Borrelia multiplex: a bead-based multiplex assay for the simultaneous detection of Borrelia specific IgG/IgM class antibodies.

BACKGROUND: Lyme borreliosis (LB) is the most common tick-borne infectious disease in the northern hemisphere. The diagnosis of LB is usually made by clinical symptoms and subsequently supported by serology. In Europe, a two-step testing consisting of an enzyme-linked immunosorbent assay (ELISA) and an immunoblot is recommended. However, due to the low sensitivity of the currently available tests, antibody detection is sometimes inaccurate, especially in the early phase of infection, leading to underdiagnoses. METHODS: To improve upon Borrelia diagnostics, we developed a multiplex Borrelia immunoassay (Borrelia multiplex), which utilizes the new INTELLIFLEX platform, enabling the simultaneous dual detection of IgG and IgM antibodies, saving further time and reducing the biosample material requirement. In order to enable correct classification, the Borrelia multiplex contains eight antigens from the five human pathogenic Borrelia species known in Europe. Six antigens are known to mainly induce an IgG response and two antigens are predominant for an IgM response. RESULTS: To validate the assay, we compared the Borrelia multiplex to a commercial bead-based immunoassay resulting in an overall assay sensitivity of 93.7% (95% CI 84.8-97.5%) and a specificity of 96.5% (95%CI 93.5-98.1%). To confirm the calculated sensitivity and specificity, a comparison with a conventional 2-step diagnostics was performed. With this comparison, we obtained a sensitivity of 95.2% (95% CI 84.2-99.2%) and a specificity of 93.0% (95% CI 90.6-94.7%). CONCLUSION: Borrelia multiplex is a highly reproducible cost- and time-effective assay that enables the profiling of antibodies against several individual antigens simultaneously.

Agonists and knockdown of estrogen receptor ß differentially affect invasion of triple-negative breast cancer cells in vitro.

BACKGROUND: Estrogen receptor ß (ERß) is expressed in the majority of invasive breast cancer cases, irrespective of their subtype, including triple-negative breast cancer (TNBC). Thus, ERß might be a potential target for therapy of this challenging cancer type. In this in vitro study, we examined the role of ERß in invasion of two triple-negative breast cancer cell lines. METHODS: MDA-MB-231 and HS578T breast cancer cells were treated with the specific ERß agonists ERB-041, WAY200070, Liquiritigenin and 3ß-Adiol. Knockdown of ERß expression was performed by means of siRNA transfection. Effects on cellular invasion were assessed in vitro by means of a modified Boyden chamber assay. Transcriptome analyses were performed using Affymetrix Human Gene 1.0 ST microarrays. Pathway and gene network analyses were performed by means of Genomatix and Ingenuity Pathway Analysis software. RESULTS: Invasiveness of MBA-MB-231 and HS578T breast cancer cells decreased after treatment with ERß agonists ERB-041 and WAY200070. Agonists Liquiritigenin and 3ß-Adiol only reduced invasion of MDA-MB-231 cells. Knockdown of ERß expression increased invasiveness of MDA-MB-231 cells about 3-fold. Transcriptome and pathway analyses revealed that ERß knockdown led to activation of TGFß signalling and induced expression of a network of genes with functions in extracellular matrix, tumor cell invasion and vitamin D3 metabolism. CONCLUSIONS: Our data suggest that ERß suppresses invasiveness of triple-negative breast cancer cells in vitro. Whether ERß agonists might be useful drugs in the treatment of triple-negative breast cancer, has to be evaluated in further animal and clinical studies.

Polymorphisms in the promoter region of estrogen receptor ß gene in endometrial cancer.

INTRODUCTION: The development of endometrial cancer is known to be affected by estrogens. Thus, genetic variations like single nucleotide polymorphisms (SNPs) in genes involved in estrogen biosynthesis, metabolism, and signal transduction might affect risk for endometrial cancer. In this study, we tested the hypothesis that polymorphisms in the promoter of ESR2 gene may be associated with susceptibility to this disease. METHODS: We compared the frequency of three SNPs in the promoter region of ESR2 gene (rs2987983, rs3020450, and rs3020449) in 135 women with endometrial cancer and 135 healthy women serving as controls by means of allele-specific tetra-primer PCR. RESULTS: Regarding allele frequency, allele positivity or genotype frequencies of these SNPs we did not observe any significant difference between healthy women and women with endometrial cancer. CONCLUSION: Our data clearly suggest that the tested SNPs in the promotor region of human ESR2 gene are not associated with the development of endometrial cancer.

Effects of a combined treatment with tamoxifen and estrogen receptor ß agonists on human breast cancer cell lines.

INTRODUCTION: Coexpression of estrogen receptors (ER) α and ß is present in about half of all breast cancer cases. Whereas ERα is a well-established target for endocrine therapy with the selective estrogen receptor modulator tamoxifen, the applicability of ERß as target in breast cancer therapy is unclear. In this study, we examined the effects of two synthetic ERß agonists alone and in combination with tamoxifen on ERα/ß-positive breast cancer cells. METHODS: We treated MCF-7 and T-47D breast cancer cells with the ERß agonists ERB-041 and WAY-200070 and measured the effects on cell growth. In addition, transcriptome analyses were performed by means of Affymetrix GeneChip arrays. RESULTS: When given alone, ERß agonists ERB-041 and WAY-200070 did not affect the growth of MCF-7 or T-47D cells. In contrast, addition of these drugs to tamoxifen increased its growth-inhibitory effect on both cell lines. This effect was more pronounced under serum-free conditions, but was also observed in the presence of serum in T-47D cells. Transcriptome analyses revealed a set of genes regulated after addition of ERß agonists including S100A8 and CD177. CONCLUSION: The observed enhanced growth-inhibitory effects of a combination of tamoxifen and ERß agonists in vitro encourage further studies to test its possible use in the clinical setting.

Expression of SCUBE2 gene declines in high grade endometrial cancer and associates with expression of steroid hormone receptors and tumor suppressor PTEN.

SCUBE2 (Signal peptide-CUB-epidermal growth factor-like domain-containing 2) gene codes for a cell-surface glycoprotein. In breast cancer, SCUBE2 transcript levels are part of important prognostic and predictive gene signatures and are linked to expression of estrogen receptor α (ERα). To elucidate the role of this gene in endometrial cancer, we compared SCUBE2 expression in malignant and normal endometrial tissue specimens. We then examined its correlation with steroid hormone receptors and PTEN and compared it to SCUBE2 expression in breast cancer samples. Expression of SCUBE2 was found to be decreased in G3 endometrial cancer when compared to postmenopausal endometrium or to G1 tumors (p < 0.05). In postmenopausal endometrium, SCUBE2 transcript levels were more than twice as high as in premenopausal women. In breast cancer, SCUBE2 expression was found to be notably reduced particularly in ERα-negative G3 tumors. Both in endometrial and breast cancer we observed a significant positive correlation of SCUBE2 transcript levels with expression of ERα, PR and PTEN. Our data suggest that SCUBE2, like in breast cancer, associates with ERα and might have a potential as prognostic or predictive marker in endometrial cancer.

Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test.

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different antibody isotypes because they appear at different stages of the host immune response. With Lyme borreliosis, the pathogenic agent can be one of the multiple members of the Borrelia species. Therefore, correct sample classification requires evaluating the immunoreactivity against different antigens of different Borrelia species. Additionally, anti-pathogen IgG and IgM responses can have different elicitation time courses during disease progression. Here we demonstrate the development of a two-reporter multiplex immunoassay that has utility in identifying Borrelia-specific immune response in human serum samples by simultaneously evaluating both IgG and IgM immunoreactivity against different bacterial antigens in the same reaction well. This dual-reporter approach retains the analytical performance of single-reporter methods while conserving time and resources and reducing sample size requirements. This assay allows essentially double the serological information to be generated from a blood sample in half the time.

Network analysis of icb-1 gene function in human breast cancer cells.

Icb-1 is a human gene previously described by our group to exert important functions in cancer cells of different origin. We now performed microarray-based gene expression profiling with subsequent network modeling to further elucidate the role of icb-1 in breast cancer cells. Analyzing the effect of icb-1 knockdown on the transcriptome of MCF-7 cells, we found 151 differentially expressed genes exhibiting more than twofold changes, 97 of which were up- and 54 downregulated. Most of the upregulated genes were cancer-related genes associated with poor prognosis, invasion and metastasis, building an oncogenic network of TNF target genes. On the other hand, network analysis identified the downregulated genes to be primarily involved in interferon signaling and cellular apoptosis. Confirming these network data, we observed that cells with reduced levels of icb-1 exhibited an impaired response to the apoptosis inducers tamoxifen, staurosporine, actinomycin, and camptothecin. The data of this study suggest that icb-1 might exert a tumor-suppressor function in breast cancer and that its loss might confer relative resistance of breast cancer cells to apoptotic drugs.

Effects of a combined treatment with GPR30 agonist G-1 and herceptin on growth and gene expression of human breast cancer cell lines.

Expression of G-protein-coupled receptor 30 (GPR30) is present in HER2-overexpressing breast cancer. In this study, we examined to what extent GPR30-agonist G-1 would affect the antitumoral action of trastuzumab (Herceptin). Combined treatment with both drugs exerted an additive growth-inhibitory effect on breast cancer cells which was accompanied by a significant decline of cyclin A2 expression both on the protein and the mRNA level. Combined treatment also resulted in expression changes of c-fos, cyclin D1, or p21/WAF-1. The results of our study encourage further attempts to test the relevance of these in vitro data in the clinical setting.

Expression of cysteine protease cathepsin L is increased in endometrial cancer and correlates with expression of growth regulatory genes.

Proteases contribute to tumor invasion and metastasis by degrading basement membranes and extracellular matrix (ECM). In this study, we compared gene expression levels of two proteases, cysteine protease Cathepsin L2 (CTSL2) and matrix metalloproteinase MMP11, in human endometrium and endometrial cancer. Our data demonstrate CTSL2 transcript levels to be strongly elevated in endometrial cancer, particularly in G3 tumors. Furthermore, we observed a highly significant positive correlation of CTSL2 with expression of growth regulatory genes Ki-67, cyclin B1, MYBL2, p21/WAF, and HER2 receptor tyrosine kinase. Our data suggest that CTSL2 might be involved in progression of endometrial cancer.

Role of estrogen receptor ß in gynecological cancer.

Estrogen receptor ß is expressed in normal and neoplastic ovarian and endometrial tissues. Recent studies indicate that levels of this receptor decline in ovarian tumorigenesis, like in breast or prostate cancer. Furthermore, ERß expression has been associated with good prognosis in ovarian cancer. In contrast, previous studies on the role of this receptor in endometrial cancer suggested that ERß might play different roles in the carcinogenesis of the ovary and endometrium. Besides its possible role as a prognostic factor, ERß might be a potential target for the treatment of ovarian and endometrial cancer.

Diminished neutralization responses towards SARS-CoV-2 Omicron VoC after mRNA or vector-based COVID-19 vaccinations.

SARS-CoV-2 variants accumulating immune escape mutations provide a significant risk to vaccine-induced protection against infection. The novel variant of concern (VoC) Omicron BA.1 and its sub-lineages have the largest number of amino acid alterations in its Spike protein to date. Thus, they may efficiently escape recognition by neutralizing antibodies, allowing breakthrough infections in convalescent and vaccinated individuals in particular in those who have only received a primary immunization scheme. We analyzed neutralization activity of sera from individuals after vaccination with all mRNA-, vector- or heterologous immunization schemes currently available in Europe by in vitro neutralization assay at peak response towards SARS-CoV-2 B.1, Omicron sub-lineages BA.1, BA.2, BA.2.12.1, BA.3, BA.4/5, Beta and Delta pseudotypes and also provide longitudinal follow-up data from BNT162b2 vaccinees. All vaccines apart from Ad26.CoV2.S showed high levels of responder rates (96-100%) towards the SARS-CoV-2 B.1 isolate, and minor to moderate reductions in neutralizing Beta and Delta VoC pseudotypes. The novel Omicron variant and its sub-lineages had the biggest impact, both in terms of response rates and neutralization titers. Only mRNA-1273 showed a 100% response rate to Omicron BA.1 and induced the highest level of neutralizing antibody titers, followed by heterologous prime-boost approaches. Homologous BNT162b2 vaccination, vector-based AZD1222 and Ad26.CoV2.S performed less well with peak responder rates of 48%, 56% and 9%, respectively. However, Omicron responder rates in BNT162b2 recipients were maintained in our six month longitudinal follow-up indicating that individuals with cross-protection against Omicron maintain it over time. Overall, our data strongly argue for booster doses in individuals who were previously vaccinated with BNT162b2, or a vector-based primary immunization scheme.

Exploring beyond clinical routine SARS-CoV-2 serology using MultiCoV-Ab to evaluate endemic coronavirus cross-reactivity.

The humoral immune response to SARS-CoV-2 is a benchmark for immunity and detailed analysis is required to understand the manifestation and progression of COVID-19, monitor seroconversion within the general population, and support vaccine development. The majority of currently available commercial serological assays only quantify the SARS-CoV-2 antibody response against individual antigens, limiting our understanding of the immune response. To overcome this, we have developed a multiplex immunoassay (MultiCoV-Ab) including spike and nucleocapsid proteins of SARS-CoV-2 and the endemic human coronaviruses. Compared to three broadly used commercial in vitro diagnostic tests, our MultiCoV-Ab achieves a higher sensitivity and specificity when analyzing a well-characterized sample set of SARS-CoV-2 infected and uninfected individuals. We find a high response against endemic coronaviruses in our sample set, but no consistent cross-reactive IgG response patterns against SARS-CoV-2. Here we show a robust, high-content-enabled, antigen-saving multiplex assay suited to both monitoring vaccination studies and facilitating epidemiologic screenings for humoral immunity towards pandemic and endemic coronaviruses.

Next generation sequencing as second-tier test in high-throughput newborn screening for nephropathic cystinosis.

Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disorder, which causes loss of renal proximal tubular function and progressive loss of glomerular function, finally leading to end stage renal failure at school age. In the course of the disease most patients will need kidney transplantation if treatment has not been started before clinical manifestation. With an effective treatment available, a newborn screening assay is highly demanded. Since newborns with cystinosis usually do not show symptoms within the first months of life and no biochemical markers are easily detectable, a DNA-based method seems to be an obvious tool for early diagnosis. Screening was performed using high-throughput nucleic acid extraction followed by 384-well qPCR and melting analysis for the three most frequent variants (57 kb deletion NC_000017.11:g.3600934_3658165del (GRCh38); c.18_21del GACT; c.926dupG) responsible for the defective lysosomal membrane protein cystinosin (CTNS). To increase sensitivity, all heterozygous samples identified in qPCR assay were verified and screened for additional variants by applying next generation sequencing. From January 2018 to July 2019 nearly 292,000 newborns were successfully screened. We identified two newborns with a homozygous 57 kb deletion and a second one with heterozygous 57 kb deletion and a G>C substitution at position c.-512 on the second allele. Cystinosis is an example for diseases caused by a limited number of high prevalence and a high number of low prevalence variants. We have shown that qPCR combined with NGS can be used as a high throughput, cost effective tool in newborn screening for such diseases.

icb-1 Gene counteracts growth of ovarian cancer cell lines.

Human gene icb-1 has been originally identified to be involved in differentiation processes of cancer cells. To examine the function of icb-1 in ovarian cancer, we knocked down its expression in three ovarian cancer cell lines and performed microarray-based gene expression profiling with subsequent gene network modeling. Loss of icb-1 expression accelerated proliferation of SK-OV-3, OVCAR-3 and OAW-42 cells and led to upregulation of ovarian cancer biomarkers like KLK10 and CLDN16. Most of the upregulated genes were part of oncogenic pathways regulated by ERα or TNF. Our data suggest that icb-1 gene inhibits growth and progression of ovarian cancer cells.

Estrogen receptor ß agonists affect growth and gene expression of human breast cancer cell lines.

Expression of estrogen receptor ß (ERß) has been described to reduce growth of cancer cell lines derived from hormone-dependent tumors, like breast cancer. In this study we tested to what extent two ERß agonists, androgen derivative 3ß-Adiol and flavonoid Liquiritigenin, would affect growth and gene expression of different ERß-positive human breast cancer cell lines. Under standard cell culture conditions, we observed 3ß-Adiol to inhibit growth of MCF-7 cells in a dose-dependent manner, whereas growth of BT-474 and MCF-10A cells was suppressed by the maximum concentration (100 nM) only. When treated in serum-free medium, all cell lines except of MDA-MB-231 were responsive to 1 nM 3ß-Adiol, and ZR75-1 cells exhibited a dose-dependent antiproliferative response. Providing putative mechanisms underlying the observed growth-inhibitory effect, expression of Ki-67 or cyclins A2 and B1 was downregulated after 3ß-Adiol treatment in all responsive lines. In contrast, treatment with lower doses of Liquiritigenin did not affect growth. In MCF-7 cells, the highest dose of this flavonoid exerted proliferative effects accompanied by increased expression of cyclin B1, PR and PS2, indicating unspecific activation of ERα. In conclusion, the ERß agonists tested exerted distinct concentration-dependent and cell line-specific effects on growth and gene expression. The observed inhibitory effects of 3ß-Adiol on breast cancer cell growth encourage further studies on the potential of this and other ERß agonists as targeted drugs for breast cancer therapy.

A polymorphism in the nuclear receptor coactivator 7 gene and breast cancer susceptibility.

The nuclear receptor coactivator 7 (NCoA7) gene codes for an estrogen receptor-associated protein that plays a significant role in the cellular response to estrogens. Given that NCoA7 is expressed in the mammary gland, alterations in this gene may affect breast cancer risk. In this study, we compared the genotype and allele frequencies of the missense single nucleotide polymorphism (SNP) rs1567, located in the coding region of the NCoA7 gene and resulting in an amino acid exchange from asparagine to glutamine, in 305 women with sporadic breast cancer and 346 women without any malignancy. Statistical analysis of the observed frequencies did not reveal a significant difference between the cancer and control groups, nor did a comparison between histological breast cancer subgroups. In conclusion, the results of our phenotype-genotype association study indicate that NCoA7 SNP rs1567 does not affect breast cancer susceptibility.

Estrogen receptor ß transcript variants associate with oncogene expression in endometrial cancer.

The human ESR2 gene codes for estrogen receptor ß1 and for multiple splice variants, which are suggested to exert distinct functions in the cellular estrogen response. Given that the function of ERß in endometrial cancer remains unclear, we examined the expression of ERß1, ERß2 and various further ERß transcript variants and their association with selected cancer-related genes in 74 human endometrium samples and endometrial cancer specimens by means of RT-qPCR. Additionally, we knocked down ERß expression in HEC-1A endometrial adenocarcinoma cells by means of siRNA transfection. Expression of four ERß transcript variants was significantly elevated in cancer tissue or in G3 tumors compared to postmenopausal endometrium. Expression of ERß1, ERß2, ERß5 and five further variants was associated with the oncogenes MYBL2 or HER2 in endometrial cancer. In addition, siRNA-triggered knockdown of ERß expression led to a significant decline of MYBL2 mRNA and protein levels in endometrial cancer cells. Our observation of increased ERß transcript levels in cancer tissue and particularly their correlation with the expression of oncogenes, as well as the results of our knockdown studies, suggest a role of ERß in endometrial carcinogenesis.

Single nucleotide polymorphisms in the regulatory region of gonadotropin-releasing hormone receptor gene and breast cancer susceptibility.

It is known that exposure to estrogens affects the pathophysiology of breast cancer. The key role of gonadotropin-releasing hormone (GnRH) in the regulation of female steroid hormone metabolism raises the question of whether polymorphisms in its receptor, GnRHR, might influence breast cancer risk. To test this hypothesis, we analyzed three single nucleotide polymorphisms (SNPs) in the 5'-regulatory region of the GnRHR gene in a total of 565 women, 254 women with breast cancer and 311 women without any malignancy by allele-specific PCR. No significant differences were observed between the breast cancer and control group in terms of genotype, allele frequency or allele positivity. In contrast, different frequencies of the SNPs rs13138607, rs12644822 and rs3756159 were observed after sub-grouping the breast cancer cases according to tumor grading. Our data suggest a potential role of GnRHR gene polymorphisms in the development of breast cancer.

A review of the costs associated with depression and treatment noncompliance: the potential benefits of online support.

This systematic review examines the economic and human costs of depression and the potential savings associated with improvement in patient adherence to treatment with antidepressants through the use of enhanced-care programs. A MEDLINE search was conducted for papers published on the health economics and costs of depression and compliance, adherence, and persistence. Compliance data collected through the online antidepressant compliance support website iCAN (www.ican.co.uk) were compared with data for patients with depression from the IMS Disease Analyzer UK database. Depression frequently causes unemployment, absenteeism, and presenteeism, which results in significantly reduced productivity. Indirect costs of depression accounted for more than $50 billion, whereas direct costs resulted in expenditure of $26 billion, in the US in 2000. Improving patients' compliance with their antidepressant medication results in improved outcomes and prolongs remission from depression, increasing work productivity, and thus reducing overall costs. The implementation of remote enhanced-care programs may improve compliance and reduce overall costs. Novel methods for delivering enhanced-care programs to assist in maintaining compliance have the potential to further reduce costs and should be a focus of future research. In conclusion, depression is a common disorder with a high economic impact. Enhanced-care programs may lower costs associated with depression and improve patients' lives.