Incidence and time trends of brain metastases admissions among breast cancer patients in Sweden.

BACKGROUND: While treatment for breast cancer has been refined and overall survival has improved, there is concern that the incidence of brain metastases has increased. METHODS: We identified patients in Sweden with incident breast cancer 1998-2006 in the National Cancer Register, and matched these to the National Patient Register to obtain information on hospital admissions for distant metastases. Hazard ratios (HRs) and 95% confidence intervals (CIs) were computed with Cox regression as estimates of relative risk. RESULTS: Among 50 528 breast cancer patients, 696 (1.4%) were admitted with brain metastases during median 3.5 years of follow-up. Admissions for other metastases were found in 3470 (6.9%) patients. Compared with the period 1998-2000, patients diagnosed with breast cancer 2004-2006 were at a 44% increased risk of being admitted with brain metastases (HR 1.44, 95% CI 1.13-1.85). CONCLUSION: The incidence of admissions with brain metastases in breast cancer patients was increasing in the mid-2000s in Sweden. These findings support a true increase in incidence of brain metastases among breast cancer patients.

A unifying hypothesis on the development of type 1 diabetes and celiac disease: gluten consumption may be a shared causative factor.

This paper presents a hypothesis of the aetiology of the increasing incidence of type 1 diabetes (T1D). This together with the global increased incidence of celiac disease (CD) and that these increases cannot be explained by genetic factors suggest a common environmental factor for these two diseases. Even though enterovirus (EV) infections are believed to trigger T1D and gluten is the trigger of CD, the increasing intake of gluten containing products all over the world could be the trigger for both diseases directly and indirectly. It has been shown that the duration of exposure to gluten is related to the prevalence of T1D. It has also been shown that T1D patients at onset have an inflammatory reaction in the gut. Hence, early diagnose of CD followed by elimination of dietary gluten will lead to a decreased incidence of T1D.

Coxsackie-adenovirus receptor expression is enhanced in pancreas from patients with type 1 diabetes.

OBJECTIVES: One of the theories connecting enterovirus (EV) infection of human islets with type 1 diabetes (T1D) is the development of a fertile field in the islets. This implies induction of appropriate proteins for the viral replication such as the coxsackie-adenovirus receptor (CAR). The aim of this study was to investigate to what extent CAR is expressed in human islets of Langerhans, and what conditions that would change the expression. DESIGN: Immunohistochemistry for CAR was performed on paraffin-embedded pancreatic tissue from patients with T1D (n=9 recent onset T1D, n=4 long-standing T1D), islet autoantibody-positive individuals (n=14) and non-diabetic controls (n=24) individuals. The expression of CAR was also examined by reverse transcription PCR on microdissected islets (n=5), exocrine tissue (n=5) and on explanted islets infected with EV or exposed to chemokines produced by EV-infected islet cells. RESULTS: An increased frequency of patients with T1D and autoantibody-positive individuals expressed CAR in the pancreas (p<0.039). CAR staining was detected more frequently in pancreatic islets from patients with T1D and autoantibody-positive subjects (15/27) compared with (6/24) non-diabetic controls (p<0.033). Also in explanted islets cultured in UV-treated culture medium from coxsackievirus B (CBV)-1-infected islets, the expression of the CAR gene was increased compared with controls. Laser microdissection of pancreatic tissue revealed that CAR expression was 10-fold higher in endocrine compared with exocrine cells of the pancreas. CAR was also expressed in explanted islets and the expression level decreased with time in culture. CBV-1 infection of explanted islets clearly decreased the expression of CAR (p<0.05). In contrast, infection with echovirus 6 did not affect the expression of CAR. CONCLUSIONS: CAR is expressed in pancreatic islets of patients with T1D and the expression level of CAR is increased in explanted islets exposed to proinflammatory cytokines/chemokines produced by infected islets. T1D is associated with increased levels of certain chemokines/cytokines in the islets and this might be the mechanism behind the increased expression of CAR in TID islets.

Enterovirus infection of human islets of Langerhans affects ß-cell function resulting in disintegrated islets, decreased glucose stimulated insulin secretion and loss of Golgi structure.

AIMS/HYPOTHESIS: In type 1 diabetes (T1D), most insulin-producing ß cells are destroyed, but the trigger is unknown. One of the possible triggers is a virus infection and the aim of this study was to test if enterovirus infection affects glucose stimulated insulin secretion and the effect of virus replication on cellular macromolecules and organelles involved in insulin secretion. METHODS: Isolated human islets were infected with different strains of coxsackievirus B (CVB) virus and the glucose-stimulated insulin release (GSIS) was measured in a dynamic perifusion system. Classical morphological electron microscopy, large-scale electron microscopy, so-called nanotomy, and immunohistochemistry were used to study to what extent virus-infected ß cells contained insulin, and real-time PCR was used to analyze virus induced changes of islet specific genes. RESULTS: In islets infected with CVB, GSIS was reduced in correlation with the degree of virus-induced islet disintegration. The expression of the gene encoding insulin was decreased in infected islets, whereas the expression of glucagon was not affected. Also, in islets that were somewhat disintegrated, there were uninfected ß cells. Ultrastructural analysis revealed that virus particles and virus replication complexes were only present in ß cells. There was a significant number of insulin granules remaining in the virus-infected ß cells, despite decreased expression of insulin mRNA. In addition, no typical Golgi apparatus was detected in these cells. Exposure of islets to synthetic dsRNA potentiated glucose-stimulated insulin secretion. CONCLUSIONS/INTERPRETATION: Glucose-stimulated insulin secretion; organelles involved in insulin secretion and gene expression were all affected by CVB replication in ß cells.

Impaired Ca2+ response to glucose in mouse beta-cells infected with coxsackie B or Echo virus.

Five strains of Coxsackie B4 virus and one of Echo 11 virus were tested with regard to their ability to replicate in pancreatic mouse beta-cells and interfere with the alterations of the cytoplasmic Ca2+ concentration ([Ca2+]i) induced by glucose. All strains except one both multiplied and caused cytopathic effect. In a control group 68% of the beta-cells responded to 11 mM glucose with large amplitude oscillations of [Ca2+]i. After inoculation with the infectious strains these oscillations appeared in only 5% of the beta-cells, whereas the non-infectious strain did not modify the glucose effect on [Ca2+]i. Despite the virus interference with the glucose response, [Ca2+]i was increased after depolarization with excessive extracellular K+ and the oscillations were induced in most beta-cells when glucose was combined with the insulin-releasing sulfonylurea tolbutamide.

Differences in inhibition of replication between Coxsackie B4 virus strains in various cell lines by antibodies to some cell surface proteins.

Monoclonal antibodies that interact with the decay accelerating factor (DAF, CD55), the lymphocyte homing receptor (CD44) or the intercellular adhesion molecule I (ICAM- 1) were found to inhibit the replication of different strains of Coxsackievirus serotype B4 (CBV-4) to various extent. By adding antibodies to CD55 the replication of two (V345 and VD2921) of seven strains in HeLa cells, three (V89-4557, VD2921 and T318) of seven in A549-10C cells and one (VD2921) of five strains in RD cells was blocked totally. Consequently, the replication of one strain (VD2921) was blocked in all cells indicating that this strain uses CD55 as a receptor or as a co-receptor on all cell lines and is unable to use another cell surface protein. The binding of this strain to the cell surface was inhibited by the antibodies to CD55. None of the CBV-4 strains was blocked totally by adding antibodies to CD44 to HeLa and A549-10C cells, whereas in RD cells the replication of one (T318) of the CBV-4 strains was blocked totally. The antibodies to ICAM-1 did not inhibit totally the replication of any strain in HeLa and RD cells, but it blocked totally the replication of one strain (CBV-4-E) in A549-10C cells. In HeLa and A549-10C cells the degree of replication correlated highly with the degree of cytopathic effect (CPE). In RD cells, four of the strains replicated without CPE. The adding of antibodies to the integrin alpha(v)beta(3) led to slightly enhanced replication of three of the CBV-4 strains in all cell lines. It is concluded that the receptor usage by different strains of CBV-4 varies not only within the same cells but also between different cell lines.

Animal serum factor(s) causing adverse effects on RIAs of human enterovirus IgM.

The effects of animal sera used at various concentrations in dilution buffers for radioimmunoassays (RIAs) of human enterovirus-IgM were studied. The origin of the sera had no impact on the titres, but adverse effects on virus-specificity tests were noted when sera from some species were used. In attempts to block the binding of 35S-labelled virus by adding unlabelled virus, sera from cow, horse and lamb and from swine and man could usually not be used; instead of a blocking effect, an increase in bound labelled virus was noted in most cases. When fetal or newborn calf serum or sera from chicken were used, this phenomenon did not occur. The factor(s) causing the enhancement of virus binding could not be identified, but it was evident that it was not present in all sera from the same species and it was very probable that immunoglobulins were not involved.

Stabilization of the antigenicity of coxsackie B procapsids by disoxaril.

Disoxaril added to cell cultures infected with coxsackie B virus serotypes 3 or 4 increased the yield and stabilized the antigenicity of the procapsids. In the absence of disoxaril, the procapsids lost their broad antigenicity after a short period of time. Prevention of this loss by disoxaril was observed in radioimmunoassays of IgM in sera from patients infected with various enteroviruses. The procapsids are useful in serological diagnosis of enterovirus infections, and the addition of disoxaril facilitates this technique.

The replication of certain Coxsackie B virus strains in CHO cells.

Cell surface molecules that can act as viral receptors may exert an important selective pressure on RNA viral quasi-species. Coxsackie-Adenovirus Receptor and Decay Accelerating Factor (DAF, CD55) have been identified as receptors for Coxsackie B virus. In studies of viral replication using different strains of Coxsackievirus serotype 4 (CBV-4), it was found that despite lack of expression of these cell surface molecules on Chinese Hamster Ovary (CHO) cells and despite their common use as negative controls in Coxsackie B virus receptor assays, two strains were able to replicate, one (V89-4557) without cytopathic effect (CPE), and the other (T318) with strong CPE. A weak signal was obtained using antibodies against enterovirus, visualized with FITC-conjugated antibodies, when the Coxsackievirus B4 strain V89-4557 was inoculated on CHO cells compared to no signal with the non-replicating Coxsackievirus B4 strain VD2921, indicating some degree of binding of the former to the cells.

Enterovirus IgM detection: specificity of mu-antibody-capture radioimmunoassays using virions and procapsids of Coxsackie B virus.

A predominantly type-specific mu-capture radioimmunoassay (RIA) of IgM antibodies to Coxsackie B1-B5 (CB1-CB5) viruses was previously described (Frisk et al., 1984). The present study is concerned with the specificity of this assay, using as antigen different strains of one serotype (CB5) and procapsids of two serotypes (CB3 and CB5). Eight strains of CB5 virions were tested against acute and/or convalescent sera from 10 patients from whom CB5 had been isolated. Seven patients' sera were tested against their own strain. The frequency of IgM-positive patients varied from 9 of 10 (90%) to 5 of 10 (50%). In three cases the highest titres were obtained with the patients' own strain. When sera from patients with other enterovirus infections were tested against the CB5 strains, heterotypic titres were obtained to a certain extent (0-15.6%). The strains giving a high frequency of homotypic titres varied concerning heterotypic reactions. It is concluded that the choice of strain is important if a high frequency of homotypic titres with no or only a few heterotypic reactions is to be obtained. When procapsids were used as antigen, both homotypic and heterotypic titres were seen to a large extent. All patients with homotypic IgM against CB3 or CB5 virions showed IgM against the CB3 or the CB5 procapsids, respectively. When sera from patients with other enterovirus infections were tested, IgM was found in 54 of 93 patients (58%) with use of the CB3 procapsid and in 52 of 87 patients (60%) with the CB5 procapsid. It was often not the same patients who showed IgM against the two different procapsids. When both procapsids were used, IgM positivity was found in 62 of 81 patients (77%) with other enterovirus infections. It is concluded that the use of two or more procapsids in combination is of value for the diagnosis of a recent or current enterovirus infection.

A novel serological technique: polymerase chain reaction enhanced immunoassay. Application to enterovirus IgM diagnosis.

The polymerase chain reaction (PCR) method is a sensitive, specific and rapid technique for virus detection. The principles of a PCR enhanced immunoassay (PIA) are described. The method combines solid phase serological techniques with the PCR, providing a versatile and sensitive method for antibody detection. By linking the antigenicity of virus particles with their content of nucleic acid, the method provides new possibilities for virus serology: for example, antibody specificity can be coupled to viral sequence in patients with chronic infections caused by highly variable viruses such as HIV and HCV. An application of the PIA technique is described for the detection of anti-enterovirus IgM. IgM is captured to anti-human IgM-coated microwell plates. The anti-enterovirus IgM is allowed to bind crude enterovirus antigen. Bound virus is heat denatured and the released RNA is used as a template for reverse transcription PCR (RT-PCR) amplification. Amplicons are detected by hybridisation to an affinity labelled probe in a microwell colorimetric assay. In a pilot study, 18 serum specimens from patients with enterovirus infections were examined. Using a mixture of ten crude enterovirus antigens, the frequency of IgM positivity was 6/18 (33%). Titres between 1/500 and 1/100,000 were recorded. Predominantly type-specific antibodies were detected. The results were compared with a procapsid enterovirus radioimmunoassay (RIA). After further optimisation, the PIA has the potential to be a clinically useful assay for the detection of antiviral antibodies.

Increased frequency of coxsackie B virus IgM in women with spontaneous abortion.

IgM antibodies to Coxsackie B virus serotypes 1-5 (CBV 1-5) were studied by radioimmunoassay (RIA) of blood samples from women who had undergone a spontaneous abortion or given birth to a stillborn child. Results were compared with those of controls comprising healthy pregnant women who had not suffered such experiences. Among 48 women with abortions, 16 (33%) had CBV-IgM, while among the controls only three of 37 (8%) had these antibodies, a statistically significant difference (P less than 0.025). Among 21 women with a stillborn child, 11 (52%) were CBV-IgM positive, while the corresponding proportion in their controls was four of 18 (22%), a difference which is not statistically significant. It is concluded that CBV may be an important causative agent in spontaneous abortions and possibly also in stillbirths.

Antibody responses to different strains of coxsackie B4 virus in patients with newly diagnosed type I diabetes mellitus or aseptic meningitis.

Considerable differences in antibody responses measured by capture-IgM RIA and neutralization tests (NT) were seen in children with newly diagnosed type I (insulin-dependent) diabetes mellitus (IDDM) when five different strains of Coxsackie B4 virus (CBV-4) were used. The IgM positivity of the 160 patients varied between 3.7 and 10.0% (P < 0.05) and with the use of all strains, IgM was found in 13.2%. Matched controls showed a significantly lower frequency (P < 0.05), but there were no apparent differences between the strains, although no IgM was found against two strains. In the NT different results were also obtained in the IDDM children with use of the five strains. However, these results differed considerably from those of the RIA, indicating that different epitopes on the virus were involved. Serum specimens from 75 patients with aseptic meningitis from whom enteroviruses had been isolated, also showed varying IgM frequencies, ranging from 13.3 to 18.7%, but the differences between the strains were different to those found in the IDDM patients. We speculate that only certain strains of a serotype of CBV may be used to distinguish IDDM pathogenesis from that of other diseases.

The possible role of Coxsackie A and echo viruses in the pathogenesis of type I diabetes mellitus studied by IgM analysis.

IgM antibodies to Coxsackie B virus (CBV) have recently been observed in the serum of a relatively high proportion of children with newly diagnosed insulin-dependent diabetes mellitus (IDDM). In the present study, 108 IDDM patients below the age of 15 years, diagnosed during the period 1976 to 1985, were investigated at the onset of their disease by mu-antibody-capture radioimmunoassay (RIA) of IgM against seven different enterovirus antigens, namely virions of CBV serotypes 1-5 (CBV 1-5) and procapsids of CBV 3 and CBV 5. As has been shown the RIAs with virions give type-specific or narrow type-specific reactions, whereas procapsids react with IgM against both homotypic and heterotypic enteroviruses. The annual frequency of IgM against virions varied between 15 and 76% (mean 38%). IgM against CBV3 and CBV2 predominated, but IgM against the other serotypes was also observed. When procapsids were used as antigen, the frequency of IgM varied between 11 and 86% (mean 63%). With virions and procapsids, the corresponding variation was 44-100% (mean 70%). The total number of patients exhibiting virion-IgM was 41, whereas procapsid-IgM alone [indicating an infection with Coxsackie A virus (CAV) and/or echo virus (EV)] was detected in 36 patients. For 2 of the years, samples of serum from control groups were included. These showed a significantly lower frequency of IgM in both the virion and the procapsid RIAs. It is concluded that not only infection with CBV but also that with other enteroviruses, such as CAV and/or EV, may be involved in the pathogenesis of IDDM in children.

Tropism Analysis of Two Coxsackie B5 Strains Reveals Virus Growth in Human Primary Pancreatic Islets but not in Exocrine Cell Clusters In Vitro.

Human Enteroviruses (HEVs) have been implicated in human pancreatic diseases such as pancreatitis and type 1 diabetes (T1D). Human studies are sparse or inconclusive and our aim was to investigate the tropism of two strains of Coxsackie B virus 5 (CBV-5) in vitro to primary human pancreatic cells. Virus replication was measured with TCID50 titrations of aliquots of the culture medium at different time points post inoculation. The presence of virus particles or virus proteins within the pancreatic cells was studied with immunohistochemistry (IHC) and electron microscopy (EM). None of the strains replicated in the human exocrine cell clusters, in contrast, both strains replicated in the endocrine islets of Langerhans. Virus particles were found exclusively in the endocrine cells, often in close association with insulin granules. In conclusion, CBV-5 can replicate in human endocrine cells but not in human exocrine cells, thus they might not be the cause of pancreatitis in humans. The association of virus with insulin granules might reflect the use of these as replication scaffolds.

Recent enterovirus infection in type 1 diabetes: evidence with a novel IgM method.

Enterovirus (EV) infection has been associated with Type 1 (T1D) diabetes and on a few occasions virus could be isolated at onset of the disease. Using two such isolates as antigens in a quantitative PCR enhanced immunoassay (T1D-EV-QPIA) we have measured IgM antibodies against such potentially diabetogenic viruses in serum from 33 newly diagnosed T1D children, 24 siblings, and 27 healthy children. Sera were also analysed with regard to autoantibodies against GAD65, the cytokine TNF-alpha and the chemokine IP-10. EV-RNA detection was performed on peripheral blood mononuclear cells (PBMC). IgM antibodies against this "new" EV antigen were more frequent in serum from T1D children than in serum from siblings and/or controls (P < 0.001). EV-RNA was detected more frequently in PBMC from T1D children than in healthy control children (P < 0.001) and also compared to the siblings (P < 0.003). The cytokine TNF-alpha was less frequently detected in serum from the T1D children compared with serum from siblings and/controls (P < 0.001). A positive correlation was found between the results obtained with the T1D-EV-QPIA and the EV-PCR (P < 0.001). These findings are in line with earlier findings of an increased frequency of enteroviral infections in newly diagnosed T1D patients. In addition, we found that T1D children at onset of the disease had lower frequencies of the chemokine TNF-alpha in their serum than age- and sex-matched controls had, suggesting an impaired immune response.

Induction of the chemokine interferon-gamma-inducible protein-10 in human pancreatic islets during enterovirus infection.

AIMS/HYPOTHESIS: Enterovirus infections have long been suspected to be environmental factors that may cause type 1 diabetes, but the pathways leading from infection to beta cell destruction are still unknown. We therefore examined whether enterovirus infection of human islets leads to upregulation of interferon-gamma-inducible protein (IP-10, now known as chemokine [C-X-C motif] ligand 10 [CXCL10]), a chemokine important for the induction of insulitis. METHODS: Isolated human islets were infected with three different strains of Coxsackie B4 virus. IP-10 expression and secretion from the infected human islets were then measured using RT-PCR and ELISA at several time points. RESULTS: IP-10 was clearly upregulated in and secreted from human islets during enterovirus infection. This was demonstrated with three different strains of Coxsackie B4 virus, two of which are lytic to islets and one which is non-lytic and can establish a persistent infection in human islets. CONCLUSIONS/INTERPRETATION: We propose that enterovirus-induced upregulation of IP-10 during infection of the islets in vivo is the first step towards destructive insulitis. Our findings support the idea that enterovirus infection triggers immune-mediated beta cell destruction, and for the first time suggest a possible mechanism behind enterovirus-induced diabetes.

Mechanisms of chronic enteroviral persistence in tissue.

Although the association remains controversial, enteroviruses have been implicated in the aetiology of several chronic diseases in humans. Investigations in vitro lead to better understanding of virus-cell interactions, and improve our knowledge of the molecular factors that are involved in the establishment and maintenance of these infections. Recent findings suggest that the most important factor in the establishment of a persistent infection is receptor usage. Studies of the mechanisms that are at work in these in-vitro models of viral infection have shown that there is frequently a co-evolution of mutant cells with higher resistance to viral infection and of virus variants with increased virulence (i.e. variants with the ability to utilize other cell-surface molecules as receptors).

Tissue culture of isolated human pancreatic islets infected with different strains of coxsackievirus B4: assessment of virus replication and effects on islet morphology and insulin release.

The aim was to study whether different strains of Coxsackievirus B4 (CBV-4) are able to infect human pancreatic islet cells in vitro and cause morphological and functional damages. Isolated islets maintained in tissue culture were infected with five well- characterised strains of CBV-4. Aliquots of the culture medium were analysed with regard to virus replication and insulin content. Infected and uninfected islets were examined by light microscopy to determine the degree of virus-induced cytopathic effect (CPE). The results showed that the islet cells were susceptible to infection by all the strains of CBV-4 although the outcome of the infection differed. The virus titres obtained at 48 and 72 hours post infection differed significantly between all the CBV-4 strains (p<0.001), indicating different ability to replicate in islet cells. Pronounced to weak CPE, which was partly due to the origin (donor) of the islets, was induced by four of the five CBV-4 strains. One strain (VD2921) replicated without causing CPE despite high virus titres. One (V89-4557) of the CBV-4 strains always revealed pronounced CPE. Infection by this strain also caused functional impairment that significantly affected insulin response to high glucose at 48 hours post infection (p<0.001). Replication of another CBV-4 strain (JVB) in the islet cells significantly increased the release of insulin compared to non-infected control cells (p<0.001) indicating damage of the beta-cells leading to leakage of insulin.