodies to BZLF1, the product of the homonymous early lytic gene, have been found to be related to risk of nasopharyngeal carcinoma, but have not been previously measured in MS studies. METHODS: We examined whether high levels of anti-BZLF1 IgG antibodies also predict MS risk in a nested case-control study among women in the Nurses Health Study and Nurses Health Study II cohorts. RESULTS: Results of this prospective study suggest that antibody titers to EBNAs are the strongest predictor of MS risk. CONCLUSION: Little further contribution may be provided by measuring anti-BZLF1 antibodies in regard to MS risk.

Isolation and characterization of a novel epithelium-specific transcription factor, ESE-1, a member of the ets family.

We report here the isolation of a novel, highly tissue-restricted member of the ets transcription factor/oncogene family, ESE-1 (for epithelium-specific Ets), which has features distinct from those of any other ets-related factor. ESE-1 contains two putative DNA binding domains: an ETS domain, which is unique in that the 5' half shows relatively weak homology to known ets factors, and an A/T hook domain, found in HMG proteins and various other nuclear factors. In contrast to any known ets factors, ESE-1 is expressed exclusively in epithelial cells. ESE-1 expression is induced during terminal differentiation of the epidermis and in a primary human keratinocyte differentiation system. The keratinocyte terminal differentiation marker gene, SPRR2A, is a putative target for ESE-1, since SPRR2A expression during keratinocyte differentiation correlates with induction of ESE-1 expression, and ESE-1 binds with high affinity to and transactivates the ets binding site in the SPRR2A promoter. ESE-1 also binds to and transactivates the enhancer of the Endo A gene, a potential target for ESE-1 in simple epithelia. Due to the important role that other ets factors play in cellular differentiation, ESE-1 is expected to be a critical regulator of epithelial cell differentiation.

Exposure to particulate matter air pollution and risk of multiple sclerosis in two large cohorts of US nurses.

BACKGROUND: Air pollution is thought to raise the risk of neurological disease by promoting neuroinflammation, oxidative stress, glial activation and cerebrovascular damage. Multiple Sclerosis is a common auto-immune disorder, primarily affecting young women. We conducted, to a large prospective study of particulate matter (PM) exposure and multiple sclerosis (MS) risk in two prospective cohorts of women: the Nurses Health Study (NHS) and the Nurses Health Study II (NHS II). METHODS: Cumulative average exposure to different size fractions of PM up to the onset of MS was estimated using spatio-temporal models. We used multivariable Cox proportional hazards models to estimate the hazard ratios (HR) and 95% confidence intervals (CI) of MS associated with each size fraction of PM independently. Participants were followed from 1998 through 2004 in NHS and from 1988 through 2007 for NHS II. We conducted additional sensitivity analyses stratified by smoking, region of the US, and age, as well as analyses restricted to women who did not move during the study. Analyses were adjusted for age, ancestry, smoking, body mass index at age 18, region, tract level population density, latitude at age 15, and UV index. RESULTS: We did not observe significant associations between air pollution and MS risk in our cohorts. Among women in the NHS II, the HRs comparing the top vs. bottom quintiles of PM was 1.11 (95% Confidence Intervals (CI): 0.74, 1.66), 1.04 (95% CI: 0.73, 1.50) and 1.09 (95% CI: 0.73, 1.62) for PM10 (≤10µm in diameter), PM2.5 (≤2.5µm in diameter), and PM2.5-10 (2.5 to 10µm in diameter) respectively, and tests for linear trends were not statistically significant. No association between exposure to PM and risk of MS was observed in the NHS. CONCLUSIONS: In this study, exposure to PM air pollution was not related to MS risk.

Human papillomavirus type 16 E7 oncoprotein-induced abnormal centrosome synthesis is an early event in the evolving malignant phenotype.

Genomic instability is a hallmark of malignant growth that frequently involves mitotic defects associated with centrosome abnormalities. However, the question of whether abnormal centrosomes cause genomic instability or develop secondary to other changes has not been conclusively resolved. Here we show that human papillomavirus (HPV)-16 E7 can induce abnormal centrosome synthesis before the development of extensive nuclear abnormalities. In contrast, expression of HPV-16 E6 is associated with marked nuclear atypia and concomitant accumulation of centrosomes. Our results demonstrate that HPV-16 E7-induced centrosome abnormalities represent an early event during neoplastic progression potentially driving genomic destabilization.

Re-expression of endogenous p16ink4a in oral squamous cell carcinoma lines by 5-aza-2'-deoxycytidine treatment induces a senescence-like state.

We have previously reported that a set of oral squamous cell carcinoma lines express specifically elevated cdk6 activity. One of the cell lines, SCC4, contains a cdk6 amplification and expresses functional p16ink4a, the other cell lines express undetectable levels of p16ink4a, despite a lack of coding-region mutations. Two of the cell lines, SCC15 and SCC40 have a hypermethylated p16ink4A promoter and a third cell line, SCC9, has a mutation in the p16ink4a promoter. Using the demethylation agent 5-aza-2'-deoxycytidine, we showed that the p16ink4a protein was re-expressed after a 5-day treatment with this chemical. One cell line, SCC15 expressed high levels of p16ink4a. In this line, cdk6 activity was decreased after 5-aza-2'deoxycytidine treatment, and the hypophosphorylated, growth suppressive form of the retinoblastoma tumor suppressor protein pRB was detected. Expression of p16ink4a persisted, even after the drug was removed and the cells expressed senescence-associated beta-galactosidase activity. Ectopic expression of p16ink4a with a recombinant retrovirus in this cell line also induced a similar senescence-like phenotype. Hence, it was possible to restore a functional pRB pathway in an oral squamous cell carcinoma line by inducing re-expression of endogenous p16ink4a in response to treatment with a demethylating agent.

The HPV E7 oncoprotein inhibits tumor necrosis factor alpha-mediated apoptosis in normal human fibroblasts.

Tumor necrosis factor-alpha (TNF) is a cytokine that induces programmed cell death, apoptosis, in a number of cell types and is employed by cytotoxic T cells to eliminate virus infected cells. Consequently, many viruses have acquired mechanisms to undermine these host cell defense mechanisms and cause resistance to TNF-mediated apoptosis. Here we show that normal human diploid fibroblasts that express the human papillomavirus type 16 E7 oncoprotein have a decreased propensity to undergo apoptosis in response to TNF treatment. The ability of E7 to undermine TNF-mediated apoptosis correlates with cellular transformation. While E7 does not generally subvert signaling by tumor necrosis factor receptor 1, pro-caspase 8 activation is decreased in E7-expressing cells. E7 also provides some protection from apoptosis caused by stimulation of the TNF receptor 1-related cytokine receptor Fas, where induction of apoptosis occurs much slower in this cell type. Hence, E7-expressing normal human fibroblasts exhibit a specific defect that obstructs cytokine-mediated activation of pro-caspase 8 and apoptosis.

Biological activities and molecular targets of the human papillomavirus E7 oncoprotein.

The human papillomavirus (HPV) E7 protein is one of only two viral proteins that remain expressed in HPV-associated human cancers. HPV E7 proteins share structural and functional similarities with oncoproteins encoded by other small DNA tumor viruses such as adenovirus E1A and SV40 large tumor antigen. The HPV E7 protein plays an important role in the viral life cycle by subverting the tight link between cellular differentiation and proliferation in normal epithelium, thus allowing the virus to replicate in differentiating epithelial cells that would have normally withdrawn from the cell division cycle. The transforming activities of E7 largely reflect this important function.

The human papillomavirus-16 E6 oncoprotein decreases the vigilance of mitotic checkpoints.

The E6 and E7 proteins of the high risk human papillomaviruses (HPVs) are consistently expressed in HPV-positive cervical carcinomas. We investigated the ability of HPV-16 E6 and E7 to disrupt mitotic checkpoints in normal diploid human cells. Acute expression of HPV-16 E6, but not HPV-16 E7, decreased the fidelity of multiple checkpoints controlling entry into and exit from mitosis. After irradiation, nearly 50% of cells containing HPV-16 E6 readily entered mitosis as opposed to less than 10% of control cells. Consistent with this, asynchronous populations of cells expressing HPV-16 E6 had increased cdc2-associated histone H1 kinase activity relative to control populations. In addition, HPV-16 E6 increased sensitivity to chemically-induced S-phase premature mitosis and decreased mitotic spindle assembly checkpoint function relative to control populations. HPV-16 E6 mutants with a reduced ability to target p53 for degradation were unable to abrogate mitotic checkpoints, suggesting a possible mechanism by which HPV-16 E6 disrupts mitotic checkpoints. Expression of a mutant p53 gene yielded an intermediate phenotype relative to HPV-16 E6, generating moderate increases in sensitivity to chemically-induced S-phase PCC and mitotic spindle disruption and a heightened propensity to enter mitosis after irradiation.

Centrosome abnormalities, genomic instability and carcinogenic progression.

Centrosome abnormalities are a frequent finding in various malignant tumors. Since centrosomes form the poles of the mitotic spindle, these abnormalities have been implicated in chromosome missegregation and the generation of aneuploid cells which is commonly found in many human neoplasms. It is a matter of debate, however, whether centrosome alterations can drive cells into aneuploidy or simply reflect loss of genomic integrity by other mechanisms. Since these two models have fundamentally different implications for the diagnostic and prognostic value of centrosome abnormalities, we will discuss the relevance of abnormal centrosomes in the context of different oncogenic events as exemplified by high-risk human papillomavirus-associated carcinogenesis.

The human papillomavirus E7 protein as a transforming and transactivating factor.

The HPV proteins encoded by the early viral genes, including E6 and E7, are thought to subvert the normal regulatory pathways of infected cells to accommodate viral replication. Mechanistically some of this is accomplished by protein-protein interactions between viral proteins and a number of key cellular regulatory proteins that include tumor suppressor gene products. By undermining cellular regulatory pathways the HPV oncogenes cause hyperproliferation and the perturbation of normal cellular differentiation pathways. Although expression of the high-risk HPV-encoded E6 and E7 oncoproteins may be important prerequisites for cellular transformation, it is very likely that additional cellular changes are necessary for carcinogenic progression. The elucidation of the role of the early HPV genes in the initiation and/or maintenance of carcinogenic progression will continue to be a fascinating area of investigation and may reveal new opportunities for antiviral therapy and antitumor intervention.

Human papillomaviruses and associated malignancies.

The human papillomaviruses (HPVS) are small DNA tumor viruses that infect epithelial cells and induce proliferative lesions. Substantial epidemiologic data along with in vitro and in vivo studies have led to the implication of particular HPVs with the development of epithelial malignancies. Greater than 90% of all cervical carcinomas are positive for HPV infection. Most of these lesions are caused by infection with mucosal-associated high-risk HPV subtypes. Much work has been undertaken in basic science laboratories to determine the molecular basis for HPV-associated malignancies. Although many significant advances have been made in understanding the biologic properties of these viruses using in vitro analyses, the field has been greatly hindered until recently by the inability to propagate the virus in culture. In this review, we discuss the basic biologic properties of HPVs and the current understanding of the mechanisms of cellular transformation by malignancy-associated viral subtypes. We place particular emphasis on discussion of the HPV oncogenes, E6 and E7. We also discuss premalignant and malignant disorders of squamous and mucosal epithelia, which have been associated with HPV infections, and the current understanding of the mechanism of HPV-associated carcinogenesis in these settings. We focus these discussions on cervical carcinogenesis and briefly review the particulars regarding HPV-associated malignancies in normal and immunocompromised hosts. We end with a discussion of potential targeted molecular therapies for HPV-associated malignancies that may result from the current knowledge of HPV-related cellular growth dysregulation and carcinogenesis.

The migration of hematopoietic cells: an in vitro study system.

An in vitro migration assay system for the study of hematopoietic cell migration was established. Using a large well modification of earlier described migration chambers, it was found that, in the presence of whole murine serum from either normal or aplastic mice, a heterogeneous population of cells was stimulated to migrate through limited size pores (8 mu) in a thin (5 mu) polycarbonate filter. In dilution studies, serum obtained from animals that had been rendered aplastic by total body irradiation provided a stronger migration stimulus than serum from normal animals. This observation is consistent with observations of hematopoietic cell migration in vivo. Primitive spleen colony-forming cells and in vitro granulocyte/macrophage colony-forming cells were present in the migrating population at a comparable fraction to that found in untreated bone marrow. These studies demonstrate the feasibility of studying hematopoietic stem cell migration under controlled experimental conditions.

Ki-67, cyclin E, and p16INK4 are complimentary surrogate biomarkers for human papilloma virus-related cervical neoplasia.

Prior studies of Ki-67, cyclin E, and p16 expression have suggested that these biomarkers may be preferentially expressed in cervical neoplasia. This study examined and compared the distribution of staining for these three antigens in 1) normal and reactive epithelial changes, 2) diagnostically challenging cases (atypical metaplasia and atypical atrophy), 3) squamous intraepithelial lesions (SIL), and 4) high-and low-risk human papilloma virus (HPV) type-specific SIL. One hundred four epithelial foci from 99 biopsies were studied, including low-grade squamous intraepithelial lesions (LSIL; 24), high-grade squamous intraepithelial lesions (HSIL; 36), mature or immature (metaplastic) squamous epithelium (29), and atrophic or metaplastic epithelium with atypia (15). Cases were scored positive for Ki-67 expression if expression extended above the basal one third of the epithelium, for cyclin E if moderate to strong staining was present, and for p16 if moderate to strong diffuse or focal staining was present. HPV status was scored by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of extracted DNA. Immunohistochemical findings were correlated with histologic and viral data. Overall, a histologic diagnosis of SIL correlated strongly with all of the biomarkers used (p <0.001). Positive scores for Ki-67, cyclin E, and p16 were seen in 68.4%, 96.7%, and 100% of LSILs and 94.7%, 91.6%, and 100% of HSILs, respectively. Positive predictive values of these three biomarkers for HPV were 82.4%, 89.5%, and 91.4%, respectively. The positive predictive value for HPV of either cyclin E or p16 was 88.7%. Strong diffuse staining for p16 was significantly associated with high-risk HPV-associated lesions. Normal or reactive epithelial changes scored positive for the three biomarkers in 7.7%, 8.0%, and 12%, respectively. Limitations in specificity included minimal or no suprabasal staining for Ki-67 in immature condylomas and occasional suprabasal staining of reactive epithelial changes (10%), diffuse weak nuclear cyclin E staining in some normal or metaplastic epithelia, and diffuse weak basal p16 staining and occasional stronger focal positivity in normal epithelia. Ki-67, cyclin E, and p16 are complementary surrogate biomarkers for HPV-related preinvasive squamous cervical disease. (Because cyclin E and p16 are most sensitive for LSIL and HSIL [including high-risk HPV], respectively, use of these biomarkers in combination for resolving diagnostic problems, with an appreciation of potential background staining, is recommended.)

The effects of surgery and acute rejection on glomerular hemodynamics in the transplanted rat kidney.

The effects of surgery and acute rejection on glomerular hemodynamics in the transplanted rat kidney are examined. Kidneys were transplanted from Munich-Wistar (MW) rats to syngeneic controls and MHC-incompatible PVG strain recipients. We report on 4 groups of animals: (1) two-kidney control MW rats; (2) unilaterally nephrectomized MW rats (UNX); (3) renal transplantation from MHC-identical MW littermates and removal of native kidneys (SYN); and (4) transplantation from MW donors to MHC-incompatible PVG rats and removal of native kidneys (ALLO). Glomerular filtration rate (GFR), single-nephron (SN)GFR, and glomerular capillary pressure (PGC) in SYN kidneys were depressed as compared to those in UNX (GFR, 1.41 +/- 0.08 vs. 0.80 +/- 0.08 ml/min; SNGFR, 67.2 +/- 4.8 vs. 44.7 +/- 6.6 nl/min; and PGC, 67 +/- 2 vs. 48 +/- 4 mmHg, UNX vs. SYN, respectively, P < 0.05). GFR and SNGFR in ALLO kidneys, however, were depressed even further (GFR, 0.40 +/- 0.05 ml/min; SNGFR, 13.8 +/- 1.8 nl/min; P < 0.05 for UNX vs. ALLO and SYN vs. ALLO). Afferent arteriolar resistance (RA) was increased greater than 4-fold (UNX, 1.01 +/- 0.15 10(10) dyn.sec.cm-5; SYN, 1.37 +/- 0.36 10(10) dyn.sec.cm-5; ALLO, 4.76 +/- 0.74 10(10) dyn.sec.cm-5 [P < 0.05, UNX vs. ALLO and SYN vs. ALLO]). This led to a precipitous fall in initial capillary flow rate in ALLO rats. These studies reveal the presence of moderate reductions in SNGFR and PGC in the nonrejecting transplanted kidney, which may relate to as yet unidentified consequences of the transplant surgery. More significantly, the principal mechanism leading to the reduced GFR characteristic of acute allograft rejection is identified as severe preglomerular vasoconstriction.