Expert-level detection of M-proteins in serum protein electrophoresis using machine learning.

OBJECTIVES: Serum protein electrophoresis (SPE) in combination with immunotyping (IMT) is the diagnostic standard for detecting monoclonal proteins (M-proteins). However, interpretation of SPE and IMT is weakly standardized, time consuming and investigator dependent. Here, we present five machine learning (ML) approaches for automated detection of M-proteins on SPE on an unprecedented large and well-curated data set and compare the performance with that of laboratory experts. METHODS: SPE and IMT were performed in serum samples from 69,722 individuals from Norway. IMT results were used to label the samples as M-protein present (positive, n=4,273) or absent (negative n=65,449). Four feature-based ML algorithms and one convolutional neural network (CNN) were trained on 68,722 randomly selected SPE patterns to detect M-proteins. Algorithm performance was compared to that of an expert group of clinical pathologists and laboratory technicians (n=10) on a test set of 1,000 samples. RESULTS: The random forest classifier showed the best performance (F1-Score 93.2 %, accuracy 99.1 %, sensitivity 89.9 %, specificity 99.8 %, positive predictive value 96.9 %, negative predictive value 99.3 %) and outperformed the experts (F1-Score 61.2 ± 16.0 %, accuracy 89.2 ± 10.2 %, sensitivity 94.3 ± 2.8 %, specificity 88.9 ± 10.9 %, positive predictive value 47.3 ± 16.2 %, negative predictive value 99.5 ± 0.2 %) on the test set. Interestingly the performance of the RFC saturated, the CNN performance increased steadily within our training set (n=68,722). CONCLUSIONS: Feature-based ML systems are capable of automated detection of M-proteins on SPE beyond expert-level and show potential for use in the clinical laboratory.

Expression of combinatorial immunoglobulins in macrophages in the tumor microenvironment.

Recent evidence indicates the presence of macrophage subpopulations that express the TCRαß in chronic inflammatory diseases such as tuberculosis and atherosclerosis and in the tumor microenvironment. Here, we demonstrate that a second subpopulation of macrophages expresses rearranged heavy and light chain immunoglobulins. We identify immunoglobulin expression in human and murine monocytes, in ex vivo differentiated macrophages and macrophages from the tumor microenvironment of five randomly selected distinct human tumor entities. The immunoglobulin heavy and light chains are expressed in a small macrophage subfraction (~3-5%) as combinatorial and individual-specific immune receptors. Using Sanger sequencing and deep sequencing, we routinely find markedly restricted Ig repertoires in monocytes/macrophages compared to normal B cells. Furthermore, we report the complete Ig heavy and light chain sequences of a fully functional immunoglobulin from a single tumor-associated macrophage. These results demonstrate that Ig expression is a defining feature of monocytes and also macrophages in the tumor microenvironment and thus reveal an as yet unrecognized modus operandi of host defense in professional phagocytes.

A combinatorial αß T cell receptor expressed by macrophages in the tumor microenvironment.

Recent evidence indicates the presence of macrophage subpopulations that express the TCRαß in two major inflammatory diseases, tuberculosis and atherosclerosis. Inflammation is also a well-established attribute of cancer progression and macrophages are one of the major immune cells that infiltrate tumors. Here, we demonstrate that the macrophage-TCRαß is expressed in the tumor microenvironment of human and murine malignancies. We identify TCRαß+ macrophages in each case of four randomly selected distinct human tumor entities. In human tumor tissues, the TCRαß expressed by macrophages in the tumor microenvironment is a combinatorial and individual-specific immune receptor. Furthermore, we routinely find TCRαß+ macrophage subpopulations in experimental tumors (TS/A, mammary adenocarcinoma) which we induced both in normal mice and mice deficient in the macrophage receptor stabilin-1. Expression of the combinatorial murine tumor macrophage TCRαß is individual-specific and independent of stabilin-1. These results demonstrate that TCRαß expression is a characteristic feature of macrophages in the tumor microenvironment and identify an as yet unrecognized flexible element in the macrophage-based host response to tumors.

The macrophage-TCRαß is a cholesterol-responsive combinatorial immune receptor and implicated in atherosclerosis.

Recent evidence indicates constitutive expression of a recombinatorial TCRαß immune receptor in mammalian monocytes and macrophages. Here, we demonstrate in vitro that macrophage-TCRß repertoires are modulated by atherogenic low density cholesterol (LDL) and high-density cholesterol (HDL). In vivo, analysis of freshly obtained artery specimens from patients with severe carotid atherosclerosis reveals massive abundance of TCRαß(+) macrophages within the atherosclerotic lesions. Experimental atherosclerosis in mouse carotids induces accumulation of TCR bearing macrophages in the vascular wall and TCR deficient rag(-/-) mice have an altered macrophage-dependent inflammatory response. We find that the majority of TCRαß bearing macrophages are localized in the hot spot regions of the atherosclerotic lesions. Advanced carotid artery lesions express highly restricted TCRαß repertoires that are characterized by a striking usage of the Vß22 and Vß16 chains. This together with a significant degree of interindividual lesion repertoire sharing suggests the existence of atherosclerosis-associated TCRαß signatures. Our results implicate the macrophage-TCRαß combinatorial immunoreceptor in atherosclerosis and thus identify an as yet unknown adaptive component in the innate response-to-injury process that underlies this macrophage-driven disease.

A second combinatorial immune receptor in monocytes/macrophages is based on the TCRγδ.

Recent evidence indicates that monocytes and macrophages express T cell receptor (TCR)αß-like combinatorial immune receptors. Here, we demonstrate the presence of a second recombinatorial immunoreceptor, which is structurally based on the TCR γ- and δ-chains, in human and murine monocytes and differentially activated macrophages (referred to here as TCRL(m)γδ). In vitro, infection of macrophages with mycobacteria and gram positive or gram negative bacteria induced expression of donor-specific and differential TCRL(m)Vδ repertoires indicating that the novel immunoreceptor represents a dynamic flexible host defense system that responds to bacterial challenge. In vivo, we find that TCRL(m)γδ bearing macrophages, which express highly restricted repertoires of the antigen-binding Vδ chain, accumulate in the cerebrospinal fluid in acute bacterial meningitis and in advanced lesions of atherosclerosis. These results identify an as yet unrecognized monocyte/macrophage subpopulation that bears combinatorial TCRL(m)γδ immune receptors, and is associated with both acute and chronic inflammatory diseases. Moreover, they indicate that the monocytic lineage uses the same bipartite system of TCRαß/TCRγδ-based combinatorial immune receptors that is present in T cells. Our findings suggest specific roles of monocytes/macrophages in various inflammatory conditions and lend further evidence that flexible immune recognition in higher vertebrates operates on a broader cellular basis than previously thought.

On the horizon: flexible immune recognition outside lymphocytes.

Since decades there is consensus among immunologists that in jawless and jawed vertebrates flexible immune recognition is strictly confined to the lymphoid lineage. In jawed vertebrates the adaptive immune system is represented by two lineages of lymphocytes, B cells and T cells that express recombinatorial antigen receptors of enormous diversity known as immunoglobulins and the T cell receptor (TCR). The recent identification of recombined immune receptors that are structurally based on the TCR in subpopulations of neutrophils and eosinophils (referred to here as TCR-like immunoreceptors, "TCRL") provides unexpected evidence for the existence of flexible host defense mechanisms beyond the realm of lymphocytes. Consistent with this, subpopulations of monocytes and macrophages from humans and mice now have also been shown to constitutively express recombined TCR-like immunoreceptors. Available in vitro evidence suggests that the TCRL in macrophages may exert functions as facilitators of phagocytosis and self-recruitment. More importantly, our recent findings that the macrophage-TCRL is implicated in granuloma formation in tuberculosis and the neutrophil-TCRL is associated with autoimmune hemolytic anemia establish for the first time a link between myeloid recombinatorial immune receptors and clinical disease. The discovery of recombined TCR-like immune receptors in granulocytes and macrophages extends the principle of combinatorial immune recognition to phagocytic cells. Conceptually, this unifies the two hitherto disparate cardinal features of innate and adaptive immunity, phagocytic capacity and recombinatorial immune recognition on a common cellular platform. Moreover, it strongly suggests that flexible host defense in vertebrates may operate on a broader cellular basis than currently thought.

A TNF-regulated recombinatorial macrophage immune receptor implicated in granuloma formation in tuberculosis.

Macrophages play a central role in host defense against mycobacterial infection and anti- TNF therapy is associated with granuloma disorganization and reactivation of tuberculosis in humans. Here, we provide evidence for the presence of a T cell receptor (TCR) αß based recombinatorial immune receptor in subpopulations of human and mouse monocytes and macrophages. In vitro, we find that the macrophage-TCRαß induces the release of CCL2 and modulates phagocytosis. TNF blockade suppresses macrophage-TCRαß expression. Infection of macrophages from healthy individuals with mycobacteria triggers formation of clusters that express restricted TCR Vß repertoires. In vivo, TCRαß bearing macrophages abundantly accumulate at the inner host-pathogen contact zone of caseous granulomas from patients with lung tuberculosis. In chimeric mouse models, deletion of the variable macrophage-TCRαß or TNF is associated with structurally compromised granulomas of pulmonary tuberculosis even in the presence of intact T cells. These results uncover a TNF-regulated recombinatorial immune receptor in monocytes/macrophages and demonstrate its implication in granuloma formation in tuberculosis.

Comparison of two different methods for CA19-9 antigen determination.

BACKGROUND: This study was designed to investigate the clinical performance of the Access GI Monitor (Beckman Coulter) on the UniCel DxI 800, a method for CA19-9 antigen determination, and to compare with CA19-9 assay on the AxSYM system (Abbott). METHODS: 1,063 serum samples from unselected patients with different underlying diagnoses were tested with both methods. Passing-Bablok regression analysis and Bland Altman analysis was performed. In addition, using ROC analysis, the distribution of Access GI Monitor and AxSYM CA19-9 antigen levels was tested in patients with pancreatic cancer (n = 50), acute inflammatory disease (n = 20), and with chronic inflammation of the pancreatic gland (n = 18). Furthermore, four patients with pancreatic cancer were monitored individually in their courses of the disease (before, during, and after therapeutic procedures) to compare their CA19-9 values with regard to inter-method concordance. RESULTS: Passing-Bablok analysis showed a systematic difference with R = 0.93, slope 0.75, and intercept -1.0. Bland Altman analysis showed a wide scatter of relative differences between both methods, especially in the low end measuring range. In the selected group of patients with pancreatic diseases the analysis of concordance revealed 95.5 % agreement between both methods with a comparable area under the ROC curves (0.73 vs. 0.76). A clear concordance was found for all four selected patients. CONCLUSIONS: Although we found significant systematic measuring variations in the global analysis, the two different automated methods for the quantitative determination of CA19-9 antigen were comparable with respect to their clinical accuracy and applicability to support decision making in the management of pancreatic cancer.

Prevalence of OPG and IL-1 gene polymorphisms in chronic periodontitis.

AIM: To investigate the association of polymorphisms in the osteoprotegerin (OPG) and interleukin 1 (IL-1) genes with chronic periodontitis (CP). MATERIAL AND METHODS: One hundred and ninety-four individuals (97 CP patients, 97 controls) were genotyped for the OPG polymorphisms Lys3Asn and Met256Val and for the IL-1 polymorphisms IL-1A (-889C/T) and IL-1B (+3953C/T). RESULTS: The homozygous variants coding for Lys3 were present at a higher frequency, whereas Asn3 and Met256 were present at a lower frequency in CP patients/controls (Lys3: 31%/25%, Asn3: 23%/32% and Met256: 66%/73%). Heterozygosity for Lys3Asn was observed at a higher frequency in CP patients/controls (46%/43%). Homozygosity for the Val256 genotype was observed in two CP patients (one in controls). Met256Val heterozygosity was more prevalent in CP patients/controls (32%/20%). All differences were statistically not significant between CP patients and controls. In contrast, both IL-1 polymorphisms were statistically significant. The heterozygous variant for IL-1A was present in 32% of the CP patients and in 20% of the controls (homozygosity (patients/controls) CC: 10%/21% and TT: 55%/33%). Heterozygosity for IL-1B was observed in 37% of the CP patients versus 34% in the controls (homozygosity (patients/controls) CC: 26%/57% and TT: 37%/9%). CONCLUSION: While the association between the IL-1 polymorphisms and CP was confirmed, no association between the OPG polymorphisms and CP could be found.

Homozygosity for the 168His variant of the minor histocompatibility antigen HA-1 is associated with reduced risk of primary Sjögren's syndrome.

The genes for the human ATP-binding cassette (ABC) transporter ABCA7 and the minor histocompatibility antigen HA-1 are juxtaposed in close proximity on chromosome 19p13.3. The multispan transmembrane protein ABCA7 contains an extracellular domain that is recognized by antisera from patients with Sjögren's syndrome ("Sjögren-epitope"). Recent work from our laboratory demonstrating the involvement of ABCA7 in cellular ceramide and phosphatidylserine export suggests a role for this transporter in programmed cell death. In HA-1, a protein of unknown function, a His/Arg polymorphism (His168Arg), which constitutes the immunologic target for HA-1-specific cytotoxic T cells, has been causatively linked to graft-versus-host disease after allogeneic stem cell transplantation. Because these findings suggest a potential implication of ABCA7 and HA-1 in immune processes, we tested the hypothesis that allelic variants in both genes are associated with autoimmune disorders. We identified a total of 31 exonic single-nucleotide polymorphisms (SNP) in the ABCA7/HA-1 gene complex, nine of which represent non-synonymous nucleotide alterations. Genotypes of ABCA7 and HA-1 SNP were determined in three distinct Caucasian populations of patients with primary Sjögren's syndrome and ethnically matched controls. Comparison of allele frequencies between these groups revealed that the incidence of the HA-1 168His allele is significantly lower in Sjögren's syndrome patients than in controls (p<0.003). In contrast, the frequencies of all ABCA7 allelic variants and additional HA-1 polymorphisms were similar in patients and controls. In cohorts of patients with systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis, no significant differences in the frequencies of ABCA7 and HA-1 allelic variants were observed relative to controls. Our results suggest that the HA-1 168His variant is associated with reduced susceptibility to primary Sjögren's syndrome.

Adenosine triphosphate binding cassette (ABC) transporters are expressed and regulated during terminal keratinocyte differentiation: a potential role for ABCA7 in epidermal lipid reorganization.

Central aspects of the cellular lipid trafficking mechanisms that occur during keratinocyte differentiation are still not well understood. In the past years, evidence has accumulated to suggest that members of the superfamily of adenosine triphosphate binding cassette (ABC) transporters are critically involved in the transmembrane transport of cellular lipids. To test the hypothesis that ABC molecules are potentially involved in the epidermal transport of sphingolipids, glycerophospholipids, cholesterol, and fatty acids, we performed mRNA expression profiling of all currently known ABC molecules during in vitro differentiation of human keratinocytes and HaCaT cells. We identified six ABC molecules that displayed significant regulation during differentiation of these cells. The recently cloned transporter ABCA7 was highly expressed in keratinocytes and HaCaT cells and upregulated during differentiation. Overexpression of ABCA7 in HeLa cells resulted in increased expression of intracellular and cell surface ceramide and elevated intracellular phosphatidylserine levels. Given the observation that during terminal keratinocyte differentiation intracellular and surface ceramide levels are increased, our results render ABCA7 a candidate regulator of ceramide transport in this process. In addition to ABCA7, the cholesterol transporters ABCB1 and ABCG1 and the glutathione/glucuronide sulfate transporters ABCC1, ABCC3, and ABCC4, were strongly upregulated during keratinocyte and HaCaT cell differentiation. These findings support the notion that ABCB1 and ABCG1 are potentially implicated in cholesterol transport, whereas ABCC1, ABCC3, and ABCC4 are candidate regulators of the translocation of sulfated lipids during stratum corneum keratinization. Our results suggest specific biologic functions for members of the ABC transporter family in epidermal lipid reorganization during terminal keratinocyte differentiation.

ABCA10, a novel cholesterol-regulated ABCA6-like ABC transporter.

We recently identified several novel members of the A subclass of ABC transporters. In this study, we report the cloning of an additional ABC A subfamily transporter, denoted ABCA10, from macrophages. The coding sequence of ABCA10 is of 4.6 kb size and codes for a 1543-amino acid protein that bears the structural features of a full-size ABC transporter. Intriguingly, ABCA10 contains a PEST sequence downstream of the N-terminal transmembrane domain which may be potentially involved in the control of its turnover rate by proteasomal degradation. Several distinct ABCA10 transcripts are expressed in human macrophages that predict the existence of various truncated forms of the novel transporter. Moreover, we identified seven single nucleotide polymorphisms in ABCA10 transcripts. ABCA10 displays high amino acid sequence homology with ABCA6 (63%), ABCA8 (62%), and ABCA9 (63%), respectively, known members of the subgroup of ABCA6-like transporters. Like other transporters of this subfamily, ABCA10 mRNA is ubiquitously expressed and highest gene expression levels are detectable in heart, brain, and the gastrointestinal tract. Analysis of the gene structure demonstrated that the ABCA10 gene consists of 40 exons that extend across a genomic region of approximately 97kb size (Chr. 17q24.3). ABCA10 mRNA is expressed in similar quantities in monocytes and M-CSF differentiated macrophages. Importantly, ABCA10 expression is suppressed by cholesterol import into macrophages, indicating that it is a cholesterol-responsive gene. Our results identify ABCA10 as a novel member of the group of ABCA6-like transporters and suggest its involvement in macrophage lipid homeostasis.

Blockade of platelet-derived growth factor or its receptors transiently delays but does not prevent fibrous cap formation in ApoE null mice.

Platelet-derived growth factor (PDGF) is a potent stimulant of smooth muscle cell migration and proliferation in culture. To test the role of PDGF in the accumulation of smooth muscle cells in vivo, we evaluated ApoE -/- mice that develop complex lesions of atherosclerosis. Fetal liver cells from PDGF-B-deficient embryos were used to replace the circulating cells of lethally irradiated ApoE -/- mice. One month after transplant, all monocytes in PDGF-B -/- chimeras are of donor origin (lack PDGF), and no PDGF-BB is detected in circulating platelets, primary sources of PDGF in lesions. Although lesion volumes are comparable in the PDGF-B +/+ and -/- chimeras at 35 weeks, lesions in PDGF-B -/- chimeras contain mostly macrophages, appear less mature, and have a reduced frequency of fibrous cap formation as compared with PDGF-B +/+ chimeras. However, after 45 weeks, smooth muscle cell accumulation in fibrous caps is indistinguishable in the two groups. Comparison of elicited peritoneal macrophages by RNase protection assay shows an altered cytokine and cytokine receptor profile in PDGF-B -/- chimeras. ApoE -/- mice were also treated for up to 50 weeks with a PDGF receptor antagonist that blocks all three PDGF receptor dimers. Blockade of the PDGF receptors similarly delays, but does not prevent, accumulation of smooth muscle and fibrous cap formation. Thus, elimination of PDGF-B from circulating cells or blockade of PDGF receptors does not appear sufficient to prevent smooth muscle accumulation in advanced lesions of atherosclerosis.

Molecular structure of a novel cholesterol-responsive A subclass ABC transporter, ABCA9.

We recently identified a novel ABC A subclass transporter, ABCA6, in human macrophages. Here, we report the molecular cloning of an additional ABC A subfamily transporter from macrophages denoted ABCA9. The identified coding sequence is 4.9 kb in size and codes for a 1624 amino acid protein product. In accordance with the proposed nomenclature, the novel transporter was designated ABCA9. The putative full-length ABC transporter polypeptide consists of two transmembrane domains and two nucleotide binding folds and thus conforms to the group of full-size ABC transporters. We identified alternative ABCA9 mRNA variants in human macrophages that predict the existence of three truncated forms of the novel transporter. Among the human ABC A subfamily transporters, ABCA9 exhibits the highest amino acid sequence homology with ABCA8 (72%) and ABCA6 (60%), respectively. The striking amino acid sequence similarity between these transporter molecules supports the notion that they represent an evolutionary more recently emerged subgroup within the family of ABC A transporters, which we refer to as "ABCA6-like transporters." ABCA9 mRNA is ubiquitously expressed with the highest mRNA levels in heart, brain, and fetal tissues. Analysis of the genomic structure revealed that the ABCA9 gene consists of 39 exons that are located within a genomic region of approximately 85 kb size on chromosome 17q24.2. In human macrophages, ABCA9 mRNA is induced during monocyte differentiation into macrophages and suppressed by cholesterol import indicating that ABCA9, like other known ABC A subfamily transporters, is a cholesterol-responsive gene. Based on this information, ABCA9 is likely involved in monocyte differentiation and macrophage lipid homeostasis.

Leukocyte ABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues.

The ATP-binding cassette transporter 1 (ABCA1) has recently been identified as a key regulator of high-density lipoprotein (HDL) metabolism, which is defective in familial HDL-deficiency syndromes such as Tangier disease. ABCA1 functions as a facilitator of cellular cholesterol and phospholipid efflux, and its expression is induced during cholesterol uptake in macrophages. To assess the role of macrophage ABCA1 in atherosclerosis, we generated low-density lipoprotein (LDL) receptor knockout (LDLr(-/-)) mice that are selectively deficient in leukocyte ABCA1 (ABCA1(-/-)) by using bone marrow transfer (ABCA1(-/-) --> LDLr(-/-)). Here we demonstrate that ABCA1(-/-) --> LDLr(-/-) chimeras develop significantly larger and more advanced atherosclerotic lesions compared with chimeric LDLr(-/-) mice with functional ABCA1 in hematopoietic cells. Targeted disruption of leukocyte ABCA1 function did not affect plasma HDL cholesterol levels. The amount of macrophages in liver and spleen and peripheral blood leukocyte counts is increased in the ABCA1(-/-) --> LDLr(-/-) chimeras. Our results provide evidence that leukocyte ABCA1 plays a critical role in the protection against atherosclerosis, and we identify ABCA1 as a leukocyte factor that controls the recruitment of inflammatory cells.

ATP-binding cassette (ABC) transporters in atherosclerosis.

Macrophages play a central role in the initiation and progression of atherosclerotic lesions. In the nascent lesion, macrophages transform into foam cells through the excessive accumulation of cholesteryl esters. Dysfunctional lipid homeostasis in macrophages and foam cells ultimately results in the breakdown of membrane integrity and cell death. Studies within the past 2 years have implicated a defined subset of multispan transmembrane proteins, the ATP-binding cassette (ABC) transporters, in macrophage lipid homeostasis. The recent finding that ABCA1, beyond its function as a major regulator of plasma high-density lipoprotein metabolism, exerts significant antiatherosclerotic activities has provided the first direct evidence for the role of an ABC transporter in the pathogenesis of atherosclerosis.

Identification of sterol-independent regulatory elements in the human ATP-binding cassette transporter A1 promoter: role of Sp1/3, E-box binding factors, and an oncostatin M-responsive element.

The ATP-binding cassette transporter A1 (ABCA1) shows a differentiation-, cAMP-, and sterol-dependent up-regulation in human monocytes. As part of an ongoing study, we investigated the proximal promoter regions that are highly conserved between the human and murine ABCA1 genes. Using reporter gene assays, we show here that a TATA box 24 bp upstream of the transcription initiation site is essential for promoter activity in RAW 264.7 and HepG2 cells, whereas further enhancement of transcriptional activity is mediated by the -175 bp promoter region. Gel shift assays revealed in vitro binding of Sp1 to a -91 GnC motif as well as binding of Sp1 and Sp3 to a -157 GnC promoter region. In co-transfection experiments using Drosophila S2 cells, we demonstrate that Sp3 competes with Sp1 for binding to the -157 GnC motif and acts as a repressor. On the other hand, overexpression of Sp1 increased ABCA1 mRNA expression in HeLa cells and enhanced cellular cholesterol and phospholipid efflux in RAW 246.7 macrophages. We also show here that the conserved E-box at position -140 binds upstream stimulatory factors 1 and 2 and hepatic nuclear factor 1alpha and that mutagenesis of the E-box enhanced constitutive ABCA1 expression in RAW 264.7 cells, implying a role for this element in silencing ABCA1 expression. Besides the functional importance for basal gene expression, we have identified that the core promoter region (-175 to +224) is also responsible for the induction of ABCA1 by the cytokine oncostatin M, resulting in a rapid increase in ABCA1 mRNA levels in HepG2 cells. Interestingly, this oncostatin M-induced expression is not dependent on the currently known sequence motifs in the ABCA1 promoter. In conclusion, a functional complex of cis-elements within the proximal human ABCA1 promoter associated with the transcription factors Sp1/3, upstream stimulatory factors 1 and 2, and hepatic nuclear factor 1alpha has been characterized, which allows a subtle tissue-specific regulation of ABCA1 gene expression.