Salivary free light chains as a new biomarker to measure psychological stress: the impact of a university exam period on salivary immunoglobulins, cortisol, DHEA and symptoms of infection.

INTRODUCTION: Measurement of immunoglobulin free light chains (FLCs) in saliva can serve as a non-invasive biomarker in health and behavioural research. FLCs have been explored in relation to physiological stress but FLC responses to psychological stress and their relationship with infections remain unknown. This study aimed to investigate the impact of exam period stress on salivary FLCs alongside other established biomarkers of stress and whether FLCs relate to symptoms of infection. METHODS: 58 healthy adults studying at university completed saliva samples and questionnaires in a period without exams (baseline), and again prior to the start of an exam period. Saliva samples were assessed for FLCs, IgA, cortisol and dehydroepiandrosterone (DHEA). Measures of life events stress, perceived stress, anxiety and depression were completed. Students also reported incidence and severity of symptoms of infection and rated general well-being at baseline, prior to, during and after the exam period. Exercise, sleep and alcohol consumption were also assessed at both timepoints. RESULTS: FLCs secretion rates were significantly lower at the exam period compared to baseline (p < .01), with reductions of 26% and 25% for κ FLC and λ FLC, respectively. In agreement, salivary IgA secretion rate was lower at exams (non-significant trend, p = .07). Cortisol concentration significantly increased at exams (p < .05) while DHEA did not change, leading to an increase in the cortisol:DHEA ratio (p = .06). Depression (p < .05) and anxiety increased from baseline to exams and life stress reported in the build up to the exam period was higher compared with baseline (p < .001). Well-being significantly decreased from baseline to exams (p < .01). The proportion of participants reporting infection symptoms (70%) was unchanged between baseline and prior to exams. No significant relationships were found between FLCs or other saliva parameters and infection symptoms, well-being or stress/psychological measures. Changes in saliva parameters between timepoints were independent of health behaviours. CONCLUSIONS: Salivary FLCs are responsive to life events stress and corroborate with IgA. This preliminary study highlights the potential utility of FLCs as a new salivary biomarker in stress research.

Free light chains and autoimmunity.

The study of free light chains (FLCs) has grown as area of enormous interest for many clinicians with the aim of disclosing the exact biological role and potential use of FLCs in the clinical routine. Moreover, the attention given to immunological functions of FLCs has sparked a new light into their pathogenic contribution in different chronic autoimmune-based inflammatory diseases. The release of intracellular antigens following cell death or ineffective clearance of apoptotic debris, modification of self-antigens, and molecular mimicry may trigger the production of immunoglobulins after activation and polyclonal expansion of B cells, by which FLCs are released. The discovery of polyclonal FLCs as potential biomarkers started with the observation of their increased concentrations in a variety of biological fluids related to patients with autoimmune diseases. This review deals with the use of polyclonal FLCs for identifying severity and monitoring outcome after treatment in some autoimmune diseases, namely systemic lupus erythematosus, myasthenia gravis, systemic sclerosis, rheumatoid arthritis and Sjögren's syndrome, as supported by the fact that levels of FLCs correlate with both B cell activation markers and other specific markers of disease activity. In a near future, following the evidence shown, FLCs might probably work as early prognostic markers of severity and also as indicators of response to treatment or early assessment of relapse in selected autoimmune diseases.

Isolated Pericardial Infiltration Without Myocardial Involvement in Light-Chain-Related Amyloidosis.

Light-chain-related amyloidosis is a systemic disease characterized by continuous accumulation of insoluble fibrillar proteins in different organs. Cardiac involvement is frequent in this condition. However, atypical presentations and unusual amyloid deposits localization may be encountered making the diagnosis challenging. We present here a case of a light-chain-related pericardial amyloidosis without evidence of myocardial involvement and emphasize the difficulty and importance of amyloidosis typing before starting treatment.

Treatment of Immunoglobulin Light Chain Amyloidosis: Mayo Stratification of Myeloma and Risk-Adapted Therapy (mSMART) Consensus Statement.

Immunoglobulin light chain amyloidosis (AL amyloidosis) has an incidence of approximately 1 case per 100,000 person-years in Western countries. The rarity of the condition not only poses a challenge for making a prompt diagnosis but also makes evidenced decision making about treatment even more challenging. Physicians caring for patients with AL amyloidosis have been borrowing and customizing the therapies used for patients with multiple myeloma with varying degrees of success. One of the biggest failings in the science of the treatment of AL amyloidosis is the paucity of prospective trials, especially phase 3 trials. Herein, we present an extensive review of the literature with an aim of making recommendations in the context of the best evidence and expert opinion.

New insights and modern treatment of AL amyloidosis.

Systemic amyloidosis is a rare disease that is rarely cured. Systemic immunoglobulin light-chain amyloidosis (AL) is the most common type, usually the result of monoclonal light chains produced by a relatively indolent small plasma cell clone in the bone marrow. In AL, the direct toxicity of light chains, their misfolded intermediates, and deposition as amyloid fibrils in vital organs cause organ dysfunction and death. Often the diagnosis is delayed and the disease is advanced at presentation. Early diagnosis is possible with vigilance in clinical situations such as for patients with monoclonal gammopathy of undetermined significance who develop albuminuria or elevated cardiac biomarkers. Treatment is aimed at eradicating the clonal disease and restoring organ function; options include high dose melphalan followed by autologous stem cell transplantation, oral melphalan and dexamethasone, bortezomib-based combination chemotherapy and immunomodulatory agents such as lenalidomide or pomalidomide combined with dexamethasone. Cardiac involvement at baseline and the free light-chain hematologic response to therapy determine overall survival. Following measures of organ disease with cardiac and other biomarkers and of hematologic disease with serum free light chains is necessary to gauge organ and hematologic responses to therapy.

Pivotal role of apoptosis signal-regulating kinase 1 in monoclonal free light chain-mediated apoptosis.

Renal failure, a major complication associated with multiple myeloma, is usually related to deposition of monoclonal immunoglobulin free light chains (FLCs) and directly contributes to morbidity and mortality in this disease. The present study focused on the cytotoxic effects of monoclonal FLCs. Human proximal tubular epithelial cells (HK-2) were examined after incubation with two human monoclonal FLCs (termed κ2 and λ3). Incubation of HK-2 cells for 24 and 48 hours with either FLCs at 1 mg/mL promoted activation of caspase-9 and caspase-3 and increased the rate of apoptosis. Because prior studies demonstrated that FLCs generated intracellular oxidative stress, our studies focused on the redox-sensitive mitogen-activated protein kinase kinase kinase known as apoptosis signal-regulating kinase 1 (ASK1). A time-dependent increase in phosphorylation of ASK1 at T845, indicating activation of this enzyme, was observed. Small interfering RNA designed to reduce ASK1 expression in HK-2 cells successfully decreased ASK1, which was confirmed by Western blot analysis. Incubation of ASK1-depleted HK-2 cells with the two FLCs prevented the increase in apoptosis while pretreating HK-2 cell with nontargeting small interfering RNA did not prevent FLCs-mediated apoptosis. The combined data demonstrate that monoclonal FLCs activated the intrinsic apoptotic pathway in renal epithelial cells by activation of ASK1.

The role of serum immunoglobulin free light chain in response and progression in waldenstrom macroglobulinemia.

INTRODUCTION: The serum free light chain (sFLC) has been widely used in the assessment of response in patients with multiple myeloma and other plasma cell dyscrasias. However, its use in Waldenstrom macroglobulinemia (WM) has not been previously assessed. We sought to examine the role of sFLC in response and progression of patients with WM. METHODS: This study was conducted in a cohort of 48 patients with a diagnosis of WM, untreated (n = 20) or relapsed/refractory (n = 28), prospectively treated on a bortezomib and rituximab trial. RESULTS: Involved FLC (iFLC) response occurred in 79% patients versus 60% by M-spike protocol criteria. The median time to response was shorter with iFLC than per protocol (2.1 and 3.7 months; P = 0.05). Progression defined using iFLC also correlated well to progression in the protocol (κ = 0.63). However, the median time to progression (TTP) was more rapid by iFLC than per protocol (13.7 and 18.9 months). We also confirmed that a flare in iFLC in post-rituximab therapy did not correlate with lack of response or shorter TTP. CONCLUSION: Involved sFLC may be a useful marker of tumor measurement, showing earlier response and progression compared with IgM or M-spike measurements.

Light chain-mediated tubulopathies.

Immunoglobulin light chains are low molecular weight proteins that are filtered through the glomerulus and reabsorbed into the proximal tubular epithelium by binding initially to a heteromeric receptor complex composed of megalin and cubilin. Saturation of this receptor-mediated endocytotic process results in the presence of free light chains in the distal nephron and urine. In the course of metabolism of monoclonal light chains, nephrotoxicity can occur, resulting in clinical manifestations that can include acute kidney injury and progressive chronic kidney disease. Patterns of tubulopathic renal injury include proximal tubular epithelial cell cytotoxicity, tubulointerstitial nephritis and cast nephropathy (also known as 'myeloma kidney'). Research efforts over the past two decades have refined understanding of the molecular mechanisms involved in light chain-mediated tubular injury and are the subject of this review.

Molecular and functional characterization of cynomolgus monkey IgG subclasses.

Studies for vaccine and human therapeutic Ab development in cynomolgus monkeys (cynos) are influenced by immune responses, with Ab responses playing a significant role in efficacy and immunogenicity. Understanding the nature of cyno humoral immune responses and characterizing the predominant cyno IgG types produced and the Fc-FcγR interactions could provide insight into the immunomodulatory effects of vaccines. Anti-drug Ab responses against human IgG therapeutic candidates in cynos may affect efficacy and safety assessments because of the formation of immune complexes. There is, however, limited information on the structure and function of cyno IgG subclasses and how they compare with human IgG subclasses in Fc-dependent effector functions. To analyze the functional nature of cyno IgG subclasses, we cloned four cyno IgG C regions by using their sequence similarity to other primate IgGs. The four clones, cyno (cy)IGG1, cyIGG2, cyIGG3, cyIGG4, were then used to construct chimeric Abs. The sequence features of cyno IgG subclasses were compared with those of rhesus monkey and human IgG. Our data show that rhesus monkey and cyno IgG C regions are generally highly conserved, with differences in the hinge and hinge-proximal CH2 regions. Fc-dependent effector functions of cyno IgG subclasses were assessed in vitro with a variety of binding and functional assays. Our findings demonstrate distinctive functional properties of cyno IgG subclasses. It is notable that human IgG1 was less potent than cyno IgG1 in cyno FcγR binding and effector functions, with the differences emphasizing the need to carefully interpret preclinical data obtained with human IgG1 therapeutics.

Immunoglobulin light chains activate nuclear factor-κB in renal epithelial cells through a Src-dependent mechanism.

One of the major attendant complications of multiple myeloma is renal injury, which contributes significantly to morbidity and mortality in this disease. Monoclonal immunoglobulin free light chains (FLCs) are usually directly involved, and tubulointerstitial renal injury and fibrosis are prominent histologic features observed in myeloma. The present study examined the role of monoclonal FLCs in altering the nuclear factor κ light chain enhancer of activated B cells (NF-κB) activity of renal epithelial cells. Human proximal tubule epithelial cells exposed to 3 different human monoclonal FLCs demonstrated Src kinase-dependent activation of the NF-κB pathway, which increased production of monocyte chemoattractant protein-1 (MCP-1). Tyrosine phosphorylation of inhibitor of κB kinases (IKKs) IKKα and IKKß and a concomitant increase in inhibitor of κB (IκB) kinase activity in cell lysates were observed. Time-dependent, Src kinase-dependent increases in serine and tyrosine phosphorylation of IκBα and NF-κB activity were also demonstrated. Proteasome inhibition partially blocked FLC-induced MCP-1 production. These findings fit into a paradigm characterized by FLC-induced redox-signaling events that activated the canonical and atypical (IKK-independent) NF-κB pathways to promote a proinflammatory, profibrotic renal environment.

Immunoglobulin light chains activate tubular epithelial cells through redox signaling.

The renal proximal tubule metabolizes circulating low-molecular-weight proteins such as Ig free light chains. In the setting of plasma cell dyscrasias, the burden of filtered protein can be very high. Endocytosis of certain nephrotoxic light chains induces H(2)O(2) production and monocyte chemoattractant protein-1 (MCP-1) release, leading to recruitment of inflammatory cells and interstitial fibrosis, but how these processes are linked mechanistically is not well understood. This study investigated the relationship between H(2)O(2) generated after light chain endocytosis by human proximal tubular (HK-2) cells and activation of c-Src, a redox-sensitive tyrosine kinase. HK-2 cells exposed to two different light chains upregulated c-Src activity, which increased the production of MCP-1. In parallel, we observed a time-dependent oxidation of c-Src. Inhibition of c-Src activity and silencing c-Src expression abrogated the light chain-induced MCP-1 response, but had no effect on H(2)O(2), indicating that production of H(2)O(2) is upstream of c-Src in the signaling cascade. Silencing megalin and cubilin expression inhibited the MCP-1 response, whereas extracellular catalase did not, indicating that endocytosis is required and that intracellular generation of reactive oxygen species activates c-Src. These data show that intracellular H(2)O(2) induced by endocytosis of monoclonal free light chains oxidizes and activates c-Src, which promotes release of MCP-1.

Free immunoglobulin light chains as a risk factor in renal and extrarenal complications.

Immunoglobulin light chains (IgLCs) are part of intact immunoglobulins and contribute to antigen recognition. However, IgLCs are synthesized by B cells slightly in excess of Ig heavy chains and are found in the plasma of healthy people at low concentrations. IgLCs are metabolized primarily by the kidney. In B-cell lymphoproliferative disorders, e.g., multiple myeloma, the serum concentration of monoclonal IgLCs markedly increases and the reabsorptive capacity of the proximal tubuli is exceeded. As a consequence, monoclonal IgLCs appear as Bence Jones proteins (BJPs) in the urine and can give rise to IgLC deposits in the kidney that may result in renal insufficiency. In patients with chronic kidney disease (CKD) of various origins, reduced excretion leads to increased serum levels of polyclonal IgLCs. Free IgLCs interfere with essential functions of neutrophils, cells of the first-line nonspecific immune defense, and therefore contribute to the disturbed immune function in CKD patients. IgLCs attenuate the coordinated apoptotic cell death of neutrophils and therefore may interfere with the normal resolution of inflammation and contribute to the chronic inflammatory state found in CKD patients. Recently it was shown that IgLCs may confer mast cell-dependent hypersensitivity and thereby play an important role in the development of contact sensitivity.

Immunoglobulin-free light chains mediate antigen-specific responses of murine dorsal root ganglion neurons.

Immunoglobulin-free light chains (IgLC) secreted by B lymphocytes, have been shown to mediate hypersensitivity by inducing antigen-specific mast cell activation. Although both mast cells and sensory neurons contribute to the hypersensitivity response, the role of IgLC in relation to sensory neurons is unknown. We therefore aimed to investigate the effects of IgLC on cultures of murine dorsal root ganglion (DRG) neurons. Immunohistochemistry demonstrated that IgLC and IgE could specifically bind to DRG neurons, on which the presence of FcepsilonRI, the specific receptor for IgE, was demonstrated by western blotting. Further, optical recordings with Fluo-4 showed that application of the corresponding antigen to IgLC- or IgE-sensitized DRG neurons induces a sustained increase in intracellular Ca(2+) in about half of these neurons. These results show that IgLC and IgE can mediate antigen-specific responses in murine neurons. Our findings present a novel way of antigen-specific neuronal activation.

IgE and immunoglobulin free light chains in allergic disease: new therapeutic opportunities.

The prevalence of allergy and associated diseases has dramatically increased during the last few decades. These hypersensitivity reactions can be subdivided into IgE- and non-IgE-dependent diseases. Anti-IgE approaches, such as omalizumab, have provided a novel treatment for allergic diseases, and have demonstrated benefit in severe allergic asthma and seasonal allergies. Nevertheless, this approach does not eliminate disease and many patients do not respond to treatment. The newly described mechanism for allergic diseases based on mast cell sensitization by Ig free light chains is exciting and may provide another approach to the treatment of allergic diseases. The peptide inhibitor F-991 has demonstrated remarkable biological activity in a number of animal models of allergic diseases, and promise for the potential treatment of allergic diseases in humans.

Removal of the BiP-retention domain in Cmicro permits surface deposition and developmental progression without L-chain.

Nascent, full length, immunoglobulin (Ig) heavy (H)-chains are post-translationally associated with H-chain-binding protein (BiP or GRP78) in the endoplasmic reticulum (ER). The first constant (C) domain, CH1 of a C gene (Cmu, Cgamma, Calpha), is important for this interaction. The contact is released upon BiP replacement by conventional Ig light (L)-chain (kappa or lambda). Incomplete or mutated H-chains with removed variable (VH) and/or C(H)1 domain, as found in H-chain disease (HCD), can preclude stable BiP interaction. Progression in development after the preB cell stage is dependent on surface expression of IgM when association of a micro H-chain with a L-chain overcomes the retention by BiP. We show that IgM lacking the BiP-binding domain is displayed on the cell surface and elicits a signal that allows developmental progression even without the presence of L-chain. The results are reminiscent of single chain Ig secretion in camelids where developmental processes leading to the generation of fully functional H-chain-only antibodies are not understood. Furthermore, in the mouse the largest secondary lymphoid organ, the spleen, is not required for H-chain-only Ig expression and the CD5 survival signal may be obsolete for cells expressing truncated IgM.

Free immunoglobulin light chains: a novel target in the therapy of inflammatory diseases.

In recent years, novel therapeutic strategies have become available for the treatment of chronic inflammatory disease. Neutralizing proinflammatory mediators such as leukotrienes and TNF-alpha, in addition to anti-IgE therapies (Omaluzimab) that target higher in the inflammatory cascade, have shown success in the treatment of allergic or autoimmune disorders. Free immunoglobulin light chains, which are produced by B lymphocytes and secreted into serum, might play a crucial role in the pathogenesis of inflammatory disease. Concentrations of free light chains are significantly increased under diverse pathological conditions in humans, and have been linked to the progression and severity of immune diseases. Here we discuss the importance of free immunoglobulin light chains as a potential therapeutic target in the treatment of chronic inflammatory disease.

Peptidomimics of antigen are present in variable region of heavy and light chains of anti-idiotypic antibody and function as surrogate antigen for perpetuation of immunological memory.

Peptide antigens composed of relevant B cell and T cell epitopes, capable of inducing protective immune response against the whole pathogen, are potentially safe, alternative vaccine antigens for prevention of wide range of diseases. Here, we show that short peptides derived from internal image sequences of anti-idiotypic antibody (peptidomimics) can function as both B and T cell epitopes and perpetuate antigen specific immunological memory. We have sequenced the variable regions of heavy and light chains of the anti-idiotypic antibody specific to rinderpest virus hemagglutinin protein and predicted T cell epitopes in these sequences by an immuno-informatics approach. We have studied the interaction of these epitopes with MHC class I by in vitro assays and in silico analysis by molecular modeling of the idiopeptide-MHC complexes as well as antigen-derived peptide-MHC complexes. The functional capacity of anti-idiotypic antibody derived peptides to stimulate antigen specific T cells in vitro was tested. The ability of peptidomimics to proliferate the immune splenocytes in vitro was 10 times more when compared with that of a control peptide taken from the constant region of immunoglobulin. Similarly three- to fivefold more amounts of IL-2 and IFN-gamma were secreted by immune splenocytes in response to in vitro re-stimulation with peptidomimics. Further, we have provided evidence for the generation of antibodies against peptidomimics in memory response generated on antigen or anti-idiotypic antibody immunizations. In summary, our experiments suggest that peptidomimics are generated in the body after antigen immunization and may have important roles in vivo in regulating antigen specific immunological memory.

Structural insight into pre-B cell receptor function.

The pre-B cell receptor (pre-BCR) serves as a checkpoint in B cell development. In the 2.7 angstrom structure of a human pre-BCR Fab-like fragment, consisting of an antibody heavy chain (HC) paired with the surrogate light chain, the "unique regions" of VpreB and lambda5 replace the complementarity-determining region 3 (CDR3) loop of an antibody light chain and appear to "probe" the HC CDR3, potentially influencing the selection of the antibody repertoire. Biochemical analysis indicates that the pre-BCR is impaired in its ability to recognize antigen, which, together with electron microscopic visualization of a pre-BCR dimer, suggests ligand-independent oligomerization as the likely signaling mechanism.