A Targeted Mass Spectrometry Strategy for Developing Proteomic Biomarkers: A Case Study of Epithelial Ovarian Cancer.

Protein biomarkers for epithelial ovarian cancer are critical for the early detection of the cancer to improve patient prognosis and for the clinical management of the disease to monitor treatment response and to detect recurrences. Unfortunately, the discovery of protein biomarkers is hampered by the limited availability of reliable and sensitive assays needed for the reproducible quantification of proteins in complex biological matrices such as blood plasma. In recent years, targeted mass spectrometry, exemplified by selected reaction monitoring (SRM) has emerged as a method, capable of overcoming this limitation. Here, we present a comprehensive SRM-based strategy for developing plasma-based protein biomarkers for epithelial ovarian cancer and illustrate how the SRM platform, when combined with rigorous experimental design and statistical analysis, can result in detection of predictive analytes.Our biomarker development strategy first involved a discovery-driven proteomic effort to derive potential N-glycoprotein biomarker candidates for plasma-based detection of human ovarian cancer from a genetically engineered mouse model of endometrioid ovarian cancer, which accurately recapitulates the human disease. Next, 65 candidate markers selected from proteins of different abundance in the discovery dataset were reproducibly quantified with SRM assays across a large cohort of over 200 plasma samples from ovarian cancer patients and healthy controls. Finally, these measurements were used to derive a 5-protein signature for distinguishing individuals with epithelial ovarian cancer from healthy controls. The sensitivity of the candidate biomarker signature in combination with CA125 ELISA-based measurements currently used in clinic, exceeded that of CA125 ELISA-based measurements alone. The SRM-based strategy in this study is broadly applicable. It can be used in any study that requires accurate and reproducible quantification of selected proteins in a high-throughput and multiplexed fashion.

The circulating immunoglobulins negatively impact on the parasite clearance in the liver of Leishmania donovani-infected mice via dampening ROS activity.

Visceral leishmaniasis, the most severe form of leishmaniasis, is caused by Leishmania donovani and L. infantum. Immunity to Leishmania infection has been shown to depend on the development of Th1 cells; however, the roles of B cells and antibodies during infection remain unclear. In the present study, we showed that AID and µs double-deficient mice (DKO), which have B cells but not circulating immunoglobulins (cIgs), became resistant to L. donovani infection, whereas µs or AID single-deficient mice did not. This resistance in DKO mice occurred in the liver from an early stage of the infection. The depletion of IFN-γ did not affect the rapid reduction of parasite burden, whereas NADPH oxidases was up-regulated in the livers of infected DKO mice. The inhibition of the reactive oxygen species pathway in vivo by apocynin, a NADPH oxidase inhibitor, resulted in a significant increase in the parasite burden in DKO mice. These results indicate that a circulating Ig deficiency induces a protective response against L. donovani infection by elevating IFN-γ-independent NADPH oxidase activity, and also that cIgs play a regulatory role in controlling L. donovani infection in mice.

Identification of Liver Epithelial Cell-derived Ig Expression in µ chain-deficient mice.

Growing evidence indicates that B cells are not the only source of immunoglobulin (Ig). To investigate this discovery further, we used µMT mice, which have a disruption of the first transmembrane exon of the µ heavy chain and do not express the membrane form of IgM. These mice lack mature B cells and thus serve as a good model to explore Ig expression by liver epithelial cells. We found that Ig heavy chains (µ, δ, γ and α) and light chains (κ and λ) were expressed in sorted liver epithelial cells of µMT mice. Surprisingly, each heavy chain class showed its respective variable region sequence characteristics in their variable region, instead of sharing the same VDJ usage, which suggests that class switching does not occur in liver epithelial cells. Moreover, the γ and α chains, but not the µ and δ chains, showed mutations in the variable region, thus indicating that different classes of Ig have different activities. Our findings support the concept that non-B cells, liver epithelial cells here, can produce different classes of Ig.

Test based on subtype-specific µ-capture IgM immunoassay can distinguish between infections of European and Siberian subtypes of tick-borne encephalitis virus.

BACKGROUND: In many European countries (including Finland, Estonia, Latvia and Russia) two subtypes of tick-borne encephalitis virus (TBEV) occur with overlapping geographic distribution yet with apparently different severity and persistence of symptoms. However, it has not usually been possible to distinguish these infections in the laboratory, as TBEV RNA or sequences have rarely been retrieved from patients seeking medical care in the second phase of infection when the neurological symptoms occur, and serological tests have so far not been able to discriminate between the subtype-specific responses. OBJECTIVES: The aim of this study was to assess the applicability of a µ-capture enzyme immunoassay (EIA) based on TBEV prME subviral particles produced in mammalian cells from Semliki-Forest virus replicons (SFV-prME EIA) to distinguish reactivity to European and Siberian strains of TBEV. STUDY DESIGN: Altogether 54 TBEV IgM positive acute human serum samples and 6 positive cerebrospinal fluid (CSF) samples from different regions of Finland were tested in EIA with subtype-specific antigens and TBEV-IgM subtype-specific index ratios were determined. RESULTS: All 30 samples from patients whose transmission had occurred in foci where only Siberian subtype of TBEV is occurring had an index ratio of more than 1.8, whereas all 30 acute TBE samples from an area where only European subtype circulates had an index ratio below 1.5. CONCLUSIONS: We conclude that the assay is a useful tool to distinguish between acute infections of European and Siberian strains of TBEV, and should help in further studies of the clinical outcome of these two subtypes.

Serum immune-related proteins are differentially expressed during hibernation in the American black bear.

Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α2-macroglobulin, complement components C1s and C4, immunoglobulin µ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α2-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears.

[Primary amyloidosis associated with IgD-lambda M-proteinemia].

We describe here a case of primary AL amyloidosis associated with IgD monoclonal gammopathy of undetermined significance. A 73-year-old man was referred to our hospital with suspected multiple myeloma due to renal failure and urinary Bence Jones protein. Although serum electrophoresis revealed IgDlambda monoclonal protein, the bone marrow did not showed plasma cell proliferation. Systemic bone survey disclosed no lytic bone lesions. Because the patient had macroglossia and multiple ecchymosis in the face and neck, primary amyloidosis was suspected. Skin biopsy revealed extensive deposition of amyloid which was positively stained by Congo red dye. A diagnosis of primary AL amyloidosis associated with IgD monoclonal gammopathy was made. The patient was also complicated renal failure that eventually needed hemodialysis. To our knowledge, this is the first report of primary AL amyloidosis associated with IgD monoclonal gammopathy with undetermined significance.

Mu-heavy chain disease associated with systemic amyloidosis.

mu-heavy chain disease (HCD) is very rare, with only 30 cases reported in the literature. We report a patient with mu-HCD associated with systemic amyloidosis. The diagnosis of mu-HCD was based on findings of mu-heavy chain fragments in the serum, Bence Jones proteinuria and vacuolated plasma cells in the bone marrow. To our knowledge, this is the third case in which systemic amyloidosis led to the patient's death.

[Immunochemical properties of free mu-chain protein in a patient with mu-heavy chain disease].

We report a case of mu-heavy chain disease. A 56-year-old woman presented with anemia and hemorrhagic diathesis. The serum of the patient was found to have free mu-heavy chain. The patient also had a kappa type-Bence Jones protein in serum and urine. Immunoelectrophoresis showed an abnormal precipitin line in the alpha 2-globulin region which reacted with antiserum to mu-chain but not with antiserum for light chains. The molecular weight of the monomer of the patient's mu-heavy chain protein was approximately 67,000 daltons less than that of the normal mu-heavy chain protein.

Ouabain as a mammalian hormone.

Endogenous ouabain changes rapidly in humans and dogs upon physical exercise and is under the control of epinephrine and angiotensin II. Hence, the steroid acts as a rapidly acting hormone. A search for a specific binding globulin for cardiac glycosides in bovine plasma resulted in the identification of the d allotype of the micro chain of IgM whose hydrophobic surfaces interact with cardiotonic steroids and cholesterol. Such IgM complexes might be involved in the hepatic elimination of cardiotonic steroids. Thus, differences in the signaling cascade starting at Na(+),K(+)-ATPase must explain any differences in the action of ouabain and digoxin in the genesis of arterial hypertension.

[A case of Sjögren's syndrome with marked lacrimal gland enlargement, atypical onset and IgA-M-proteinemia].

Since July, 1999, a 66 year-old man had been complaining of dry cough. He noticed submandibular swelling, lacrimal gland enlargement and dry eye. Keratoconjuctivitis sicca was detected by an ophthalmologist. Sjögren's syndrome was diagnosed based on microscopic findings of a labial salivary gland biopsy although anti-SS-A and anti-SS-B antibodies were negative. Hypergammaglobulinemia (IgG 3916 mg/dl) and IgA-M-proteinemia were pointed out. Swelling of mediastinal and abdominal lymph nodes was detected together with enlargement of salivary and lacrimal glands. We suspected the existence of malignant lymphoma, but a biopsy of lacrimal glands showed only lymphocytic and plasma cell infiltration and immunohistochemical analysis denied monoclonality of lymphoid line. An administration of corticosteroids caused rapid diminution in size of lacrimal and submandibular glands and lymph nodes. Clinical symptoms were also improved, but IgA-M proteinemia remains. The characteristics of our case were enlargement of lacrimal glands, the negativity of anti SS-A and SS-B antibodies, atypical onset and M-proteinemia. We discussed about these characteristics of Sjögren's syndrome in our case.

Prevention of scrapie pathogenesis by transgenic expression of anti-prion protein antibodies.

Variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy are initiated by extracerebral exposure to prions. Although prion transmission from extracerebral sites to the brain represents a potential target for prophylaxis, attempts at vaccination have been limited by the poor immunogenicity of prion proteins. To circumvent this, we expressed an anti-prion protein (anti-PrP) mu chain in Prnp(o/o) mice. Transgenic mice developed sustained anti-PrP titers, which were not suppressed by introduction of Prnp+ alleles. Transgene expression prevented pathogenesis of prions introduced by intraperitoneal injection in the spleen and brain. Expression of endogenous PrP (PrP(C)) in the spleen and brain was unaffected, suggesting that immunity was responsible for protection. This indicates the feasibility of immunological inhibition of prion disease in vivo.

Ontogeny of the murine Ig joining chain gene and protein.

We used Northern blot analysis in order to investigate the ontogeny of the murine joining (J)-chain gene. No J-chain expression was detected in embryonic tissues, including liver, spleen and intestine, but an expression of mu heavy chain was detected in foetal liver at day 17. J-chain expression was detected in the spleen at day 9 and in the intestine at day 15 after birth. Western blot analysis was carried out in order to compare the protein levels of J and mu heavy chains in serum from day 8 to day 24 after birth, using antihuman J chain and antimouse mu chain antibodies. Although mu chain protein could be detected in serum from day 8, J-chain protein was detectable only at day 24. These results suggest that the expression of J chain is a later event than the mu chain in the mouse, which thus differs in embryogenesis from humans.

Cast nephropathy in mu heavy chain disease.

The occurrence of kidney diseases was very rarely reported in heavy chain diseases (HCD). At variance with gamma and alpha HCD in which there is no free light chain secretion, about two-thirds of mu HCD patients have urinary Bence Jones (BJ) proteins. We report on a 66 year-old man affected with typical mu HCD who developed renal failure after a 3-year follow-up. He had presented with chronic lymphocytic leukemia with bone marrow vacuolated plasma cells, serum mu HCD protein and serum and urine BJ protein. After an apparent hematological remission following fludarabine therapy, anemia and blood hyperlymphocytosis recurred together with microscopic hematuria, proteinuria and increased creatininemia. Kidney biopsy showed numerous tubular eosinophilic casts which stained for kappa chain determinants by immunofluorescence and an interstitial infiltration by lymphocytes and plasma cells. The hematological and renal condition improved after reinitiation of chemotherapy. This appears to be the first documented report of a light chain-dependent visceral complication in HCD.

In vivo depletion of xenoreactive natural antibodies with an anti-mu monoclonal antibody.

Hyperacute rejection of vascularized discordant xenografts, such as pig-to-primate kidney or heart xenotransplants, is thought to be mediated by xenoreactive natural antibodies (XNA) of the IgM isotype and the activation of the classic pathway of complement. Using the guinea pig-to-rat discordant xenograft model, we have developed a potential therapeutic protocol leading to long-term depletion of circulating IgM in adult animals. This protocol consists of the injection into adult LOU/C rats of an antirat IgM MAb (MARM-7) after splenectomy, plasma exchange, and the administration of an anti-B cell immunosuppressant, mycophenylate mofetil (RS61443). Splectomized plasma exchanged adult rats receiving RS61443 showed strongly decreased IgG and IgM serum concentrations for a relatively short period during which these isotypes remained nevertheless detectable by a sensitive ELISA technique. In contrast to IgM, IgG in serum returned, shortly after the end of this treatment, to normal concentrations. Splenectomy alone was able to significantly decrease, for a long period (more than 70 days), IgM but not IgG serum concentrations in these rats. During this treatment, IgM XNA concentration mirrored total IgM. The injection of MARM-7 MAb to adult LOU/C rats was able to deplete circulating IgM and IgM XNA for a period of several weeks during which IgM was undetectable by a sensitive ELISA technique. Depletion time was dose-dependent--the higher the dose of injected MARM-7, the longer the period for which IgM and IgM XNA remained undetectable. Moreover depletion of circulating IgM was correlated with the detection in the serum of these rats of noncomplexed, free MARM-7. Finally, MARM-7 administration was significantly more efficacious in rats that had decreased levels of circulating IgM after splenectomy, plasma exchange, and administration of RS61443. These experiments suggest that the anti-mu approach may allow depletion of IgM XNA for a sufficiently long period to test the hypothesis of "accommodation" in other xenograft models such as the pig-to-primate xenograft or even in ABO-incompatible allografts.

IgM gammopathy and polyneuropathy react with an antigenic glycolipid present in human central nervous system.

An immunological mechanism may be responsible for the peripheral neuropathies associated with the IgM monoclonal gammopathies. Myelin-associated glycoprotein, and sulfated glucuronic acid-glycosphingolipids of the paragloboside type specific of peripheral nervous system (PNS), have been found to be targets for these antibodies. We report here on the presence of a glycolipid antigen present in central nervous system white matter in 7 of 12 patients with IgM monoclonal gammopathy and neuropathy whose IgM reacts also with the specific sulfated glycolipids of PNS. Immunodetection of the antibodies was performed on thin-layer plates after separation of the glycosphingolipids from peripheral nerve and white matter. The lipid antigen had a migration on thin-layer chromatography close to sulfogalactosylceramide but the sulfatide standard did not react with the monoclonal IgM.

IgM binding to sialosyllactosaminylparagloboside in a patient with polyradiculoneuropathy due to Mycoplasma pneumoniae infection.

IgM in serum without paraprotein in a patient with polyradiculoneuropathy due to a Mycoplasma pneumoniae infection reacted specifically with a ganglioside, sialosyllactosaminylparagroboside (SLPG), in a human peripheral nerve on a thin-layer chromatogram plate by an immunostaining technique. This finding suggests the possibility that anti-SLPG antibody in the patient's serum may play a role in the pathogenesis of neuropathy.

Serum antibodies to gangliosides in Guillain-Barré syndrome.

To determine whether antibodies to acidic glycolipids of nervous tissue are present in patients with Guillain-Barré syndrome (GBS), sera from patients with GBS and appropriate control subjects were tested by a thin-layer chromatogram overlay technique. Chromatograms on which the whole ganglioside fractions from peripheral nerve and brain had been separated were overlaid with appropriate dilutions of the patients' sera (1:100 or greater), and antibody binding was revealed with a radiolabeled or peroxidase-labeled second antibody. Antibodies to ganglioside antigens were detected in 5 of 26 patients with GBS. IgG antibodies in 1 patient reacted strongly with LM1 (sialosyl paragloboside), the major ganglioside of human peripheral nervous system myelin, and its hexaose analog (sialosyl lactosaminyl paragloboside), a minor ganglioside of human peripheral nervous system myelin. The antibody titer in this patient fell 8-fold over 6 weeks coincident with clinical improvement. IgG from 2 other patients with GBS reacted with GD1b ganglioside, and the antibody titers in these patients also decreased substantially with clinical improvement. IgM antibodies in the sera from 2 other patients reacted with GD1a and GT1b gangliosides, which have a shared terminal carbohydrate sequence. Antibodies to gangliosides were not detected in the sera from 19 patients with other neurological diseases or from 10 normal subjects, and the frequency with which antiganglioside antibodies occurred in the patients with GBS was significantly greater than that in the combined control subjects (p less than 0.01). The results demonstrate relatively high levels of antibodies to gangliosides in some GBS patients.

Serum and CSF humoral immunity in Guillain-Barré syndrome: clinical correlations.

Paired samples of CSF and serum obtained from 29 patients affected with Guillain-Barré syndrome (GBS) were analyzed for various protein levels, including immunoglobulins and complement components. An attempt was made to correlate these findings to the clinical stage, severity, and duration of the disease. Intrathecal IgG synthesis was detected and quantified by means of a previously reported formula. It is practically constant in the GBS during the stage of stabilized paralysis, and is significantly greater in this stage than in the stage of progressive paralysis. Moreover, it increases with severity and duration (longer than 3 months) of the disease. Evidence of intrathecal C3 consumption is also presented.

An analysis of clinical and laboratory features of acute lymphocytic leukemias with emphasis on 35 children with pre-B leukemia.

In 35 of 191 patients with acute lymphocytic leukemia (ALL) malignant cells were similar in phenotype to B-lymphocyte precursors. Both these patients' lymphoblasts and normal pre-B-cells contain cytoplasmic immunoglobulin (Ig) mu heavy chains, but have no surface Ig. In patients with pre-B leukemias, lymphoblasts containing cytoplasmic mu chains alone were often accompanied by cells of identical morphology that expressed no Ig and less frequently by lymphoblasts bearing scant amounts of surface mu. This spectrum of cellular Ig expression suggests that "null," pre-B, and intermediate pre-B/B ALLs represent closely related malignancies with complete or partial arrests at different stages of maturation. When pre-B, B, T, and "null" cell categories of ALL were compared for 22 different clinical and laboratory features, including remission rate and short-term remission duration, no statistical differences were observed between the pre-B and "null" groups. These early results suggest that pre-B-cell leukemias represents a relatively good prognostic subclass of ALL, do not require more intensive treatment than that proven to be effective for "null" cell ALL, and should be distinguished from the less common, but more clinically aggressive, B-cell subclass of ALL. Longer follow-up will be required to confirm these preliminary conclusions.