Kappa Light Chain Research Articles

Monoclonal gammapathies: Epidemiological, immunochemical and etiological analysis of a series of 50 cases

During the period from September 2022 to January 2023, 50 cases of monoclonal gammopathy (GM) were collected at the Avicenne military hospital in Marrakech. The average age of the patients was 62 years. Thirty patients were male and 20 were female. The etiological diagnosis could be established for the 50 observations: 5 were classified as GM of undetermined significance (GMSI), 42 were classified as myeloma, two case of Waldenström's disease and one case of AL amyloidosis. The clinical symptomatology was dominated by bone pain in 78% of patients with a deterioration in general condition in 43%. The most frequently found biological abnormalities were anemia in 62% of patients, renal failure in 48% of patients and 19% had hypercalcemia. A monoclonal peak was found in the electrophoreses of 45 patients (90%), of whom 6 migrated to the beta zone and 39 migrated to the gamma zone. The IgG Kappa isotype was the most frequent with a rate of 56%, followed by IgG Lambda (27%), IgA Kappa (8%), IgA Lambda (3%), IgM Lambda (3%) and lambda free light chains (3%).

Free Kappa light chain in a sample of Egyptian multiple sclerosis patients (a pilot study)

BackgroundMultiple sclerosis is a chronic autoimmune-mediated demyelinating disease of the central nervous system that is usually associated with varying degrees of progressive disability. Free Kappa light chain (FKLC) has attracted growing attention as a significant diagnostic marker of MS. Our aim was to study the diagnostic utility of cerebrospinal fluid free Kappa light chain and related indices in a sample of Egyptian MS patients vs. CSF IgG oligoclonal bands. It was a prospective case–control study of MS patients carried in our hospital during the period from January 2021 till January 2022. Our study carried on 30 patients with multiple sclerosis and other inflammatory neurologic diseases and 20 age and sex matched controls. The study measured FKLC in the CSF and serum sample pairs of patients and control group. Indices calculated using FKLC measured in CSF and serum included; FKLC index, FKLC intrathecal fraction and quotient of FKLC. Indices were used to assess intrathecal synthesis of FKLC considering blood–CSF barrier function. Receiver operating characteristic curve analysis was used to determine diagnostic performance of FKLC and related indices in comparison to CSF–OCB testing.ResultsMeasured FKLC levels as well as its calculated indices have shown statistically significant higher values among MS patients against OIND patients and healthy control group. Both FKLC index and FKLC IF were similarly showing 100% diagnostic sensitivity and 100% diagnostic specificity for MS diagnosis.ConclusionsFKLC biomarkers are proposed to be highly sensitive and easy to detect first-line markers of intrathecal immunoglobulin synthesis with accurate performance and low cost that might prove to be promising diagnostic markers of MS.

Immunoglobulin free light chain-kappa, a novel biomarker of inflammation and cardiovascular risk, decreases after smoking cessation in association with NT-proBNP

Abstract Introduction Inflammation plays an important role in the development and progression of cardiovascular diseases. Unhealthy diet, infection, and smoking coupled with genetic factors induce inflammation in the heart and blood vessels by activating nuclear factor kappa B, which regulates the transcription of immunoglobulin free light chain (FLC)-κ in B-cells and the production of multiple inflammatory molecules. FLCs are novel biomarkers of inflammation, and their levels are associated with overall mortality in a general population and with cardiovascular outcomes. Purpose This study aimed to investigate the effect of smoking cessation (SC) on levels of FLCs as well as of the inflammation and heart failure markers. Methods Inclusion criterion was first-visit smokers with desire to quit smoking at our outpatient clinic. Smoking patients with nicotine dependence were treated with transdermal nicotine patches or orally administrated varenicline. Various clinical parameters, including inflammatory markers such as FLC levels, were measured on their first visit to our outpatient clinic for SC and after 3 months of successful and unsuccessful SC. Paired t-test or Wilcoxon signed-rank test was performed to evaluate changes in these parameters before and after SC. Results Out of 96 patients who participated in this study, 76 patients (55 males and 21 females) succeeded in SC over a period of 3 months. Body mass index (BMI) significantly increased from baseline to 3 months after initiating SC (from 23.8 to 24.2, p=0.004) in patients with successful SC. On the other hand, levels of various inflammatory markers; FLC-κ, FLC-κ/FLC-λ, and the neutrophil-to-lymphocyte ratio) significantly decreased from baseline to 3 months (from 27.5 to 24.7 mg/L, p = 0.042; from 1.2 to 1.2 mg/L, p = 0.010; and from 2.0 to 1.9, p = 0.024, respectively). In contrast, no significant changes occurred in these parameters among patients with unsuccessful SC. Percent changes of FLC-κ levels from baseline to 3 months were significantly correlated with those of CRP (r=0.217, p=0.034) and NT-proBNP (r=0.335, p<0.001) levels. Conclusion The FLC-κ level, a novel inflammatory and cardiovascular risk biomarker, decreased in smoking patients after their SC despite the increase in body weight, suggesting that the beneficial effect of SC overcomes potential risks by body weight increase. The results also suggest that a decrease in the FLC-κ level after SC is associated with a reduction of load on the heart as well as decrease in inflammation.

MRD Detection in B-Cell Non-Hodgkin Lymphomas Using Ig Gene Rearrangements and Chromosomal Translocations as Targets for Real-Time Quantitative PCR and ddPCR.

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell non-Hodgkin lymphomas (B-NHL), B-cell chronic lymphocytic leukemia (CLL), and multiple myeloma and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. Real-time quantitative PCR (RQ-PCR) targeting circulating lymphoma cells is still the gold standard for MRD detection in indolent B-NHL and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). Alternatively, droplet digital PCR (ddPCR) can be used for MRD monitoring in multiple myeloma, mantle cell lymphoma, CLL, and FL with comparable sensitivity, accuracy, and reproducibility.The most broadly applicable MRD target in B-NHL is the junctional regions of the rearranged immunoglobulin heavy (IGH) and light chain genes. Complete and incomplete IGH and additionally IG kappa light chain rearrangements can be used as targets for MRD. Next-generation sequencing (NGS) of IG-rearrangements (IG-NGS) as new sequencing-based technology can overcome the limitation of PCR-based approaches and has a potential for higher sensitivity. Chromosomal translocations like the t(14;18)(q32;q21) translocation associated with IGH::BCL2 fusion in FL and t(11;14)(q13;q32) translocation in MCL leading to the IGH::CCND1 fusion can be used as MRD target in selected lymphoma subtypes. In patients with CLL, both flow-cytometry and RQ-PCR are equally suited for MRD assessment as long as a sensitivity of 10-4 is achieved.MRD diagnostics targeting the IG loci is complex and requires extensive knowledge and experience because the junctional regions of each clonal rearranged gene have to be identified before the patient-specific PCR assays can be designed for MRD monitoring. In addition, the presence and load of somatic hypermutation within the rearranged IGH gene occurring during B-cell development of germinal center and post-germinal center B-cell lymphomas may hamper appropriate primer binding leading to false-negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IGH joining region of chromosome 14. PCR-based methods using allele-specific primers can reach a high sensitivity of up to 10-5. This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IG gene rearrangements and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results. This is available by the EuroMRD network ( https://euromrd.org ), a subgroup of ESHLO ( https://eslho.org ).

An Unusual Case of Lymphoplasmacytic Variant of Multiple Myeloma

Abstract Introduction/Objective Multiple myeloma is characterized by unregulated growth of monoclonal plasma cells with overt production of nonfunctional intact immunoglobulins or immunoglobulin chains. Immunohistochemically, the plasma cells are positive for CD138 and CD38, and are rarely positive for CD20 or cyclin D1. CD20 positivity and plasmacytoid morphology in our case pointed towards a lymphoplasmacytic variant of multiple myeloma. The translocation t(11;14) has been detected in this variant, which can explain the positive cyclin D1 immunostaining. The differential diagnosis of this myeloma variant includes lymphoplasmacytic lymphoma. Methods/Case Report We present a case of a 75-year-old female with no significant past medical history who suffered a fall and hesitated to get evaluated until her pain persisted. Initial ER evaluation revealed she had a compression fracture of the T10 and T11 vertebrae with no other abnormality identified. Subsequently, she underwent kyphoplasty, and bone biopsies were performed. The biopsies showed prominent monotonous lymphocytes and some plasmacytoid cell proliferation. Immunostains showed the tumor cells were positive for CD20, Pax-5, Bcl-2, cyclin D1, CD138, and kappa light chain, weakly positive for CD23; and negative for CD5, CD3, CD10, Sox11, and lambda light chain. Based on the morphology and immunoprofile, current findings were considered suggestive of a low-grade B cell lymphoplasmacytic proliferation with cyclin D1 expression. The differential diagnosis includes a lymphoplasmacytic lymphoma or lymphoplasmacytic variant of multiple myeloma. This case was forwarded to a higher-level care facility for consultation and the diagnosis of plasmacytoma, kappa light chain restricted, was rendered. Results (if a Case Study enter NA) NA Conclusion Misdiagnosis of lymphoma can be made in lesions showing classic plasma cells, numerous lymphoplasmacytic cells, and cyclin-D1 positivity on immunohistochemistry if additional testing is not performed. We recommended further testing, including but not limited to bone marrow biopsy, molecular testing, FISH, and genetic testing for a definitive diagnosis.

PLASMA CELL NEOPLASM IN BREAST: A RARE CASE REPORT

Abstract Introduction/Objective Breast carcinoma continues to be the most common malignancy in women however plasma cell neoplasms are rare in breast tissue. Most of these recorded cases are from patients with Multiple myeloma (1). We present a rare case of a new breast lesion in a 69 year female incidentally found on imaging. She had a history of adenocarcinoma of lung with brain metastasis undergoing treatment. Methods/Case Report The patient’s breast lesion presentation was unusual without any pain or associated symptoms, but a new right breast mass was incidentally discovered on CT-Chest Abdomen-Pelvis. Mammogram revealed a 1.4 cm circumscribed mass in lower, outer breast with scattered calcifications. With the suspicion for a primary breast malignancy or metastatic disease from her lung cancer the patient’s breast lesion was biopsied. On histology, abnormal proliferation of cells with ovoid nuclei, vesicular chromatin and variable cytoplasm was noted with a background of mature adipose tissue. Immunohistochemistry showed neoplastic cells positive for plasma cell associated markers including CD38 (patchy), CD56, CD138, and MUM1 with a kappa restriction on ISH. The Ki67 showed 20% proliferation index. EBER was negative and SMARC4 and INI were preserved. Other immunohistochemical stains were performed to rule out differentials and were all negative. The morphology and IHCs were consistent with the diagnosis of Plasma cell neoplasm with kappa-restriction. Subsequently this patient developed a shoulder lesion which on biopsy showed a plasma cell neoplasm expressing plasma cell markers and kappa light chain overexpression. Results (if a Case Study enter NA) NA Conclusion It is critical to distinguish a breast plasma cell neoplasm from other primary and metastatic breast lesions in order to avoid unnecessary surgery and cytotoxic chemotherapy. Rarely, plasma cell neoplasm can manifests as a breast lesion, which in this case turned out to be the early manifestation of multiple myeloma.

B-311 Relation of Free Immunoglobulin Ligh Chain Concentration to Overflow Proteinuria

Abstract Background Free immunoglobulin light chains are small proteins that undergo substantial glomerular filtration. When high serum concentrations of free light chains occur in multiple myeloma, the high filtered load of light chains exceeds the uptake capacity of proximal tubules, and overflow proteinuria ensues. Massive excretion of light chains can lead to cast formation and renal injury. The present study sought to determine the relation of serum light chain concentration to overflow proteinuria. Methods Results for patients with plasma cell disorders were reviewed as approved by an institutional review board. A Binding Site Optilite analyzer measured serum free light chain concentrations. Urine and serum protein electrophoresis were performed on Helena agarose gels. A total of 170 paired serum and 24-hour urine specimens were evaluated for 36 patients with increased kappa light chains and 26 patients with increased lambda light chains. Results Overflow proteinuria with either immunoglobulin light chain presented with light chain as the major urinary protein and limited increases of urinary albumin. Light chain excretion exceeded albumin excretion when light chain excretion surpassed 100 mg/d. Similar results were obtained for cases with increased kappa and lambda free light chains. There was a general relation of increased serum kappa and lambda light chains and urinary excretion of the corresponding light chain. Limited urinary excretion of light chains (<120 mg/d) was seen when the serum light chain concentration was < 250 mg/L, representing an approximate threshold for substantial overflow proteinuria. Overflow proteinuria exceeding 2,000 g/d was seen only when serum free light chain concentrations exceeded 800 mg/L. However, a few patients with high serum light chain values (>1,000/L) had low urinary light chain excretion that was discordant from the relation of serum light chain concentration to light chain excretion. Serum light chain values >4,000 mg/L (N = 10) were all associated with overflow proteinuria of > 1,500 mg/d and, in 7 cases, >10,000 mg/d. In a few cases, there was evidence that serum light chain assays overestimated light chain concentrations. In one case, the serum free light chain value was twice the total protein and about 12-fold higher than the serum M-spike, suggestive of substantial overestimation. Conclusions Defining the relation between serum free light chain concentrations and the magnitude of overflow proteinuria helps identify patients at low and high risk of massive overflow proteinuria that can cause renal injury. Limited overflow proteinuria (<120 mg/d) occurred when serum light chain concentration was < 250 mg/L, and substantial overflow proteinuria occurred when serum light chains exceeded 4,000 mg/L Serum free light chain concentrations above 250 mg/L were incompletely predictive of urinary free light chain excretion; a few discordant cases with high measured serum light chain concentrations (>1,000 mg/L) and low excretion of light chains were observed. Further investigation is indicated for cases with possible overestimation of free light chains or discordance between serum light chain values and urinary light chain excretion. Unexpected results in some cases could relate to polymerization of free light chains as previously reported.

B-165 The EXENT System reliably distinguishes daratumumab from endogenous monoclonal immunoglobulins in multiple myeloma patients

Abstract Background The use of therapeutic monoclonal antibodies (t-mAbs) in the treatment of multiple myeloma (MM) has created challenges in the clinical laboratory. Co-migration of t-mAbs (predominantly IgGK) and the endogenous M-protein during serum protein electrophoresis can result in inability to distinguish between a patient’s M protein and the t-mAb. This can hinder accurate assessment of therapeutic responses or may lead to extensive workup with immunofixation electrophoresis and gel-shift assays for accurate confirmation of a complete response, or to exclude the emergence of a new clone. There is a need for an analytically specific routine method to identify t-mAb interference. MALDI-TOF mass spectrometry is an especially promising tool, as due to its ability to identify M proteins’ molecular mass, it has the potential to recognize any t-mAbs with known amino acid composition. We have used the EXENT® Immunoglobulin Isotypes (GAM) Assay for the EXENT Analyzer (The Binding Site, part of Thermo Fisher Scientific) to assess the reproducibility of daratumumab (anti-CD38, IgGK) detection in an analytical study, and assessed the EXENT System’s ability to detect daratumumab in a cohort of MM patients. Methods The expected mass to charge ratio (m/z) of the +2-charge state of daratumumab’s kappa light chain was calculated based on published information. Daratumumab was spiked into normal human serum at 1.0 g/L, and this material was tested in 5 replicates a day over 5 days. In addition, 109 samples from 34 MM patients receiving daratumumab, carfilzomib, lenalidomide and dexamethasone (Dara-KRd) treatment were also tested. Samples were obtained after 14 or 28 days following daratumumab administration in various treatment cycles, ranging from cycle 4 to end-of-treatment. Results The expected m/z for the +2-charge state of daratumumab’s kappa light chain was calculated as 11,691.01 to 11,693.1. In the reproducibility experiment, the median m/z was 11691.3, within the expected range. The difference between the lowest and highest m/z was 1.3 across the 25 replicates. Daratumumab was detected in 103 out of 109 samples (97.3%): 51/57 (89.5%) in IgGK samples, and in all IgGL (23) and IgA (29) samples. In two IgGK patients, where daratumumab was not detected in 2 out of 4 samples and in 1 out of 3 samples, respectively, the patients’ own M proteins were in the proximity (24.8 and 27.1 m/z) of daratumumab’s m/z. In the other three cases where daratumumab was not modeled by the algorithm, the daratumumab peak overlapped with the endogenous M-protein's adduct peak (estimated m/z 11,688) in one sample; it was masked by the shoulder of a large (8.42 g/L) IgGK M-protein in another sample; and no obvious reason was identified in the third sample. There was no difference in the detection rate between 14 days and 28 days post administration. Conclusions The EXENT Immunoglobulin Isotypes (GAM) Assay has successfully identified the presence of daratumumab in approximately 90% of IgGK patient samples in addition to all other (non-IgGK) MM samples. These results demonstrate the potential of the EXENT Immunoglobulin Isotypes (GAM) Assay for recognizing known t-mAb interference.

B-319 Kappa and Lambda Free Light Chains Quantification in Serum Samples with 3 or 4 Monoclonal Components Detected by Serum Immunofixation

Abstract Background Serum free light chain (FLC) quantification is a diagnostic criterion for monoclonal gammopathy. Oligoclonal expansion is defined when two or more small bands are detected in the gamma region of serum protein electrophoresis (SPEP) or immunofixation (SIFE) within the context of infectious diseases, autoimmune diseases, or some lymphoproliferative processes. This study aims to investigate the frequency of FLC disturbances in samples with oligoclonal pattern of 3 or 4 monoclonal bands observable in SIFE. Methods We have analyzed 7553 consecutive samples during the period of January to December 2023 in which both determination of FLC and SIFE were ordered in a large central laboratory at São Paulo, Brazil. FLC quantification and SIFE analysis were performed with FreeLite assay and Optilite system (The Binding Site, UK) and SPIFE 3000 (Helena Laboratories, USA), respectively. Results In our analysis, 119 samples (50 males, 69 females, median age 69 years (range 37-90)), with 3 or 4 monoclonal bands were identified, constituting 1.57% of the overall study population. These samples were then categorized into two groups. The first group had normal FLC Ratio (range 0.26 to 1.65) and the second group had altered FLC Ratio (outside 0.26-1.65 range). Seventy-seven samples (65%) had altered FLC ratio, with kappa elevation being the most common alteration (51 samples). Within these samples, median FLC ratio was 2.99 (range 1.72-719.05). Lambda elevation was present in 26 samples, with median FLC ratio 0.04 (range 0.005-0.16). Conclusions Presence of 3 or more oligoclonal bands in SIFE analysis may be indicative of various pathological processes (benign and malignant) and also be observed in post-transplantation. In this study, we have observed that most of these samples present altered FLC ratio, which indicate monoclonal disturbances in the production of free light chains. Those disturbances may be associated with a lymphoproliferative disease (e.g. multiple myeloma) or may be transient. Clinicians should be aware of the need for follow-up of these cases.

CDR identification, epitope mapping and binding affinity determination of novel monoclonal antibodies generated against human apolipoprotein B-100

In-house generated mAbs to apolipoprotein B-100 (apoB-100) clones hLDL-E8, hLDL-2D8 and hLDL-F5 were extensively studied to determine their complementarity-determining regions (CDRs), binding epitopes and affinity. RT-PCR revealed that all mAbs consisted of kappa light chains and gamma heavy chains. DNA sequencing and bioinformatic analysis showed that the variable gene and protein sequences of their CDRs shared over 50 % identity with the existing databases. The 3D structures of the mAb variable fragments (Fv) with a QSQE score above 0.7 were constructed using the SWISS-MODEL platform. The structural accuracy was confirmed by Ramachandran plots, with 99 % of amino acid residues falling within acceptable regions. Thrombolytic cleavage of apoB-100 and Western blot analysis demonstrated that hLDL-E8 and hLDL-F5 specifically bind to the T3 fragment (aa 1297–3249), whereas hLDL-2D8 binds to the T4 fragment (aa 1–1297). These findings were supported with epitope-binding assays using inhibition ELISA, which indicated that hLDL-E8 binds at different epitopes from hLDL-2D8 and has some overlap with hLDL-F5. Lastly, the binding affinity of the mAbs was examined by indirect ELISA. The average affinity constants (Kaff) for mAbs hLDL-2D8, hLDL-E8 and hLDL-F5 are 1.51 ± 0.69 × 109 Mol−1, 7.25 ± 3.56 × 108 Mol−1 and 4.39 ± 2.63 × 106 Mol−1, respectively. Additionally, the behavior of the antibodies in the dose-response curve revealed that hLDL-F5 may recognize two epitopes of apoB-100 or have very low binding affinity. In contrast, hLDL-2D8 and hLDL-E8 each recognize a single epitope. These findings provide information that will be useful when selecting mAbs for both laboratory and clinical research purposes.

A genetically engineered mouse/human chimeric antibody targeting CD99 enhances antibody-dependent cellular phagocytosis against human mantle cell lymphoma Z138 cells

Background: Mantle cell lymphoma (MCL) is an aggressive form of B-cell nonHodgkin lymphoma. The elimination of MCL cells via phagocytosis is essential for cancer eradication. Therefore, discovering novel targeted antibodies that can induce phagocytosis is needed. We have demonstrated that our in-houseproduced mouse anti-CD99 mAb clone MT99/3 could induce potent anticancer activities against MCL cell lines in both in vitro and in vivo mouse xenograft models. Nevertheless, for use in humans, the mouse mAb needs to be transformed into a mouse/human chimeric mAb that contains a human Fc region to activate human immune effector functions, especially macrophage-mediated phagocytosis. Antibody-dependent cellular phagocytosis (ADCP) mediated by mouse/human chimeric mAb MT99/3 against MCL has not been previously reported. Objective: This study aimed to genetically engineer a mouse/human chimeric antibody against human CD99 derived from mouse mAb MT99/3 and to evaluate its effect in mediating the ADCP mechanism for eradicating MCL cells in vitro using monocyte-derived macrophages. Materials and methods: The expression plasmid to produce chimeric anti-CD99 antibody, ChAbMT99/3, was constructed by fusing the variable domains of mouse mAb MT99/3 with the constant domains of human IgG1 and the constant domains of kappa light chain. ChAbMT99/3 was expressed in the stable human expression system based on HEK293T cells. ChAbMT99/3 was purified from the culture supernatant of ChAbMT99/3-expressing HEK293T cells using Protein G chromatography. The purity and structure of ChAbMT99/3 were verified by SDS-PAGE and western blotting. The binding specificity and activity were determined by staining with cells expressing recombinant and native human CD99. The anticancer activity of ChAbMT99/3 in mediating the ADCP mechanism against MCL cell line Z138 using human monocytederived macrophages was evaluated. Results: We successfully constructed the plasmid to produce ChAbMT99/3. Human HEK293T cells stably expressing ChAbMT99/3 were established. The ChAbMT99/3- expressing HEK293T cells could secrete ChAbMT99/3 into the culture supernatant. The high purity and complete IgG structure of ChAbMT99/3 were obtained from the purification process. Crucially, this chimeric antibody retained its binding reactivity to recombinant and native human CD99. In addition, the produced ChAbMT99/3, upon binding to MCL cells, significantly enhanced ADCP against MCL cell line Z138 in a dose-dependent manner. Conclusion: The production of a mouse/human chimeric antibody against human CD99 derived from mouse mAb MT99/3 was successful. The engineered antibody could mediate ADCP activity against MCL cells. The produced ChAbMT99/3 might be a promising therapeutic candidate for MCL treatment.

Development of an immunochromatographic strip for the rapid detection of 11-dehydro-thromboxane B2 in human urine

Urinary 11-dehydro-thromboxane B2 (11-DH-TXB2) is a diagnostic marker for evaluating the effect of aspirin treatment in patients with cardiovascular disease. Therefore, it is of great clinical significance to develop a rapid detection method for 11-DH-TXB2 in urine. In this study, we prepared an ultrasensitive monoclonal antibody (mAb) against 11-DH-TXB2. The mAb belongs to the IgG1 subtype with kappa light chains. In an indirect competitive ELISA, it had a half-maximal inhibitory concentration of 3.63 ng/mL and a high affinity of 5.60 × 109 L/mol for 11-DH-TXB2. We then developed an immunochromatographic strip for point-of-care testing (POCT) of 11-DH-TXB2 in human urine. The strip provides a cut-off value and limit of detection of 500 ng/mL and 31.2 ng/mL, respectively. The result is obtained within 15 min. In summary, the proposed strip is a portable POCT tool for monitoring 11-DH-TXB2 in human urine.

Development of the PD9-9 Monoclonal Antibody for Identifying Porcine Bone Marrow-Derived Dendritic Cells.

The purpose of this study was to develop a monoclonal antibody (mAb) that can identify porcine dendritic cells (DCs) that have differentiated from bone marrow progenitor cells. Hybridoma technology was used to obtain mAbs, and bone marrow-derived DCs (BMDCs) were employed as immunogens for producing antibodies. The generated PD9-9 mAbs exhibited considerable reactivity towards porcine BMDCs with applications in flow cytometry and immunostaining. The antibody was composed of heavy immunoglobulin gamma-1 chains and light kappa chains. The PD9-9 mAb recognized fully differentiated porcine BMDCs and cells undergoing DC differentiation. In contrast, bone marrow cells and macrophages were not recognized by PD9-9. In addition, the PD9-9 mAb promoted porcine DC proliferation. Consequently, the PD9-9 mAb may be a biomarker for porcine DCs and will be advantageous for investigating porcine DC biology.

Features that characterize monoclonal light chain ("myeloma") cast nephropathy with immunofluorescence challenges and emphasis on electron microscopy.

Renal disease is a common cause of morbidity and mortality in patients with plasma cell dyscrasias. The serum-free light chain assay is used in patients, mostly older, with unexplained acute kidney injury to screen for potential myeloma cast nephropathy. This study consists of a systematic review of diagnostic features in myeloma cast nephropathy. The morphological features of tubular casts in patients with multiple myeloma have not been systematically analyzed. This study focuses on the morphology of these casts, emphasizing ultrastructural features, in a series of 23 patients with light chain ("myeloma") cast nephropathy and compared them with casts in 10 patients with various diseases. The immunofluorescence data were correlated with morphological findings to provide diagnostic assessments and practice guidelines. The ultrastructural features identified as diagnostic of casts associated with myeloma included: amyloid and crystals in the casts, multiple well-defined fracture planes forming a complex jigsaw puzzle arrangement of cast contents, indicative of the fragility of the immunoglobulin light chains involved, and reactive tubular cells lining the tubules with the casts. These features were seen in 95.2% of MCN cases and none of the casts in other renal conditions. Myeloma casts exhibited light chain monoclonality in a significant percentage of the MCN cases and often no staining for IgA or IgM. In contrast, the majority of non-myeloma casts stained for both kappa and lambda light chains, lgA, and lgM, and showed ultrastructurally a rather uniform finely to coarsely granular electron density occasionally admixed with cellular debris.

Evaluating the complement C1q levels in serum and cerebrospinal fluid in multiple sclerosis patients: Could it serve as a valuable marker in clinical practice?

Immunohistochemical studies have identified complement component C1q in MS lesions. We aimed to compare serum (sC1q) and CSF (csfC1q) levels in a large cohort of MS patients (pwMS) (n = 222) with those of healthy controls (HC, n = 52), individuals with other immune (IND, n = 14), and non-immune neurological disorders (nIND, n = 15), and to analyze their correlation with other biomarkers.pwMS were divided into three series based on their origin. CSF samples were unavailable for HC. All three pwMS cohorts had lower sC1q levels compared to HC and IND. csfC1q was higher in one pwMS cohort, with a trend in another, and correlated with IgG, Free Kappa Light Chains, GFAP, and Chitinase-3 Like Protein-1 in CSF. Our findings suggest a significant role for C1q in MS pathophysiology, potentially serving as a biomarker for disease identification.

The Monoclonal Gammopathies of Renal Significance

Abstract As a heterogeneous group of diseases, monoclonal gammopathy is typical for older age and is characterized by the presence of paraprotein in serum and urine. In addition to damage to the bone marrow, other organs are also affected, whereas in the case of monoclonal gammopathy of renal significance (MGRS), the kidneys are dominant. The clinical presentation of MGRS is diverse depending on the type of renal monoclonal gammopathy. The basis of the diagnosis is the detection of free light chains in the serum, along with electrophoresis and subsequent serum immunofixation. The kappa and lambda serum light chain ratio is evaluated in patients with MGRS within modified reference parameters. A kidney biopsy provides information on the extent of renal parenchymal involvement. The basis of therapy is chemotherapeutics with possible autologous stem cell transplantation to preserve kidney function.

Bi-specific antibody engagers for cancer immunotherapy.

Bispecific antibody engagers are fusion proteins composed of a nanobody that recognizes immunoglobulin kappa light chains (VHH kappa ) and a nanobody that recognizes either CTLA-4 or PD-L1. These fusions show strong antitumor activity in mice through recruitment of polyclonal immunoglobulins independently of specificity or isotype. In the MC38 mouse model of colorectal carcinoma, the anti-CTLA-4 VHH-VHH kappa conjugate eradicates tumors and reduces the number of intratumoral regulatory T cells. The anti-PD-L1 VHH-VHH kappa conjugate is less effective in the MC38 model, whilst still outperforming an antibody of similar specificity. The potency of the anti-PD-L1 VHH-VHH kappa conjugate was strongly enhanced by installation of the cytotoxic drug maytansine or a STING agonist. The ability of such fusions to engage the Fc-mediated functions of all immunoglobulin isotypes is an appealing strategy to further improve on the efficacy of immune checkpoint blockade, commonly delivered as a monoclonal immunoglobulin of a single defined isotype.

Translational pharmacokinetic/pharmacodynamic model for mRNA-0184, an investigational therapeutic for the treatment of heart failure.

Heart failure (HF) is a complex, progressive disorder that is associated with substantial morbidity and mortality on a global scale. Relaxin-2 is a naturally occurring hormone that may have potential therapeutic benefit for patients with HF. To investigate the therapeutic potential of relaxin in the treatment of patients with HF, mRNA-0184, a novel, investigational, lipid nanoparticle (LNP)-encapsulated mRNA therapy that encodes for human relaxin-2 fused to variable light chain kappa (Rel2-vlk) was developed. A translational semi-mechanistic population pharmacokinetic (PK)/pharmacodynamic (PD) model was developed using data from non-human primates at dose levels ranging from 0.15 to 1 mg/kg. The PK/PD model was able to describe the PK of Rel2-vlk mRNA and translated Rel2-vlk protein in non-human primates adequately with relatively precise estimates. The preclinical PK/PD model was then scaled allometrically to determine the human mRNA-0184 dose that would achieve therapeutic levels of Rel2-vlk protein expression in patients with stable HF with reduced ejection fraction. Model-based simulations derived from the scaled PK/PD model support the selection of 0.025 mg/kg as an appropriate starting human dose of mRNA-0184 to achieve average trough relaxin levels between 1 and 2.5 ng/mL, which is the potential exposure for cardioprotective action of relaxin.

Structural modification of 2-phenylquinoline-4-carboxylic acid containing SIRT3 inhibitors for the cancer differentiation therapy.

Inhibition of SIRT3 exhibited potency in triggering leukemic cell differentiation. In discovery of potent SIRT3 inhibitors for cancer differentiation therapy, structural modification was performed on the previously developed lead compound P6. A total of 33 compounds were designed and synthesized. In the enzyme inhibitory assay, several molecules S18, S26, S27 and T5 showed potent SIRT3 inhibitory activity with IC50 value of 0.53, 1.86, 5.06, and 2.88 μM, respectively. Moreover, the tested compounds exhibited SIRT3 inhibitory selectivity over SIRT1 and SIRT2. Compounds S27 and T5 were potent in inhibition the growth of MM1.S and RPMI-8226 cells in the invitro antiproliferative test. Significantly, representative compounds, especially S27 and T5, promoted differentiation of tested MM cells in the cellular morphological evaluation, accompanied by increasing the expression of differentiation antigen CD49e and human immunoglobulin light chain lambda and kappa. Additionally, molecule S18 without antiproliferative potency itself, showed significant inhibitory activity against growth factor IL-6 induced RPMI-8226 cell proliferation. Collectively, potent SIRT3 selective inhibitors with MM cell differentiation potency were developed for further discovery of anticancer drugs.

Effects of ashwagandha (Withania somnifera) root extract on aging-related changes in healthy geriatric dogs: A randomized, double-blinded placebo-controlled study.

This study aimed to explore the clinical potential of Withania somnifera/ashwagandha root extract (ARE) to mitigate age-related changes in healthy geriatric dogs. We hypothesized that ARE can reduce the effects of advancing age, including physiological changes, immune response decline and susceptibility to diseases, by its immunomodulatory effects. A randomized, double-blind, placebo-controlled trial was conducted in Telangana, India, from July 2022 to September 2022. Twenty apparently healthy dogs, aged 8 years or older, were enrolled. The dogs were divided into two groups to receive ARE (15mg/kg, once daily, orally) or a placebo control. Various parameters, including serum cortisol levels, haematological profiles, biochemical markers, antioxidant indicators and anti-inflammatory responses, were assessed at the initiation of study, day 30, and day 60. The erythrocyte count and haemoglobin levels were significantly increased with ARE (p<0.001), whereas leukocyte count decreased (p<0.05). Moreover, significant decreases in important markers of liver function (alanine aminotransferase, aspartate aminotransferase, albumin and globulin; p<0.001 at day 60), as well as kidney function markers (creatinine and blood urea nitrogen; p<0.001 at days 30 and 60), were observed in ARE-treated dogs compared to the placebo control group. In addition, the levels of markers of oxidative stress (superoxide dismutase, catalase, glutathione and malondialdehyde) were significantly modulated by ARE intervention, indicating strong antioxidant effects. Interestingly, serum cortisol levels reduced significantly with ARE (p<0.001). Compared to baseline, ARE significantly decreased key inflammatory markers, including interferon-γ, tumour necrosis factor-α, nuclear factor kappa light chain enhancer of activated B cells and interleukin-10 (p<0.001) levels at day 60. In conclusion, the findings of this study suggest that ARE has adaptogenic properties in healthy geriatric dogs by improving haematological and biochemical profiles, enhancing antioxidant defence, reducing stress and modulating inflammatory responses.