The present invention relates to a method, in particular an in vitro method for identifying IL-17 expressing T cells in a blood and/or tissue sample derived from a mammal, comprising analysing the methylation status of at least one CpG position in the gene IL-17A, wherein a demethylation of said at least one CpG position in said sample when compared to an analogous position in a non IL-17 blood cell is indicative for a IL-17 positive CD4 positive T cell. The analyses according to the invention can identify IL-17 positive T cells and distinguish them from all other cells in complex samples, such as, for example, other blood cells. The present invention furthermore provides an improved method for quantifying IL-17 positive T cells in complex samples based on a comparison of the IL-17A methylation with a methylation of at least one marker selected from the group of CD3, FOXP3, and/or GAPDH. The method can be performed without a step of purifying and/or enriching cells, preferably in whole blood and/or non-trypsinized tissue.

T helper 17 cells (Th17) are a subset of T helper cells producing interleukin 17 (IL-17) discovered in 2007. They are considered developmentally distinct from Th1 and Th2 cells and excessive amounts of the cell are thought to play a key role in autoimmune disease (Stockinger B, Veldhoen M (June 2007). "Differentiation and function of Th17 T cells". Current Opinion in Immunology 19 (3): 281-286) such as multiple sclerosis (which was previously thought to be caused by Th1 cells), but also psoriasis, autoimmune uveitis, juvenile diabetes, rheumatoid arthritis, and Crohn's disease.
Th17 cells primarily produce two main members of the IL-17 family, IL-17A and IL-17F, which are involved in the recruitment, activation and migration of neutrophils. These cells also secrete IL-21 and IL-22. Recently, Th17 polarized cells have been shown to mediate the regression of established tumors in mice (Martin-Orozco N, Muranski P, Chung Y et al. (November 2009). "T helper 17 cells promote cytotoxic T cell activation in tumor immunity". Immunity 31 (5): 787-798).
Even though almost all cells in an individual contain the exact same complement of DNA code, higher organisms must impose and maintain different patterns of gene expression in the various tissue types. Most gene regulation is transitory, depending on the current state of the cell and changes in external stimuli. Persistent regulation, on the other hand, is a primary role of epigenetics—heritable regulatory patterns that do not alter the basic genetic coding of the DNA.
As explained below, one form of epigenetic modification is the attachment of methyl groups to the base cytosine. Nevertheless, in addition to the "fifth base" 5-methylcytosine (mC), a sixth (5-hydroxymethylcytosine, hmC), seventh (5-formylcytosine, fC) and eighth (5-carboxycytosine, cC) can be found (Michael J. Booth et al. Quantitative Sequencing of 5-Methylcytosine and 5-Hydroxymethylcytosine at Single-Base Resolution Science 18 May 2012, Vol. 336 no. 6083 pp. 934-937). Both, 5-methylcytosine and 5-hydroxymethylcytosine are not bisulfite convertible.
DNA methylation is the archetypical form of epigenetic regulation; it serves as the stable memory for cells and performs a crucial role in maintaining the long-term identity of various cell types. The primary target of methylation is the two-nucleotide sequence Cytosine-Guanine (a 'CpG site'); within this context cytosine (C) can undergo a simple chemical modification to become formylated, methylated, hydroxymethylated, carboxylated. In the human genome, the CG sequence is much rarer than expected except in certain relatively dense clusters called 'CpG islands'. CpG islands are frequently associated with gene promoters, and it has been estimated that more than half of the human genes have CpG islands (Antequera and Bird, Proc Natl Acad Sci USA. 90:11995-9, 1993).
At this point, there is a very abrupt change from DNA methylation to Treg biology. Thus, one could include a bridging phrase such as: It has been shown that distinct cell types display a unique and characteristic DNA methylation pattern, i.e., an epigenetic fingerprint that can be exploited for cell type identification and quantification (DNA methylation analysis as a tool for cell typing. Baron U, Türbachova I, Hellwag A, Eckhardt F, Berlin K, Hoffmuller U, Gardina P, Olek S. Epigenetics. 2006 January-March; 1(1):55-60. Epub 2006 Feb. 25.). Moreover, regulatory T cells, establishing tolerance for self-antigens but allowing immune responses against foes, were characterized to be exclusively demethylated in the FOXP3 gene, a master transcription factor pivotal for Treg differentiation and function (Epigenetic control of the foxp3 locus in regulatory T cells. Floess S, Freyer J, Siewert C, Baron U, Olek S, Polansky J, Schlawe K, Chang H D, Bopp T, Schmitt E, Klein-Hessling S, Serfling E, Hamann A, Huehn J. PLoS Biol. 2007 February; 5(2):e38.; DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3(+) conventional T cells. Baron U, Floess S, Wieczorek G, Baumann K, Grützkau A, Dong J, Thiel A, Boeld T J, Hoffmann P, Edinger M, Türbachova I, Hamann A, Olek S, Huehn J. Eur J Immunol. 2007 September; 37(9):2378-89.)
The adoptive transfer of in vitro expanded Treg is a promising treatment option for autoimmune as well as alloantigen-induced diseases. Yet, concerns about the phenotypic and functional stability of Tregs upon in vitro culture command both careful selection of the starting population and thorough characterization of the final cell product.
Recently, a high degree of developmental plasticity has been described for murine Treg and Th17 cells. Similarly, IL-17-producing FOXP3(+) cells have been detected among the CD45RA(−) memory-type subpopulation of human Tregs ex vivo. This prompted the investigation of the predisposition of human naïve and memory Tregs to develop into Th17 cells during polyclonal in vitro expansion. Stimulation-induced DNA demethylation of RORC, which encodes the lineage-defining transcription factor for Th17 cells, occurs selectively in CD45RA(−) memory-type Tregs, irrespective of their FOXP3 expression level. On the contrary, naïve CD45RA(+) Tregs retain stable CpG methylation across the RORC locus even upon prolonged ex vivo expansion and in consequence show only a marginal tendency to express RORC and develop into IL-17-producing cells (Schmidl C, Hansmann L, Andreesen R, Edinger M, Hoffmann P, Rehli M. Epigenetic reprogramming of the RORC locus during in vitro expansion is a distinctive feature of human memory but not naïve Treg. Eur J Immunol. 2011 May; 41(5):1491-8. Epub 2011 Apr. 12).
It is commonly thought that immune cell quantification is relatively easy and fully standardized, since the non-adherent, non-matrixed cells in peripheral blood can be marked with antibodies and flow-cytometrically quantified. Providing that cells are non-adherent, single cell suspensions, intact and cell-type specific surface antigens are available, flow cytometry is indeed a highly accurate cell quantification tool.
However, for many applications in research and medical routine, the named prerequisites for such precise measurements are not given:    1. Often, the material/samples measured are not derived from peripheral blood and thus the solubility and single cell suspension property is not met. This is for example true for all biopsy analyzes, such as performed in the pathological routine.    2. Even if the analyte is peripheral blood, the prerequisite of having intact cells is difficult to meet, since—in order to maintain their structural integrity ("intactness")—these cells must not be frozen or stored as EDTA-blood for more than 6 hours, before subfractions, such as granulocytes start disintegrating.    3. In contrast to the common perception, there are not highly specific (surface) antigens for all immune cell types and hence the identification of cell types is not as unambiguous as may be hoped.    3a. Since antigen expression is not a digital process, thresholds must be defined to decide, whether cells belong to the positive or negative fraction. For T cells, this problem is particular apparent:
Hence, for many applications the current methodological approaches for a quantitative determination of immune cells remain problematic, such as for routine testing in clinical applications, which usually requires some lag times, and hence robustness and stability of the analyte. As said, the flow cytometric methods used for measurement of cells in peripheral blood are not adequate for immune cells infiltrating other tissues, including solid tissues during tumor development or at/after inflammation. Hence, flow cytometric methods are not applied in these areas and the surrogate methods (mostly immune histochemistry) are at most semi-quantitative methods.
For the purpose of definition for the present application, epigenetic modifications in the DNA sequence is referred to by the terminology of DNA methylation (5-methylcytosine (mC), 5-hydroxymethylcytosine, (hmC), 5-formylcytosine, (fC) and/or 5-carboxycytosine (cC)). Both, at least, 5-methylcytosine and 5-hydroxymethylcytosine are not bisulfite convertible and cannot be distinguished via bisulfite conversion analysis.
In the scientific literature, the status of methylation is often displayed as either "hyper" (above, more than normal, (lat.: super)) or "hypo" (below, less than normal, (lat.: sub) methylation. In the view of the inventor, these terms are inappropriate, since they suggest variance from the "normal" status. For healthy cells however, there is no such thing as non-normal, since for one cell type it is normal that it is methylated whereas the other is unmethylated.
Both signatures are perfectly normal. For the inventor, gene regions are therefore either methylated or unmethylated (also and equivalent to unmethylated: demethylated). The potential decision as to whether a region in a cell type is aberrantly methylated (hypermethylated) or aberrantly demethylated (hypomethylated) may potentially play a role in certain diseases and decision may be made based on this. However, it is not an issue that is discussed or found in the technical process of measuring the methylation status in DNA. Despite this and owing to the general rules, whenever hypermethylation and hypomethylation is written it is thought to indicate the technical classification of demethylation and methylation.
The NCBI Reference Sequence: NC_000006.11 discloses chromosome 6, including the genomic region for human IL17A (Chromosome 6: position for the mRNA is 52,051,185-52,055,436 on the forward strand).
The present inventors present a marker that can be used for the identification and quantification of IL17 positive T cells in an alternative quantitative, more efficient, robust and integral approach: the analysis of cell type-, or cell status-specific epigenetic (DNA methylation and or chromatin structure and or DNA chemical inertness) markers. In the clinical routine and the general cell biological practice, the identification of specific epigenetic markers will greatly facilitate the measurement of blood and immune cell types.
Janson et al. (in Janson P C, Linton L B, Bergman E A, Marits P, Eberhardson M, Piehl F, Malmström V, Winqvist O. Profiling of CD4+ T cells with epigenetic immune lineage analysis. J Immunol. 2011 Jan. 1;186(1):92-102. Epub 2010 Dec. 3. ) describe a method to pinpoint the location of isolated human CD4(+) T cells on any T cell effector axis based on specific CpG methylation of cytokine and transcription factor loci. They applied such a method on CD4(+) cells obtained from rheumatoid arthritis and multiple sclerosis patients and showed that synovial fluid infiltrating CD4(+) T cells are committed toward both Th1 and regulatory T cell phenotype, whereas the Th2 response is suppressed. Furthermore, they showed that the IL-17A gene is regulated by promoter methylation and that Th17 commitment is not a common feature in the inflamed joints of rheumatoid arthritis patients. They concluded that the method described in the publication thus would allow for accurate profiling of Th lineage commitment in ex vivo-isolated CD4(+) T cells.
The importance of methylation becomes particularly apparent in tumor cells. Here, the "normal" methylation patterns—that are essential for a healthy development of a cell—are lost, and the cells therefore can no longer be controlled, and become not only epigenetically aberrant. Although aberrant methylation is not the primary subject of the present invention, the apparent significance of a correct epigenetic regulation becomes apparent by the close association between cancer cells and wrong methylation. Form this, one can conclude on the importance of a correct methylation. For single genes, as was shown earlier with the FOXP3 (Baron et al, Eur J Immunol. 2007 Sep.; 37(9):2378-89; Wieczorek et al. Cancer Res. 2009 Jan. 15; 69(2):599-608) and CD3 locus (Sehouli et al. Epigenetics. 2011 Feb. 1; 6(2):236-46), the chemical/structural nature of the DNA at the given loci (such as the methylation status/imprint) coincides with the differentiation and the type of certain cells. This allows for an identification and quantification of the cell type of healthy cells based on their epigenetic fingerprint. Whether the highly inflammatory nature of Th17 cells is sufficient to cause or contribute to carcinogenesis is the subject of current debate (Wu S, Rhee K J, Albesiano E et al. (September 2009). "A human colonic commensal promotes colon tumorigenesis via activation of T helper type 17 T cell responses". Nature Medicine 15 (9): 1016-1022; Huang C, Fu Z X. Localization of IL-17+Foxp3+ T cells in esophageal cancer. Immunol Invest. 2011; 40(4):400-12. Epub 2011 Feb. 11.).
In view of the above, despite of some progresses in the art, it is an object of the present invention to provide an improved method based on accessibility to bisulfite conversion and/or DNA methylation analysis as a superior tool in order to more conveniently and reliably identify and quantify IL17 positive T cells in a given sample derived/obtained from for example animal/human blood or tissue. Measurement can advantageously be done independent of purification, storage and to quite some extend also to tissue quality.
In further view of the above, it is an object of the present invention to provide an improved method to identify not only actual IL-17 protein producing cells but also cells that are capable of producing IL-17 protein after stimulation by e.g. ionomycin and PMA (phorbol myristate acetate). Currently, common techniques of cell identification only detect cells that are currently producing the IL-17 protein (up to 0.1% of cells within whole blood). However, the proportion of cells identified by the present invention amounts to 0.1-2% encompassing cells that are capable to produce IL-17 (currently not producing IL-17), too. IL17 positive cells are cells such as Th17 cells.
In a first aspect thereof, the invention solves the above problem by providing a method for identifying IL-17 positive CD4 positive T cells (also designated herein as IL-17 expressing T cells) in a blood and/or tissue sample derived from a mammal, comprising analysing the methylation status of at least one CpG position in the gene IL-17A, wherein a demethylation of said at least one CpG position in said sample when compared to an analogous position in a non IL-17 blood cell is indicative for an IL-17 positive CD4 positive T cell, such as Th17 cells.
Preferably, said method is performed without a step of purifying and/or enriching cells, preferably in whole blood and/or non-trypsinized tissue, or any other biological sample potentially containing T cells. Most preferably, said sample is a sample for autologous cell transfer into a patient, that is, is a sample to be transplanted into a patient.
A further embodiment of the invention then comprises the inventive method, wherein said method further comprises the quantification of Th17 cells in complex tissues compared to either natural Tregs, overall T cells and/or the respective tissue, in particular blood or solid diseased tissue, or adjacent tissue irrespective of its origin. In another aspect, IL17A can be part of a "Panel" of genes/markers, such as, for example, CD3, FOXP3 and/or GAPDH, as measured in case of several cell types that are important for the immune status of the mammal.
A further embodiment of the invention then comprises the inventive method, wherein said at least one CpG position is present in the 5′ region upstream from the transcription start, promoter region, the 5′ or 3′ untranslated regions, intron, and/or exon/intron border, or in the 3′ region downstream of the transcriptional stop.
A yet further embodiment of the invention then comprises the inventive method, wherein said at least one CpG position is selected from the CpG positions located in the gene IL17A according to SEQ ID NO: 17, preferably of the IL17A amplicon 1909 according to SEQ ID NO: 1, preferably a target region according to SEQ ID No: 2 or 3, or an IL17A amplicon as amplified with a primer pair selected from the sequences according to SEQ ID NOs: 4 to 16, or 18 to 21, particularly as amplified with the primer pair according to SEQ ID NO 19 and 20.
For a particular CpG as analyzed, theoretically there are four states per cell: (both alleles methylated), (allele A methylated, allele B unmethylated), (allele A unmethylated, allele B methylated), and (both alleles unmethylated). This leads to three different results: 100% methylated, 50% methylated and 0% methylated. Thus, theoretically IL-17 positive T cells are 0% methylated, and non-IL-17 positive T cells are approximately 100% methylated. A similar situation is present for the assay analyzing the accessibility of the region as analyzed to bisulfite conversion, where IL-17 positive T cells are 0% accessible, and non-IL-17 positive T cells are approximately 100% accessible.
In practical measurements employing e.g. bisulfite sequencing, the complete, "pure" methylation patterns are rarely detected, since both slight technical deficits of the employed technologies and possibly minor biological variances blur the theoretically expected values. Therefore, methods of the invention are preferred, wherein said at least one CpG position in said region as analyzed is demethylated to more than 80%, preferably more than 90%, and most preferred more than 95%, when compared to an analogous position in a non-IL-17 positive T cell.
Furthermore, the present invention includes methods, where more than one CpG position in a region is analyzed, such as, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions (e.g. in the AMP 1909 region (SEQ ID No. 1) as above). Then, an overall methylation (or demethylation) of the region as analyzed can be determined, when compared to an analogous region in a non-IL-17 positive T cell. Therefore, also methods of the invention are preferred, wherein said region is demethylated (hypomethylated) to more than 70%, preferably more than 80%, or 90%, and most preferred more than 95% when compared to an analogous region (hyper or fully methylated) in a non-IL-17 positive T cell.
The person of skill will furthermore be able to select specific subsets of CpG positions in order to minimize the amount of sites to be analyzed, for example all sites as present on the amplicon according to SEQ ID No. 1, or any other selected sub-sequence in the IL-17 gene as analyzed, for example as described above of the 5′ region upstream from the transcription start, promoter region, the 5′ or 3′ untranslated regions, intron, and/or exon/intron border, or in the 3′ region downstream of the transcriptional stop, and/or the target regions according to SEQ ID No. 2 or 3, or an IL17 amplicon as amplified with a primer pair selected from the sequences according to SEQ ID NOs: 4 to 16, or 18 to 21, particularly as amplified with the primer pair according to SEQ ID NO. 19 and 20.
Yet another aspect relates to a method according to the present invention, wherein the analysis of the accessibility to bisulfite conversion and/or the methylation status comprises a method selected from methylation specific enzymatic digests, bisulphite sequencing, analysis selected from promoter methylation, CpG island methylation, MSP, HeavyMethyl, MethyLight, Ms-SNuPE, qPCR or other methods relying on the detection of genomic DNA, chemically or enzymatically modified DNA or amplified genomic or chemically or enzymatically modified DNA. Also preferred is an additional analysis of the marker CD4, CD3, FOXP3, and/or GAPDH.
Another embodiment of the invention relates to the above methods, wherein said identification comprises a distinction and, optionally, a further quantification, of said IL-17 positive T cells from all major peripheral blood cell types or non-blood cells, and then further comprises the step of concluding on the immune status of said mammal based on said IL-17 expressing T cells as identified. Hereby, in a sample of a mammal, including whole blood or various subfractions as well as tissues or isolated subfractions of tissues, IL-17 positive T cells can be identified and quantified due to their (unique) methylation pattern in the analyzed genes. Based on this they can also be quantitated, as the loss of methylation strictly correlates with IL-17 positive T cells. The quantification preferably comprises a quantification of the IL-17 cells in said sample based on a comparison of the IL-17 methylation with a methylation of at least one marker selected from the group of CD3, FOXP3, and/or GAPDH.
Herein, the "immune status" of a person shall mean the status of the immune system of a given person in a given situation, in any given tissue type at any given disease situation. For example, it may be important to determine the immune status in a (tumor) tissue biopsy of a person who suffers from a solid tumor. Also, it may be relevant to determine the immune status of a (presumably) healthy person in the peripheral blood in order to determine the persons health status, whereby both an increase or a decrease of the cells—as quantified by the given number of methylated and unmethylated copies of the analyzed genes, in particular IL-17—may be indicative for a disease, such as for example, the presence of a tumor at an unknown site of the body, or an autoimmune reaction or a chronic infection.
In particular, the inventors regard the herein described methods to be suitable for routine application, for example on a DNA-chip. Samples are selected from a fresh, fresh-frozen or fully prepared (such as formalin fixed paraffin embedded) sample, including mammalian body fluid, preferable human blood samples, (serum samples) or tumorous or non-tumorous solid tissue samples, organ or cell type blood sample. These samples should be mammalian, preferably mouse, rat, monkey, bovine, swine or human. Especially preferred is a mammal, most preferred a human, which suffers from or is likely to suffer from IL-17 mediated diseases and side effects of anti-IL-17 therapies, such as, for example, psoriatic diseases, psoriasis, psoriatic arthritis, rheumatic diseases as e.g. rheumatoid arthritis, multiple sclerosis, arteriosclerosis, ankylosing spondylitis, Crohn's disease, inflammatory bowel disease, inflammatory diseases, uveitis, hepatitis diseases, lupus, lung diseases (e.g. asthma), hyper IgE syndrome, anti-tumor immunity, renal injuries, infections (viral, bacterial, fungal, parasitic) endotoxic shock, and autoimmune diseases, viral or bacterial infections, transplant rejections, cancer including solid and non solid cancers, and/or allergy or any disease directly correlated to IL-17 expressing T cells. The present invention can also be used to monitor anti-IL-17 therapies in a patient and/or patient group.
WO2012/093254A1 (incorporated by reference) describes the use of lipocalin 2 (LCN2) as a biomarker for IL-17 mediated diseases and for monitoring the response of a patient to anti-IL-17 therapy, and IL-17 related diseases that are also relevant for the present invention, such as a disorder selected from the group consisting of arthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic onset juvenile idiopathic arthritis (HA), systemic lupus erythematosus (SLE), multiple sclerosis, asthma, chronic obstructive airway disease, chronic obstructive pulmonary disease, atopic dermatitis, scleroderma, systemic sclerosis, lung fibrosis, Crohn's disease, ulcerative colitis and ankylosing spondylitis and other spondyloarthropathies.
Yet another aspect relates to a method according to the present invention, further comprising the step of concluding on the number and/or amount of said IL-17 positive T cells as identified in said sample based on said identification and quantification. As the demethylation of the IL-17A gene and the amplicon according to SEQ ID No. 1 or the target regions according to SEQ ID No. 2 or 3, or an IL17 amplicon as amplified with a primer pair selected from the sequences according to SEQ ID NOs: 4 to 16, or 18 to 21, particularly as amplified with the primer pair according to SEQ ID NO. 19 and 20 are so strictly linked to the IL-17 expressing T cells, in the most convenient embodiment of said method, the number and/or amount of said IL-17 expressing T cells can be correlated directly with the results of the demethylation analysis, if normalized for the copy number in the assay and/or the gender of the subject. In other alternatives, additional control experiments (e.g. demethylated GAPDH analysis in parallel) may be applied (see also above).
In a further aspect the inventive method is useful for monitoring the level of IL-17 positive T cells in a mammal, comprising a method according to the invention, and comparing the amount of IL-17 positive T cells as identified with an earlier sample taken from the same mammal, and/or with a control sample.
Yet another aspect relates to a method according to the present invention, further comprising the step of concluding on the immune status of said mammal based on the number and/or amount of said IL17 expressing T cells as identified in said sample as described herein above.
Yet another aspect relates to a method according to the present invention, wherein said mammal suffers from or is likely to suffer from IL-17 mediated diseases and side effects of anti-IL-17 therapies, such as, for example, psoriatic diseases, psoriasis, psoriatic arthritis, rheumatic diseases as e.g. rheumatoid arthritis, multiple sclerosis, arteriosclerosis, ankylosing spondylitis, Crohn's disease, inflammatory bowel disease, inflammatory diseases, uveitis, hepatitis diseases, lupus, lung diseases (e.g. asthma), hyper IgE syndrome, anti-tumor immunity, renal injuries, infections (viral, bacterial, fungal, parasitic) endotoxic shock, and autoimmune diseases, viral or bacterial infections, transplant rejections, cancer including solid and non solid cancers, and/or allergy or any disease directly correlated to IL-17 expressing T cells.
In another aspect of the present invention, the method is also useful for measuring and/or monitoring the amount of said IL-17 expressing T cells in response to chemical and/or biological substances that are provided to said mammal.
In yet another aspect, the invention provides an amplicon according to SEQ ID NO: 1 or a target region thereof, preferably according to SEQ ID NOs. 2 or 3. The amplicon can be used as a tool in the methods according to the present invention.
The invention also provides a kit for identifying, quantifying, and/or monitoring IL-17 expressing T cells in a mammal based on the analysis of the methylation status of CpG positions in the gene IL-17, comprising materials for performing a method according to the invention as described hereinabove.
Such an inventive kit preferably comprises, but is not limited to, a) a bisulfite reagent, and b) materials for the methylation analysis of CpG positions selected from the CpG positions of the gene IL-17A according to SEQ ID NO: 17, preferably of the IL17 amplicon 1909 according to SEQ ID NO: 1, or an IL17 amplicon as amplified with a primer pair selected from the sequences according to SEQ ID NOs: 4 to 16, preferably a target region according to SEQ ID No: 2 or 3.
The present invention solves the above problem that the detection of IL-17 expressing T cells and their distinction from one another, is problematic in essentially all applications in the R&D and in particular in all clinical (routine) applications by providing a method for identifying IL-17 expressing T cells of a mammal, comprising analyzing the methylation status of at least one CpG position in one or various, for example regulatory, potentially differentially methylated regions of the gene for IL-17, wherein a demethylation, and/or accessibility to bisulfite conversion is highly specific or indicative for IL-17 expressing T cells.
In another preferred embodiment of the present invention, the inventors furthermore present a novel and more specific way in order to monitor IL-17 expressing T cells in all human body fluids, including human whole blood samples, or in any given (solid) tissue, organ or cell type.
The inventive concept is generally based on a specific demethylation and/or accessibility to bisulfite and other chemical base specific conversion of DNA of the genetic IL-17 region in IL-17 expressing T cells. Using a simple and precise quantitative PCR method, as a signal amplification method (e.g. a precise quantitative PCR method or other method allowing for the determination of copies of the nucleic acid molecules), the inventors show that the IL-17A region demethylation and/or accessibility to bisulfite conversion represents surrogate markers for IL-17 expressing T cell counts in blood or tissues. The present inventors have thus identified particular and new regions within the IL-17 gene that are functionally involved in, or reliably associated with, the existence of IL-17 expressing T cells.
The main aspect of the present invention is the distinction between and among functionally different fractions of T cells, namely the IL-17 expressing T cell fraction on one hand and other human/animal cell types on the other hand.
The inventors could demonstrate that in all IL-17 expressing T cells the CpG motifs are almost completely demethylated (i.e. to more than 70%, preferably 80%, preferably, more than 90% and most preferred more than 95%, see above), whereas the same motifs are completely methylated in all non-IL-17 expressing T cells. Determination of the methylation status of the IL-17 loci is therefore a valuable tool to identify IL-17 expressing T cells, such as will be required/or at least of some value for measuring IL-17 expressing T cells in autoimmune diseases, (viral) infections, transplant rejections, cancer, infections, and/or allergy. The assay allows measurement of IL-17 expressing T cells in "complex" biological samples (i.e. samples, such as tissue and/or blood) containing more than one type of cell, preferably more than one type of blood cell) without purification or any staining procedures. As a particularly preferred embodiment, the measurement of IL-17 expressing T cells by either of the markers described in here can be easily detected and quantified from within solid tissue samples of healthy or diseased nature, including tumorous or non-tumourous tissues. For such analysis it is possible to make the analysis either from fresh, fresh-frozen or any type of conserved (such as, for example, formalin fixed and/or paraffin-embedded) tissue. Another preferred embodiment is to determine the ratio between IL-17 expressing T cells on one hand and other T cell types on the other.
The inventors have shown that the potential to form IL-17 expressing T cell properties of mammalian immune cells coincide with epigenetic, i.e., DNA methylation based regulation in the genetic region of IL-17. DNA methylation is a biologically and chemically stable epigenetic modification, resulting in long-term gene expression changes. The inventors found demethylation and/or the accessibility of the genomic DNA to bisulfite conversion at the human IL-17 locus to be restricted to IL-17 expressing T cells when tested against all major peripheral blood cell types and a selection of different non-blood cell types/lines. These data indicated that epigenetic modifications in the IL-17A locus serve as valuable marker for the identification of cells with the phenotype of IL-17 expressing T cells, regardless of the expression of any genes.
It is well established in the art that often additional and important gene regulatory elements that are subject to gene regulation by methylation are located upstream and downstream of an open reading frame of a given gene—e.g. enhancer regions which are binding sites for indispensable transcriptional regulators. Thus, as a preferred embodiment of the present invention, an analysis of this elements is included in the methods of the present invention, which may be located within 10000 bases upstream of the transcriptional start site of IL-17A, preferably 9000 bases, 8000 bases, 7000 bases, 6000 bases, 5000 bases, 4000 bases, 3000 bases or 2000 bases upstream of IL-17A, even more preferred is a region 1000 bases upstream of the transcriptional start of IL-17A and most preferable in the first 500 bases upstream of the transcriptional start site of IL-17A. It is, however, particularly preferred that the sites as analyzed in the present invention are located within the gene promoter of IL-17A.
In another embodiment, the method according to the present invention is preferred, wherein said analysis of the methylation status comprises amplification with at least one primer of the primer pairs useful to amplify the amplicon according to SEQ ID NO: 1 or the target regions according to SEQ ID NOs 2 or 3.
Preferably, the amplification involves a polymerase enzyme, a PCR or chemical amplification reaction, or other amplification methods as known to the person of skill as described below, e.g. in the context of MSP, HeavyMethyl, Scorpion, MS-SNUPE, MethylLight, sequencing or methyl specific restriction assays. With the amplification, the amplicon of the IL-17A gene or any paralog or ortholog as described herein is produced that is a particularly preferred "tool" for performing the method(s) according to the present invention. Consequently, a primer pair for the amplification of the regions according to SEQ ID NO: 1 and SEQ ID NO: 17 and parts thereof and SEQ ID NOs 4 to 16 or 18 to 22, in particular of SEQ ID NO: 19 and SEQ ID NO: 20 constitutes a preferred embodiment of the present invention.
Furthermore, preferred is a method according to the invention, wherein above 5 mM of MgCl2 are (can be) used in the amplification reaction, preferably up to 6.4 mM MgCl2.
Furthermore, preferred is a method according to the invention, further comprising the step of analyzing the cellular markers CD3, CD4, FOXP3 and/or GAPDH. In order to analyze these additional markers, any known method to analyze expression can be used, such as methods using antibodies, and/or methylation analysis. The analysis of these markers preferably further improves the accuracy of the analysis, and might allow to identify sub-sets of cells. Thus, the method according to the present invention comprises an identification that is a distinction of said IL-17 expressing T cells from all major peripheral blood cell types or non-blood cells.
The method according to the present invention can be performed with any mammal having the above markers or orthologs or paralogs thereof, preferred is a method according to the present invention, wherein said mammal is a mouse, rat, pig or cow, monkey or human, preferably a human.
The method(s) according to the present invention can be performed in vitro and/or in vivo. In general, all biological samples can be used, as long as they contain suitable cells or suitable DNA of cells of interest. Preferred is a method wherein said sample is selected from a fresh, fresh-frozen or fully prepared sample including mammalian body fluid, preferable human whole blood samples, serum samples or a tumorous or non-tumorous solid tissue, organ or cell type blood sample, a sample of blood lymphocytes or a fraction thereof.
Another preferred aspect of the present invention then relates to the use of the method according to the present invention as above in diagnostics and the use in monitoring diseases. Thereby, in an alternative embodiment, the invention is directed at a method according to the present invention which further comprises the step of concluding on the immune status of said mammal based on said IL-17 expressing T cells as identified and/or quantified. In said method according to the invention, a demethylation of at least one CpG position in the gene IL-17A is indicative for an IL-17 expressing T cell.
Another important aspect of the present invention then relates to a method according to the present invention for monitoring the level of IL-17 expressing T cells in a mammal, comprising a method according to the invention as above, and comparing the amount of IL-17 expressing T cells as identified with an earlier sample taken from the same mammal, and/or with a control sample. Preferably, said method is performed on a sample from a mammal suffering from or is likely to suffer from autoimmune IL-17 mediated diseases and side effects of anti-IL-17 therapies, such as, for example, psoriatic diseases, psoriasis, psoriatic arthritis, rheumatic diseases as e.g. rheumatoid arthritis, multiple sclerosis, arteriosclerosis, ankylosing spondylitis, Crohn's disease, inflammatory bowel disease, inflammatory diseases, uveitis, hepatitis diseases, lupus, lung diseases (e.g. asthma), hyper IgE syndrome, anti-tumor immunity, renal injuries, infections (viral, bacterial, fungal, parasitic) endotoxic shock, and autoimmune diseases, viral or bacterial infections, transplant rejections, cancer including solid and non solid cancers, and/or allergy or any disease directly correlated to IL-17 expressing T cells.
Further preferred, said method according to the invention then further comprises measuring and/or monitoring the amount of IL-17 expressing T cells in response to chemical and/or biological substances that are provided to said mammal. That is, changes in the amount or ratio of IL-17 expressing T cells that are caused by, for example, the treatment of a disease (e.g. as described herein), and the success and/or progress of said treatment in terms of an effect on the IL-17 expressing T cells can be followed using this method. A follow-up of the methylation pattern based on the markers herein will point to changes in the cells that are due to a response to said chemical and/or biological substances, in some cases even before a phenotypic change can be observed.
In yet another aspect of the present invention, the present invention provides a method for identifying chemical and/or biological substances that selectively modulate IL-17 expressing T cells expressing the markers as described herein, comprising contacting one or more of said chemical and/or biological substance with potentially IL-17 expressing T cells, and detecting, whether said chemical and/or biological substance modulates the methylation of the CpG positions as analyzed, and/or whether said one or more of said chemical and/or biological substance selectively modulates the amount and/or ratio of marker-expressing IL-17 expressing T cells. Particularly preferred is a modulation of said IL-17 expressing T cells that increases the amount and/or ratio of said IL-17 expressing T cells.
The method can be performed in vitro and/or in a suitable animal. In this aspect, the present invention provides a method, sometimes called a "screening-method", that seeks to identify chemical and/or biological substances modulating expression of the markers as above that can be used as starting points for the development of IL-17 expressing T cell-specific medication and respective pharmaceutical compositions. The present method is based on the fact that it is well accepted that the marker genes as identified herein must play a central role for the development of IL-17 expressing T cells. Therefore, factors stimulating marker expression are interesting for the treatment of patients. Such factors, which lead to a stable modification, preferably induction, of the development/ratio/amount of IL-17 expressing T cells, can be detected with the method described in this invention.
Chemical and/or biological substances that are suitable as screening compounds are known to the person of skill and, for example, include small molecules, peptides and proteins, and antibodies or fragments thereof. Furthermore, the screening can be done using a commercially compound library, optimally together with suitable automation, such as a robot. In one preferred embodiment of the method for identifying chemical and/or biological substances, said substance provides a demethylation of the CpG positions as analyzed to at least 80%, preferably 90%, and more preferably 95%.
Another important aspect of the present invention then relates to a method according to the present invention, which further comprises the step of providing a treatment for a patient suffering from or being likely to suffer from IL-17 mediated diseases and side effects of anti-IL-17 therapies, such as, for example, psoriatic diseases, psoriasis, psoriatic arthritis, rheumatic diseases as e.g. rheumatoid arthritis, multiple sclerosis, arteriosclerosis, ankylosing spondylitis, Crohn's disease, inflammatory bowel disease, inflammatory diseases, uveitis, hepatitis diseases, lupus, lung diseases (e.g. asthma), hyper IgE syndrome, anti-tumor immunity, renal injuries, infections (viral, bacterial, fungal, parasitic) endotoxic shock, and autoimmune diseases, viral or bacterial infections, transplant rejections, cancer including solid and non solid cancers, and/or allergy or any disease directly correlated to IL-17 expressing T cells, wherein said treatment modulates, and preferably increases the amount and/or proportion of IL-17 expressing T cells in said, preferably, autoimmune or cancer patient. Preferred is a method according to the present invention, wherein said treatment is selected from providing chemical and/or biological substances that selectively stimulate IL-17 expressing T cells in said patient, or a treatment that stimulates the expression of the marker genes as above or supports the biological activity of said marker genes in said IL-17 expressing T cells in said patient. Preferred examples of such treatments are demethylating agents that provide for a reduced methylation of said genes. Other preferred examples of such treatments are agents that provide for a reduced number of IL-17 expressing T cells in case of autoimmune diseases.
Yet another preferred aspect of the present invention relates to an improved method of treatment of diseases that are related to marker gene expression and/or demethylation, IL-17 mediated diseases and side effects of anti-IL-17 therapies, such as, for example, psoriatic diseases, psoriasis, psoriatic arthritis, rheumatic diseases as e.g. rheumatoid arthritis, multiple sclerosis, arteriosclerosis, ankylosing spondylitis, Crohn's disease, inflammatory bowel disease, inflammatory diseases, uveitis, hepatitis diseases, lupus, lung diseases (e.g. asthma), hyper IgE syndrome, anti-tumor immunity, renal injuries, infections (viral, bacterial, fungal, parasitic) endotoxic shock, and autoimmune diseases, viral or bacterial infections, transplant rejections, cancer including solid and non solid cancers, and/or allergy or any disease directly correlated to IL-17 expressing T cells, comprising a method as described herein above. The term "treatment" also includes a prevention of marker gene expression and/or demethylation related diseases.
In yet another aspect of the present invention, the present invention relates to the use of an amplicon according to the present invention or a kit according to the present invention for identifying, quantifying and/or monitoring IL-17 expressing T cells in a mammal.
The present invention will now be further described in more detail in the form of preferred embodiments thereof in the following examples, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties.
FIG. 1 shows the results of the test-template as amplified according to Example 1. TpG Template: unmethylated test-DNA; CpG template: methylated test-DNA; NTC: No template control.
FIG. 2 shows A) the genomic sequence of the amplicon No. 1909 according to SEQ ID No. 1. The target-region of the qPCR-assay is underlined, CpG positions of interest are double underlined; B) the bisulfite-converted target-region 1 of the qPCR-assay-system (TpG-specific), primer and probe are underlined, CpG positions of interest are double underlined; and C) the bisulfite-converted alternative target-region 2 of the qPCR-assay-system (TpG-specific), primer and probe are underlined, the target-region is indicated in bold; CpG positions of interest are double underlined.
FIG. 3 demonstrates the specificity of the preferred TpG-specific IL17A qPCR-assay system according to Example 3 for demethylated template-DNA. Shown are the amplification profiles of serial dilutions of plasmid standards carrying the target region of the IL17A-gene (bisulfite-converted, unmethylated). In addition, the amplification profiles of unmethylated (TpG; gray curve) and methylated (CpG; dark gray curve) plasmid templates (86000 copies each) are shown. The TpG-specific IL17A qPCR-system amplifies only non-methylated template-DNA with high specificity, but no methylated template, and shows that there is no cross-reactivity from the technical point of view on the assay.
FIG. 4 demonstrates the analysis of a complex sample (e.g. blood sample) (peripheral blood) for a detection of the relative amount of IL17-positive T-cells.
The upper Figure illustrates the TpG-(demethyl-)specific IL17A qPCR-system according to the invention. Amplification profiles of the plasmid standards (50000-80 copies) are shown. A (linear) standard curve was derived from the profiles by depicting the CP-value as measured over the logarithm of the template concentration. Amplification profiles of the blood sample are depicted in light gray, and labeled with the arrow. Dark gray curve: "no template"-control (NTC).
The lower Figure illustrates the TpG-(demethyl-)specific GAPDH qPCR-system. Amplification profiles of the plasmid standards (50000-16 copies) are shown. A (linear) standard curve was derived from the profiles by depicting the CP-value as measured over the logarithm of the template concentration. Amplification profiles of the blood sample are depicted in light gray, and labeled with the (light gray) arrow. Dark gray curve: "no template"-control (NTC).
SEQ ID NO: 1 shows the nucleotide sequence of AMP 1909;
SEQ ID NO: 2 and SEQ ID NO: 3 show the nucleotide sequence of the target regions according to FIG. 2;
SEQ ID NOs: 4 to 16 show the nucleotide sequences of the primers and probes as used in the examples.
SEQ ID NO: 17 discloses the mRNA for human IL17(A).
SEQ ID NOs: 18 to 22 show the nucleotide sequences of particularly preferred primers and probes as used in example 3.

Epigenetic marker for the identification of IL17 positive T cells in complex samples


A circuit arrangement is disclosed for controlling an illuminating device, particularly light-emitting diodes, in a control device of a motor vehicle, comprised of at least one illuminating device, which is integrated in a switching circuit having a semiconductor component, and of a microprocessor, in particular having an integrated non-volatile memory, for generating a pulse-width modulated signal, whereby the pulse-width modulated signal is coupled to a control electrode of the semiconductor component, whereby the microprocessor generates at least two pulse-width modulated signals, the pulse-width modulated signals are coupled to the semiconductor component via a logical system and the logical system can be subjected to the action of an additional control signal via the microcomputer.

1. Field of the Invention
The present invention relates to a circuit arrangement for controlling an illuminating device, in particular light-emitting diodes, in a control device of a motor vehicle, comprised of at least one illuminating device integrated in a switching circuit having a semiconductor component, and a microprocessor, in particular with an integrated non-volatile memory, for generating a pulse-width modulated signal, whereby the pulse-width modulated signal is connected to the control electrode of the semiconductor component.
2. Description of the Background Art
It is known to provide control elements in motor vehicles with an illumination. In order to adapt the illumination, or rather the backlighting, of the control elements and displays to ambient light effects, the illumination is adjustable. A distinction is thereby made between a day and night illumination on the one hand, and between a locator and function illumination on the other hand. Depending on the variation of interior design in the motor vehicle, it is also known to adapt, via a photo-sensitive sensor, these different types of lighting to the incident light striking the control element directly. There are different methods known for controlling the illuminating means.
Light-emitting diodes (LEDs) are typically used for as illuminating devices in control devices. The objective thereby is to achieve a harmonious, evenly illuminated layout of the control device. The luminosity of LEDs or lamps, however, tends to be very widely scattered so that the tolerances stipulated by the automobile industry can frequently not be achieved without further measures. From DE 196 02 891 A1, it is known to determine, after the manufacture of the control device, a correction factor depending on the brightness of the illuminating means, with which the control voltage or the control current of the illuminating means is adjusted.
In order for the described types of illumination to be even continuously adjustable and controllable, if need be, it is known from the cited publication to control the illuminating means via a semiconductor component, and in particular via a pulse-width modulated signal (PWM). With the assistance of a PWM signal present at the output of a microprocessor, a transistor, for example, is clock-controlled, which allows for the brightness of the light-emitting diode to be adjusted with very great precision. Due to the modulation of the pulse width, the brightness of the light of the illuminating means can thus be altered, whereby the duty cycle, that is, the ratio of impulse duration to cycle duration, represents a direct measure of the brightness of the illuminating means.
In order to be able to regulate the illumination of a combined locator and function symbol via a PWM signal of a control device, which can be a microcomputer, for example, in dependence from incident sunlight, it is known from DE 102 09 622 A1, which corresponds to U.S. Pat. No. 7,022,961, to detect the brightness in the ambient area of the control element via an optoelectric sensor. The influence of a non-linearity of the light-current characteristic line of the respective LED, which occurs at high currents, for example, self-heating of the LEDs, can hereby be adjusted by way of a characteristic line stored in a software of a control unit, whereby the PWM ratio is preferably adapted.
It is known from patent application DE 102 54 741 to influence a pulse-width modulated signal for the activation of LEDs in the duty cycle. For this purpose, the PWM signal generated in the microcomputer in a circuit arrangement having logical connectives is influenced such that only every other impulse of the PWM signal is transmitted to the illuminating means.

Circuit arrangement for controlling illuminating means


Aspects of the present disclosure may address a virtual gaming system. The virtual gaming system may present various graphical user interfaces (GUIs) to a user, in conjunction with a device used by the user, in a client-server configuration. The virtual gaming system may facilitate play of games using at least two virtual currencies, where one virtual currency is purchased and has no monetary value and the second virtual currency may not be purchased but has monetary value. Separate sets of games may be provided for play using the at least two virtual currencies. It may also be possible, in some cases, to obtain first virtual currency, second virtual currency, or both, other than by purchase.

Electronic gaming or Internet gaming (e-gaming or i-gaming) systems have proliferated at a high rate in recent years. E-gaming systems range from sportsbooks to on-line casino games. Typically in casino-type games, a user registers with a gaming service/system and purchases virtual currency that enables the user to participate in the games offered, which may include, for example, but are not limited to, slot machines, roulette, etc.
While most of this proliferation of e-gaming has historically taken place outside of the United States, in countries where gambling is legal and individuals "buy-into" games and receive cash awards for winning, e-gaming has also gained traction in the United States, where casino-type games, e.g., virtual slot machine-based games, have been made available for use.
Typically, an individual who plays such games may receive an initial number of tokens, simply for registering to play. Some systems provide a daily allotment of additional tokens. But an individual who would like to continue to play beyond the tokens he or she has received (and without waiting for a further allotment of tokens, if any) may purchase additional tokens. Winners of such games may typically receive only tokens as prizes for winning.
More recently, e-gaming systems of this type have considered implementing other ways of playing. One way of doing this is by providing two types of virtual currency, one for purchase that permits a user to play games "for fun," i.e., with no cash or other types of non-virtual-currency prizes (i.e., more tokens, as in the preceding paragraph), and one that permits a user to play games with the hope of winning cash or prizes having cash value, with the outcome determined purely by luck/chance. The latter is often referred to as a sweepstakes, and the virtual currency associated with the sweepstakes may be called, for example, "sweepstakes coins" or "non-monetary currency." For an example of such a system, see, for example, U.S. Pat. Nos. 10,580,256 and 11,132,865, which are incorporated by reference herein.
In a system according to the aforementioned U.S. patents, two types of virtual currency exist: "gold coins" and "sweepstakes coins." Gold coins may be purchased by users and used to play a set of casino-type games, such as virtual slot machines, blackjack games, etc. Sweepstakes coins may be obtained, e.g., as a bonus for purchasing gold coins, as giveaways, as incentives, by mail-in request, in contests, etc. In these patents, a user playing a game, for example, using gold coins, is given the option to switch to playing the same game using sweepstakes coins.
Such a system, in which a player is permitted to simply switch between currencies while playing the same game, may lead the player to be confused about which currency she is using at a given time and to inadvertently use the wrong virtual currency (e.g., use sweepstakes coins or gold coins when the player intends to use the other type of coin). It also requires the system to track which currency the player is using and, accordingly, what type of award is available to that player. Therefore, alternative methods of applying the two (or more) virtual currencies may be desirable.

Electronic gaming using multiple virtual currencies


An access control unit for an electrified door lock also toggles the state of an associated alarm. The unit is arranged to unlock the door only temporarily, but the alarm state is not toggled except by user request. The system is arranged to prevent the anomalous situation of an unlocked door and an armed alarm. Another aspect is the use of standardized key-receiving nests in disparate types of access control devices. Some disparate devices employ identical key-receiving nests, while others use different forms of nests. Yet all mate with and respond to a single type of key.

The related applications detail a variety of electronic access systems. Many are characterized by a variety of disparate lock types (e.g. padlocks, door locks, vaults), each of which is operable by a common electronic key.
In accordance with one aspect of the present invention, a key nest is provided that is well suited for use on locks of disparate types. The nest forms a recess for receiving part of the key, and desirably holding same in place. One or more electrical connectors in the nest establish contact with one or more corresponding contacts on the key.
In accordance with another aspect of the invention, a door lock controller is integrated with an alarm controller, so that a user needn't separately deal with both the door lock and an alarm console when entering and leaving a building.
In many prior art systems, the door lock controller and the alarm controller are separate units. A user entering a secured facility first operates the electronic door lock. Once the door is opened, the user has a brief period (e.g. 30 seconds) within which to disarm the alarm (e.g. by entering a PIN number). On leaving the building the reverse sequence occurs. First the user re-arms the alarm (e.g. by re-entry of the PIN number). A brief delay period then follows before the alarm takes effect, allowing the user to exit the building. Once outside the building, the user re-locks the door.
One improvement to such systems is to integrate the door lock controller and the alarm controller. By such arrangement, unlocking the door also disarms the alarm. Similarly, locking the door re-arms the alarm.
A further improvement to such systems is to arrange the lock to automatically re-lock after a brief period. This improvement, however, introduces a problem: keeping the state of the alarm in synchrony with the door lock.
Consider the case of a delivery entrance to a fast food restaurant. Some suppliers may make deliveries to the restaurant before any employees arrive, and so are provided with their own keys. To track the coming and going of such suppliers, restaurants are increasingly using electronic door locks that compile a log of lock activity, detailing the particular keyholders who open the door, together with the dates and times of such accesses.
The way such a system is supposed to work is that the supplier arrives and engages the key with a lock controller mounted outside the building. So doing toggles the state of a first relay (or switching circuitry) controlling a door lock solenoid or motor, switching the door lock from locked to unlocked. So doing also toggles the state of a second relay (or switching circuitry) controlling the alarm state, switching the alarm from armed to disarmed. The supplier then makes the delivery. Prior to driving away, the supplier engages the key with the lock controller a second time. So doing toggles the first relay back to locking the door, and toggles the second relay back to arming the alarm.
While restaurants strictly admonish their suppliers to follow this procedure, it is desirable to take precautionary steps in case a supplier forgets. In particular, restaurants want to guard against the possibility of the door being left unlocked if the supplier neglects to relock the door prior to departing. To provide a fail-safe situation, such door locks can be made to automatically re-lock after a brief interval.
Unfortunately, the alarm cannot similarly be automatically re-armed. (I.e. the door lock and alarm cannot be controlled by the same relay/switching circuit). The supplier may still be unloading supplies, or may be otherwise occupied inside the premises. Automatically re-arming the alarm whenever the door is automatically re-locked would cause numerous false alarms. Accordingly, a situation can arise where the door is locked, yet the alarm is disarmed.
If a supplier leaves the restaurant in this anomalous state, and a second supplier then visits, a false alarm is likely to occur. When the second supplier engages the key with the lock, this toggles the door lock from locked to unlocked. But it also toggles the alarm from disarmed to armed. When the second supplier enters the restaurant, the motion is detected by the armed alarm system and an alarm is sounded.
In accordance with a second aspect of the present invention, the foregoing problem is solved. One embodiment detects the anomalous state and, when a key is next presented to the lock, the door lock is opened but the alarm state is not changed. In a particular embodiment, this operation is performed in two phases. In the first, the state of the alarm is toggled back to armed while the door remains locked. In the second, the states of both the lock and the alarm are toggled, e.g. to unlocked and dis-armed. By this arrangement, security is enhanced against human error, without increasing the possibility of false alarms.
The foregoing and additional features and advantages of the invention will be more readily apparent from the following detailed description, which proceeds with reference to the accompanying drawings.


The invention relates to a device for driving rotation of an aircraft wheel comprising a rim (4) mounted to rotate on an axle (2) of landing gear about an axis of rotation (X), the device comprising a drive ring gear (103) and a drive actuator (101) having an outlet pinion (102) that co-operates with the drive ring gear to drive the wheel in rotation. The drive ring gear is mounted to rotate about the axis of the rim of the wheel, the drive device including selective coupling means (150) that are operable to link the rim and the drive ring gears together in rotation, or to disconnect them.

Devices are known for driving rotation of an aircraft wheel carried by an axle of landing gear. Such devices generally comprise a drive actuator having a shaft with an outlet pinion for driving a ring gear secured to a rim of the wheel. For safety reasons, the actuator is generally provided with a coupling member that is operable to selectively couple or decouple a motor of the actuator with the outlet shaft of the actuator, such that during stages in which the wheel of the aircraft is providing drive (e.g. when landing), it cannot drive the motor.
Nevertheless, such provisions are found to be expensive and complex to manage.

Device for driving an aircraft wheel in rotation


A multilayer filter comprises an inductor stacked-layer portion and a varistor stacked-layer portion. The varistor stacked-layer portion has a varistor layer the main component of which is ZnO and a hot electrode and ground electrode positioned in opposite with the varistor layer intervening, and the region enclosed between the opposing hot electrode and ground electrode does not contain a Cu component. Because the region enclosed between the opposing hot electrode and ground electrode is a region which manifests varistor characteristics, and thus the region does not contain a Cu component, degradation of the attenuation characteristics can be suppressed.

1. Field of the Invention
The present invention relates to a multilayer filter.
2. Related Background Art
In recent years, noise filters having surge functions have been used in various electronic equipment as components to alleviate EMC. In Patent Document 1 (See for Japanese Patent Publication No. 2626143), a multilayer composite electronic component is disclosed in which a magnetic layer, in which a prescribed conductor pattern is formed internally, and a varistor layer, in which a prescribed conductor pattern is formed internally, are layered, and the magnetic layer and varistor layer are electrically connected by means of a through-hole.
However, in the above Patent Document 1 the magnetic layer and varistor layer are integrally sintered, and material components comprising the respective layers may diffuse into the other layer through the interface between the magnetic layer and the varistor layer. When diffusion of these material components occurs, the characteristics of the layers in which diffusion occurs are affected, and there are concerns that noise filter functions may be degraded. In the above Patent Document 1, an Ni—Cu—Zn system ferrite is used in the magnetic layer, but studies by these inventors have revealed that when a magnetic layer comprising such a material is integrally sintered with a varistor layer, the Cu component in the magnetic layer diffuses into the varistor layer, permeating into the region which manifests the varistor characteristics, degradation of varistor functions, and in particular of the attenuation characteristic, occurs.

Multilayer filter having an inductor portion and a varistor portion stacked with an intermediate portion


Thermomechanical and thermo-oxidative stabilities in resin composites across the range of aerospace "engineering thermoplastic" resins are improved by forming four crosslinks at each addition polymerization site in the backbone of the resin using crosslinking functionalities of the general formula: ##STR1## wherein Z= ##STR2## .beta.=the residue an organic radical selected from the group consisting of: ##STR3## R.sub.R =a divalent organic radical; X=halogen;. Me=methyl. T=allyl or methallyl.. G=--CH.sub.2 --,--S--, --CO--, --SO--, --O--, --CHR.sub.3 --, or --C(R.sub.3).sub.2 --;. i=1 or 2;. R.sub.3 =hydrogen, lower alkyl, lower alkoxy, aryl, or aryloxy; and. .theta.=--C.tbd.N, --O--C.tbd.N, --S--C.tbd.N, or --CR.sub.3 .dbd.C(R.sub.3).sub.2 --

The thermosetting resins that are commonly used today in fiber-reinforced composites cannot be reshaped after thermoforming. Because errors in forming cannot be corrected, these thermosetting resins are undesirable in many applications. In addition, these thermosetting resins, made from relatively low molecular weight monomers, are relatively low molecular weight, and often form brittle composites that have relatively low thermal stabilities.
Although thermoplastic resins are well known, practical aerospace application of high performance, fiber-reinforced thermoplastic resins is relatively new. Fiber in such composites toughens and stiffens the thermoplastic resin. While the industry is exploring lower temperature thermoplastic systems, like fiber-reinforced polyolefins or PEEKs, our focus is on high performance thermoplastics suitable, for example, for primary structure in advanced high speed aircraft including the High Speed Civil Transport (HSCT). These materials should have high tensile strength, and high glass transitions. Such materials are classified as "engineering thermoplastics." At moderate or high temperatures, the low performance, fiber-reinforced thermoplastic composites (polyolefins or PEEKS, for example) lose their ability to carry loads because the resin softens. Thus, improved thermal stability is needed for advanced composites to find applications in many aerospace situations. The oligomers of the present invention produce advanced composites.
Advanced composites should be thermoplastic, solvent resistant, tough, impact resistant, easy to process, and strong. Oligomers and composites that have high thermomechanical stability and thermo-oxidative stability are particularly desirable.
While epoxy-based composites like those described in U.S. Pat. No. 5,254,605 are suitable for many applications, their brittle nature and susceptibility to degradation often force significant design concessions when these epoxies are selected for aerospace applications. The epoxies are inadequate for applications which require thermally stable, tough composites, especially when the composites are expected to survive for a long time in a hot, oxidizing environment. Recent research has focused on polyimide composites to achieve an acceptable balance between thermal stability, solvent resistance, and toughness for these high performance applications. Still the maximum temperatures for use of the polyimide composites, such as those formed from PMR-15, can only be used at temperatures below about 600-625.degree. F. (315-330.degree. C.), since they have glass transition temperatures of about 690.degree. F. (365.degree. C.). PMR composites are usable in long term service (50,000 hours) at about 350.degree. F. (170.degree. C.). They can withstand temperatures up to about 600.degree. F. (315.degree. C.) for up to about five hundred hours.
PMR-15 prepregs, however, suffer significant processing limitations that hinder their adoption because the prepreg has a mixture of the unreacted monomer reactants on the fiber-reinforcing fabric, making them sensitive to changes in temperature, moisture, and other storage conditions, which cause the prepregs to be at different stages of cure. The resulting composites have varying, often unpredictable properties. Aging these PMR prepregs even in controlled environments can lead to problems. The reactants on the prepreg are slowed in their reaction by keeping them cold, but the quality of the prepreg depends on its absolute age and on its prior storage and handling history. And, the quality of the composite is directly proportional to the quality of the prepregs. In addition, the PMR monomers are toxic or hazardous (especially MDA), presenting health and safety concerns for the workforce. Achieving complete formation of stable imide rings in the PMR composites remains an issue. These and other problems plague PMR-15 composites.
The commercial long chain polyimides also present significant processing problems. AVIMID-N and AVIMID-KIII (trademarks of E. I. duPont de Nemours) resins and prepregs differ from PMRs because they do not include aliphatic chain terminators which the PMRs use to control molecular weight and to retain solubility of the PMR intermediates during consolidation and cure. Lacking the chain terminators, the AVIMIDs can chain extend to appreciable molecular weights. To achieve these molecular weights, however, the AVIMIDs (and their LARC cousins) rely on the melting of crystalline powders to retain solubility or, at least, to permit processing. It has proven difficult to use the AVIMIDs in aerospace parts both because of their crystalline melt intermediate stage and the PMR plague that these AVIMID prepregs also suffer aging.
So, research continues and is now turning toward soluble systems like those we described in our earlier patents, including acetylenic-terminated AVIMID-KIII prepregs of the Hergenrother (NASA-Langley) type. For these soluble systems, many different polyimide sulfone compounds have been synthesized to provide unique properties or combinations of properties. For example, Kwiatkowski and Brode (U.S. Pat. No. 3,839,287) synthesized monofunctional, maleic-capped linear polyarylimides. Holub and Evans (U.S. Pat. No. 3,729,446) synthesized similar maleic or nadic-capped, imido-substituted polyester compositions.
For imides and many other resin backbones we have shown surprisingly high glass transition temperatures, reasonable processing parameters and constraints for the prepregs, and desirable physical properties for the composites by using soluble oligomers having difunctional caps, especially those with nadic caps. Linear oligomers of this type include two crosslinking functionalities at each end of the resin chain to promote crosslinking upon curing. Linear oligomers are "monofunctional" when they have one crosslinking functionality at each end. The preferred oligomers from our earlier research were "difunctional," because they had two functional groups at each end. Upon curing, the crosslinking functionalities provide sites for chain extension. Because the crosslinks were generally the weakest portions of the resulting composite, we improved thermo-oxidative stability of the composites by including two crosslinks at each junction. We built in redundancy, then, at each weak point. We maintained solubility of the reactants and resins using, primarily, phenoxyphenyl sulfone chemistries. Our work during the past fifteen years across a broad range of resin types or chemical families is described in the following, forty-nine United States Patents (all of which we incorporate by reference):
______________________________________ INVENTOR PATENT TITLE ISSUE DATE ______________________________________ Lubowitz et al. 4,414,269 Solvent Resistant November 8, 1983 Polysulfone and Poly- ethersulfone Com- posites Lubowitz et al. 4,476,184 Thermally Stable October 9, 1984 Polysulfone Composi- tions for Composite Structures Lubowitz et al. 4,536,559 Thermally Stable August 20, 1985 Polyimide Polysulfone Compositions for Composite Structures Lubowitz et al. 4,547,553 Polybutadiene October 15, 1985 Modified Polyester Compositions Lubowitz et al. 4,584,364 Phenolic-Capped April 22, 1986 Imide Sulfone Resins Lubowitz et al. 4,661,604 Monofunctional Cross- April 28, 1987 linking Imidophenols Lubowitz et al. 4,684,714 Method for Making August 4, 1987 Polyimide Oligomers Lubowitz et al. 4,739,030 Difunctional End-Cap April 19, 1988 Monomers Lubowitz et al. 4,847,333 Blended Polyamide July 11, 1989 Oligomers Lubowitz et al. 4,851,495 Polyetherimide July 25, 1989 Oligomers Lubowitz et al. 4,851,501 Polyethersulfone July 25, 1989 Prepregs, Composites, and Blends Lubowitz et al. 4,868,270 Heterocycle Sulfone September 19, 1989 Oligomers and Blends Lubowitz et al. 4,871,475 Method for Making October 3, 1989 Polysulfone and Polyethersulfone Oligomers Lubowitz et al. 4,876,328 Polyamide Oligomers October 24, 1989 Lubowitz et al. 4,935,523 Crosslinking June 19, 1990 Imidophenylamines Lubowitz et al. 4,958,031 Crosslinking September 18, 1990 Nitromonomers Lubowitz et al. 4,965,336 High Performance October 23, 1990 Heterocycle Oligomers and Blends Lubowitz et al. 4,980,481 Pyrimidine-Based December 25, 1990 End-Cap Monomers and Oligomers Lubowitz et al. 4,981,922 Blended Etherimide January 1, 1991 Oligomers Lubowitz et al. 4,985,568 Method of Making January 15, 1991 Crosslinking Imidophenyl-amines Lubowitz et al. 4,990,624 Intermediate February 5, 1991 Anhydrides Useful for Synthesizing Etherimides Lubowitz et al. 5,011,905 Polyimide Oligomers April 30, 1991 and Blends Lubowitz et al. 5,066,541 Multidimensional November 19, 1991 Heterocycle Sulfone Oligomers Lubowitz et al. 5,071,941 Multidimensional December 10, 1991 Ether Sulfone Oligomers Lubowitz et al. 5,175,233 Multidimensional December 29, 1992 Ester or Ether Oligomers with Pyrimidinyl End Caps Lubowitz et al. 5,082,905 Blended Heterocycles January 21, 1992 Lubowitz et al. 5,087,701 Phthalimide Acid February 11, 1992 Halides Lubowitz et al. 5,104,967 Amideimide Oli- April 14, 1992 gomers and Blends Lubowitz et al. 5,109,105 Polyamides April 28, 1992 Lubowitz et al. 5,112,939 Oligomers Having May 12, 1992 Pyrimidinyl End Caps Lubowitz et al. 5,115,087 Coreactive Imido May 19, 1992 Oligomer Blends Lubowitz et al. 5,116,935 High Performance May 26, 1992 Modified Cyanate Oligomers and Blends Lubowitz et al. 5,120,819 High Performance June 9, 1992 Heterocycles Lubowitz et al. 5,126,410 Advanced Heterocycle June 30, 1992 Oligomers Lubowitz et al. 5,144,000 Method for Forming September 1, 1992 Crosslinking Oligomers Lubowitz et al. 5,151,487 Method of Preparing a September 29, 1992 Crosslinking Oligomer Lubowitz et al. 5,155,206 Amideimide Oli- October 13, 1992 gomers, Blends and Sizings for Carbon Fiber Composites Lubowitz et al. 5,159,055 Coreactive Oligomer October 27, 1992 Blends Lubowitz et al. 5,175,234 Lightly-Crosslinked December 29, 1992 Polyimides Lubowitz et al. 5,175,304 Halo- or Nitro- December 29, 1992 Intermediates Useful for Synthesizing Etherimides Lubowitz et al. 5,198,526 Heterocycle Oligomers March 30, 1993 with Multidimensional Morphology Lubowitz et al. 5,210,213 Multidimensional May 11, 1993 Crosslinkable Oligomers Lubowitz et al. 5,216,117 Amideimide Blends June 1, 1993 Lubowitz et al. 5,227,461 Extended Difunctional July 13, 1993 End-Cap Monomers Lubowitz et al. 5,239,046 Amideimide Sizing August 24, 1993 For Carbon Fiber Lubowitz et al. 5,268,519 Lightly Crosslinked December 7, 1993 Etherimide Oligomers Lubowitz et al. 5,286,811 Blended Polyimide February 15, 1994 Oligomers and Method of Curing Polyimides Lubowitz et al. 5,344,894 Polyimide Oligomers September 6, 1994 and Blends ______________________________________
The heterocycles (i.e., oxazoles, thiazoles, or imidazoles) use a processing principle more akin to the AVIMIDs than the phenoxyphenyl sulfone solubility principle of our other resins. The heterocycles have poor solubility, even with our "sulfone" chemistries, but they at least form liquid crystals or soluble crystals in strong acids. To produce non-crystalline (amorphous) composites, we capitalize on the ability of our heterocycles to melt at the same temperature range as the cure and promote crosslinking in the melt. With relatively low molecular weight, capped, heterocycle oligomers, we can autoclave process these materials. Autoclave processing is a significant achievement for these heterocycles which present to the industry, perhaps, the most challenging problems. The polybenzoxazoles we produced, in addition, are useful at temperatures up to about 750-775.degree. F. (400-413.degree. C.), since they have glass transition temperatures of about 840.degree. F. (450.degree. C.). We describe multifunctional heterocycle and heterocycle sulfones in copending U.S. patent application Ser. No. 08/327,180, which we incorporate by reference.
Some aerospace applications need composites which have even higher use temperatures than these polybenzoxazoles while maintaining toughness, solvent resistance, ease of processing, formability, strength, and impact resistance. Southcott et al. discuss the problems of the prior art imide systems and the advantages of our soluble monofunctional and difunctional nadic-capped imide systems in the article: Southcott et al., "The development of processable, fully imidized, polyimides for high-temperature applications," 6 High Perform. Polym., 1-12 (U.K. 1994). For these extremely demanding requirements, our multidimensional oligomers (i.e., oligomers that have three or more arms extending from a central organic hub to yield three-dimensional morphology) have superior processing parameters over more conventional, linear oligomers that might produce composites having these high thermal stabilities. Our multidimensional oligomers can satisfy the thermal stability requirements and can be processed at significantly lower temperatures. Upon curing the end caps, the multidimensional oligomers crosslink so that the thermal resistance of the resulting composite is markedly increased with only a minor loss of stiffness, matrix stress transfer (impact resistance), toughness, elasticity, and other mechanical properties. We can achieve glass transition temperatures above 950.degree. F. (510.degree. C.) with composites cured from our difunctional multidimensional oligomers (which we call "star-burst" oligomers). Of course, a full range of use temperatures are possible by selecting the hubs (which usually is an aromatic moiety), the backbone monomers used in the arms, end caps, and number of crosslinking functionalities per cap.
We now believe we can achieve even better properties in advanced composites by including an even higher number of crosslinking functionalities than the mono- or difunctional systems of the linear or multidimensional resins discussed in our earlier patents or patent applications. The higher density of crosslinks provide redundancy at those locations in the macromolecular, cured composite which are most susceptible to thermal degradation.

Oligomers with multiple chemically functional end caps


A silver halide photographic material is disclosed. The photographic material comprises a support having coated thereon one or more hydrophilic colloidal layers, at least one of the hydrophilic colloidal layers being a negative working silver halide emulsion layer, wherein said silver halide emulsion layer or another hydrophilic colloidal layer contains a water-soluble polymer or copolymer having a quaternary ammonium salt in a repeating unit. The process for forming a high contrast negative image using the photographic material is also disclosed.

Since in a step of photomechanical process, the formation of sharp dot images or line images is required, an image-forming system requires very high contrast photographic characteristics (in particular, gamma of at least 10). Hitherto, for this purpose, a process of processing a lith-type silver halide photographic material comprising a silver chlorobromide emulsion having a silver chloride content of over 50 mol %, and more preferably over 70 mol % with a specific developer called a "lithographic developer" containing only hydroquinone as a developing agent and having a very low free sulfite ion concentration (usually not more than 0.1 mol/liter) has been used. However, since for a lith-type silver halide photographic emulsion, a silver chlorobromide having a high silver chloride content must be used, it is difficult to attain a high sensitivity.
As other processes of obtaining high contrast negative images, there are processes using specific hydrazine derivatives disclosed in U.S. Pat. Nos. 4,168,977, 4,224,401, 4,241,164, 4,269,929, 4,311,781, 4,650,746, etc. According to these processes, by processing a surface latent image-type silver halide photographic material containing a specific hydrazine derivative (generally, an acylphenylhydrazine derivative) as a nucleating agent with a developer having pH of from 11.0 to 12.3, photographic characteristics having a superhigh contrast of gamma over 10 and a high sensitivity are obtained. Since in these processes, a silver bromide emulsion or a silver chlorobromide emulsion having a high silver bromide content can be used, a high sensitivity can be achieved as compared to the case of using lith-type silver halide emulsion.
However, it has been found that the foregoing high contrast image-forming system using the hydrazine derivative has various defects. That is, when the foregoing image-forming system is used, high contrast negative images are obtained but, at the same time, it is accompanied by the formation of pepper (black pepper), which becomes a large problem for a photomechanical process. The pepper in photography means black sesame-like spots occurring in an unexposed area, for example, an area to become an undeveloped area between a dot and a dot. The appearance of the pepper causes the trouble of greatly reducing the commercial value as a photographic light-sensitive material for a photomechanical process. Accordingly, various efforts have been made for the development of a pepper restraining technique, but the improvement in pepper susceptibility is frequently accompanied by the reduction of the sensitivity and gamma. Hence, it has been strongly desired to develop an image forming system capable of obtaining a high sensitivity and high contrast without being accompanied by the appearance of the pepper.
On the other hand, for the purpose of improving photographic performance, the technique of incorporating a certain kind of a quaternary ammonium salt polymer in a silver halide photographic material as a silver halide photographic element is disclosed in JP-A-62-9346, JP-A-62-215949, JP-A-63-13034, JP-A-1-280749, JP-A-2-144533, JP-A-2-308160, JP-A-3-36544, and JP-A-3-38637 (the term "JP-A" as used herein means an "unexamined published Japanese patent application"), European Patents 320,692 and 398,223, U.S. Pat. No. 4,914,018, etc., for static prevention, in JP-A-63-249839, JP-A-2-212834, etc., for the improvement of tone, in JP-A-2-68545 and European Patent 306,246, etc., for the improvement of image sharpness, in JP-B-55-39821 (the term "JP-B" as used herein means an "examined Japanese patent publication"), JP-A-60-122942, JP-A-3-27036, etc., for the improvement of photographic characteristics, and in JP-A-1-150130 for the improvement of the performance and the physical properties.
However, it has never been known that by adding a quaternary ammonium salt polymer to silver halide photographic material and processing the photographic material with a specific developer, the sensitivity is greatly increased and also the contrast is greatly increased, whereby negative images of a very high contrast over 10 in gamma can be obtained.


A toggle switch construction operating in conjunction with a conventional coin slide acceptor to produce a single signal each time the coin slide is reciprocated. The switch is mounted upon a support member, which, in turn, is mounted for limited pivotal movement during actuation by a member on the coin slide such that the member, after actuating the switch passes over the toggle. On the return movement of the coin slide to its initial position, the toggle is re-engaged and returned to its initial position, following which the member again passes over the toggle. The toggle can therefore be cycled only once for each reciprocation of the coin slide.

This invention relates generally to the field of coin operated machines which either vend an article of merchandise or provide a service in response to the depositing of coins by a user using a so-called coin slide, and more particularly to an improved switch construction which is operated in response to the reciprocating movement of such coin slides.
Particularly in the case of commercially operated laundrying machines including both washers and dryers which commence a cycle of operation with the completion of one or more such coin slide reciprocations, it has become increasingly necessary to monitor each such reciprocation either electronically or electro-mechanically to prevent the fraudulant use of the machine by obtaining more cyles of operations than had been paid for. Such use is often possible for the reason that the inner end of the coin slide commonly used to initiate a cycle by a mechanical tripping function has in large measure been replaced by using the same movement to close an electrical switch connected to the electronic circuitry which controls the operation of the machine. Most coin slide mechanisms are provided with a ratchet and pawl which comes into operation when the coin slide has been advanced sufficiently to allow deposited coins to fall into a coin receiving receptacle, the ratchet usually having teeth engaged by the pawl which are spaced a distance of approximately one-quarter of an inch. The last tooth on the ratchet is in engagement when the inner end of the coin slide is in position to close a switch which provides a signal input to the circuitry which, inter alia commences an operational cycle, after which the ratchet disengages to permit return of the slide under the action of a spring to its original position.
However, while the last tooth on the ratchet is engaged, it is possible to move the slide over a short path of travel approximating one-quarter inch without disengaging the ratchet, and if the slide is advanced cautiously, it is possible for an unscrupulous user to trip the initiating switch without releasing the last tooth on the ratchet. A cycle can then be initiated on a repeating basis by merely allowing the slide to remain engaged with the last tooth on the ratchet during an operational cycle, and commence a new cycle when needed thereafter by merely again advancing the slide only sufficiently to again trip the switch.
This problem is not resolved by providing a toggle type switch in place of a normally open switch wherein the toggle is operated by selective contact with two separate points on the coin slide. If the above procedure is followed, the toggle switch can be allowed to remain in closed condition, and recycling is commenced automatically, the user controlling operations by manually opening and closing the door of the machine.

Switch construction for coin operated machines


The present embodiments provide a system that facilitates lazy register window fills in a processor. During program execution, when the system encounters a restore instruction for a register window, the system determines if the restore instruction causes an underflow condition that requires the register window to be filled from a stack in memory. If so, the system completes the restore instruction by updating state information for the register window to indicate that the restore instruction is complete without actually filling the individual registers that comprise the register window from the stack. During subsequent program execution, the system lazily fills registers in the register window from the stack as the registers are accessed by the program.

Field
The disclosed embodiments generally relate to techniques for improving performance in computer systems. More specifically, the disclosed embodiments relate to a technique for improving the performance of register window operations in computer systems by performing lazy register fills.
Related Art
The use of register windows can greatly improve processor performance by eliminating the need to save and restore registers when a program makes function calls. It does so by providing multiple sets of registers, one set (called a window) for each function call, with adjacent sets being overlapped to enable parameter passing between functions. However, when a processor runs out of windows to allocate to a new function call, it has to spill the contents of one of the register windows to a stack in memory in order to make the register window available for the new function call. This condition is called a "register window overflow." Similarly, when this spilled window of registers subsequently needs to be accessed by the program, the registers have to be restored from the stack. This condition is called a "register window underflow."
In existing processors, register window overflows and underflows are typically handled via software traps (referred to as "spill traps" and "fill traps," respectively). These traps are expensive operations because they typically generate a processor pipeline flush before entering the trap handler and another flush after exiting the trap handler. Also, during a spill trap, all of the registers in the window (e.g., 16 registers in a SPARC™ architecture) are saved to the stack in memory, even though most of them are not live across the function call. Conversely, on a fill trap, all of the registers are restored from the stack even though most of them are not subsequently used. In fact, empirical results show that, for a wide range of applications, typically only four of the 16 registers are used. Moreover, register window underflows typically affect processor performance more than register window overflows. This is because the spill trap handler uses load instructions while the fill trap handler uses store instructions, and it is much more difficult for the processor to hide the latency of load instructions than to hide the latency of store instructions.
Because of the high cost of handling register window overflows and underflows, the use of register windows can degrade processor performance if an application generates too many overflows and underflows.

Register window performance via lazy register fills