Peptide specific monoclonal antibodies of Leptospiral LigA for acute diagnosis of leptospirosis.

Leptospirosis is underdiagnosed due to low sensitivity, need of specialised equipment, and expensive reagents for serological and molecular diagnosis respectively. Considering the sensitivity, rapidity, inexpensive reagents and collection of clinical samples, the monoclonal antibody based antigen detection method from urine samples has been developed and evaluated. LigA (LK90) based B-cell specific epitopes were predicted and synthesised as peptides for the production of monoclonal antibody. LK90543: SNAQKNQGNA (amino acids: 543 to 552), and LK901110: DHHTQSSYTP (amino acids: 1110 to 1119) with VaxiJen score of 1.3719 and 1.2215, respectively were used. Thirty two and 28 urine samples from confirmed and seronegative healthy human subjects, respectively were included for the evaluation of MAb-based dot blot ELISA. The specificity of the evaluated MAbs, P1B1 and P4W2 were found to be in the range of ~93-96%. Moreover, the MAbs did not show cross-reactivity with other bacterial antigens as confirmed by IgG ELISA, further validating its specificity for leptospiral antigens. These findings suggest that the developed MAb based dot blot ELISA is a simple, rapid performed in less than 8 h, inexpensive with a ICER of $8.7/QALY, and affordable in developing countries and area where laboratory facilities are limited.

Serum-free light-chain analysis in diagnosis and management of multiple myeloma and related conditions.

The introduction of the serum-free light-chain (S-FLC) assay has been a breakthrough in the diagnosis and management of plasma cell dyscrasias, particularly monoclonal light-chain diseases. The first method, proposed in 2001, quantifies serum-free light-chains using polyclonal antibodies. More recently, assays based on monoclonal antibodies have entered into clinical practice. S-FLC measurement plays a central role in the screening for multiple myeloma and related conditions, in association with electrophoretic techniques. Analysis of S-FLC is essential in assessing the risk of progression of precursor diseases to overt plasma cell dyscrasias. It is also useful for risk stratification in solitary plasmacytoma and AL amyloidosis. The S-FLC measurement is part of the new diagnostic criteria for multiple myeloma, and provides a marker to follow changes in clonal substructure over time. Finally, the evaluation of S-FLC is fundamental for assessing the response to treatment in monoclonal light chain diseases.

Differences identified between serum and urine immunofixation electrophoresis.

BACKGROUND: Different patterns between serum immunofixation electrophoresis (SIFE) and urine immunofixation electrophoresis (UIFE) happen occasionally and laboratorians should understand the mechanisms behind the differences and additional tests required for complicated cases. METHODS: We investigated a complicated multiple myeloma case that showed inconsistent patterns of SIFE and UIFE. To differentiate monoclonal proteins (M-proteins), the urine sample was treated with dithiothreitol to open up the IgA molecule and urine free light chain assay was used for free light chain analysis. RESULTS: The patient's SIFE indicated 2 IgA λ and 1 IgM κ M-proteins, while UIFE revealed monoclonal free λ light chains and a suspicious monoclonal IgA κ protein. Subsequent treatment of the urine sample with dithiothreitol and urine free light chain assay demonstrated that the suspicious monoclonal IgA κ protein was actually a monoclonal IgA λ and a free κ light chain that had similar electrophoretic mobility. CONCLUSIONS: The differences identified between SIFE and UIFE in this case are due to the limitation of immunofixation electrophoresis on different specimen types and intra-molecular disulfide bonds formation in IgA. The laboratorians must be cognizant of the strengths and limitations of the procedure being performed and the ancillary testing techniques available to solve the problem.

Decreased bone remodeling and porosity are associated with improved bone strength in ovariectomized cynomolgus monkeys treated with denosumab, a fully human RANKL antibody.

This study examined the effects of denosumab, an anti-RANKL antibody that inhibits bone resorption, on bone histomorphometry in adult ovariectomized cynomolgus monkeys (OVX cynos). A month after surgery, OVX cynos were treated with subcutaneous vehicle (OVX-Veh) or denosumab (25 or 50mg/kg/month) for 16months (n=14-20/group). Sham controls were treated with vehicle (Sham-Veh; n=17). Areal and volumetric BMD, urine NTx, and serum osteocalcin were measured at baseline and months 3, 6, 12, and 16. Double fluorochrome labels were injected prior to iliac and rib biopsies at month 6 and month 12, and prior to sacrifice at month 16. Histomorphometry was performed on these biopsies, the tibial diaphysis, the L2 vertebra, and the proximal femur. Strength of humeral cortical beams, femur diaphysis, femur neck, and trabecular cores of L5-L6 vertebrae was determined by destructive biomechanical testing. There was no evidence of woven bone, osteomalacia, or other bone histopathologic changes with OVX or with denosumab. OVX-Veh animals exhibited significantly greater bone remodeling at all skeletal sites relative to Sham-Veh controls. Both doses of denosumab markedly inhibited bone remodeling at all sites, including significant reductions in trabecular eroded surfaces (48-86% lower than OVX-Veh controls), cortical porosity (28-72% lower), and dynamic parameters of bone formation (81-100% lower). Decreased fluorochrome labeling with denosumab was related to reductions in cortical porosity and trabecular eroded surfaces, and regression analyses suggested that these reductions contributed to denosumab-related increments in BMD and bone strength. Denosumab-treated animals with the lowest levels of fluorescent labeling exhibited the greatest structural bone strength values at each site. Intracortical remodeling had no relationship with material properties including ultimate strength, elastic modulus or toughness (r(2)=0.00-0.01). These data suggest that remodeling inhibition with denosumab improved structural strength without altering material properties under these experimental conditions. Greater structural strength in the denosumab-treated animals can be primarily explained by the combined effects of increased trabecular and cortical bone mass, and reductions in trabecular eroded surfaces and cortical porosity.

Membranoproliferative glomerulonephritis secondary to monoclonal gammopathy.

BACKGROUND AND OBJECTIVES: Membranoproliferative glomerulonephritis (MPGN) is an immune complex-mediated glomerulonephritis characterized by subendothelial and mesangial deposition of immune complexes. Autoimmune diseases and chronic infections, such as hepatitis C, are commonly recognized causes of MPGN; however, monoclonal gammopathy is a less widely recognized cause of MPGN. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We reviewed all renal biopsies of MPGN in Mayo Clinic patients during a 6-year period to determine the association of monoclonal gammopathy with MPGN. Results were correlated with electrophoresis studies and bone marrow biopsies to clarify the relationship between MPGN and gammopathies. RESULTS: Of 126 patients with MPGN, 20 did not have workup for hepatitis B or C. Of the remaining 106 patients, 25 (23.5%) were positive for hepatitis B or C. Of the 81 hepatitis-negative patients, 13 were not evaluated for gammopathies. Of the remaining 68 patients, 28 (41.1%) had serum and/or urine electrophoresis studies positive for monoclonal gammopathy. Serum immunofixation electrophoresis was the most sensitive method for diagnosing monoclonal gammopathy. Renal biopsy showed a membranoproliferative pattern of injury; immunofluorescence microscopy was often instrumental in diagnosing the underlying gammopathy. On the basis of the bone marrow biopsy, monoclonal gammopathy of undetermined significance was the most common entity associated with MPGN. Other, less common causes included multiple myeloma, low-grade B cell lymphoma, and chronic lymphocytic leukemia. CONCLUSIONS: Monoclonal gammopathy is an important and common cause of MPGN; therefore, all patients with a diagnosis of MPGN should be evaluated for an underlying monoclonal gammopathy.

Serum free light chain analysis may miss monoclonal light chains that urine immunofixation electrophoreses would detect.

BACKGROUND: A nephelometric assay for identifying free light chains (FLC) of immunoglobulin in serum is commercially available. Although this assay is not specific for monoclonal FLC, an abnormal kappa/lambda ratio is thought to presumptively identify monoclonal FLCs. It has been proposed that serum FLC assays can replace urine immunofixation electrophoresis (IFE) for routine detection. This proposal seems to be based upon testing of samples retrospectively drawn from patients mostly with myeloma or known primary amyloidosis-conditions that are less frequently observed in general laboratory practice. METHODS: Prospective analysis of 5 patient samples by standard care techniques with serum/urine IFE compared with serum FLC nephelometric assay. Rather than classical myeloma, cases were selected on the basis of unusual or equivocal atypical serum protein electrophoresis patterns. RESULTS: In all 5 cases, the nephelometric method for free light chains was negative by virtue of serum FLC kappa/lambda ratio being within the reference range, contrasting with monoclonal proteins or free light chains detected by serum/urine IFE. CONCLUSIONS: Since peculiar or unusual patterns are more commonly observed in general practice, these cases illustrate the pitfalls of relying solely on the serum free light chain analysis method in order to establish the presence of monoclonal FLC.

Improvement of cast nephropathy with plasma exchange depends on the diagnosis and on reduction of serum free light chains.

Cast nephropathy is the most common cause of renal disease in multiple myeloma, however, treatment with plasma exchange remains controversial even after 3 randomized controlled studies. We sought to determine the importance of diagnostic confirmation and goal directed therapy in the treatment of cast nephropathy in forty patients with confirmed multiple myeloma and renal failure who underwent plasma exchange. A positive renal response was defined as a decrease by half in the presenting serum creatinine and dialysis independence. No baseline differences were noted between eventual renal responders and non-responders. Three quarters of the patients with biopsy proven cast nephropathy resolved their renal disease when the free light chains present in the serum were reduced by half or more but there was no significant response when the reduction was less. The median time to a response was about 2 months. In patients without cast nephropathy, renal recovery occurred despite reductions in free light chain levels of the serum. No association was found between free light chains in the serum, urinary monoclonal proteins, overall proteinuria and cast nephropathy. We found that the relationship between renal recovery and free light chain reduction was present only in patients with biopsy proven cast nephropathy showing the importance of extracorporeal light chain removal in this disease.

Cancer therapeutic antibodies come of age: targeting minimal residual disease.

Ten years after the first clinical application of Rituximab, an anti-CD20 recombinant monoclonal antibody, immunotherapy has become common practice in oncology wards. Thanks to the great diversity of the immune system and the powerful methodology of genetic engineering, the pharmacologic potential of antibody-based therapy is far from exhaustion. The recent application of Trastuzumab, an antibody to a receptor tyrosine kinase, in adjuvant breast cancer therapy marks the beginning of a new phase in cancer treatment. Here we discuss molecular mechanisms of antibody-based therapy, the emerging ability to target minimal disease and the therapeutic potential of combining antibodies with other modalities.

Toward critical evaluation of the role(s) of molecular biomarkers in the management of bladder cancer.

Among genitourinary malignancies, bladder cancer is a common, potentially dangerous and exceedingly costly disease entity. Predicated on substantial gains in our understanding of the natural history and molecular biology of bladder cancer, recent years have seen an explosion of new applied technologies to aid clinicians in the management of bladder cancer. Herein we present a systematic overview of general conceptual issues and specific strategies of potential relevance to the clinical evaluation of patients with bladder cancer. A number of basic epidemiological issues provide the relevant background within which we should consider candidate biomarkers. Within this framework, we highlight a number of important recent research findings representative of the large number of candidate bladder cancer biomarkers that have emerged. With several markers already having obtained regulatory approval for clinical use, this topic is of clear relevance not only to researchers but also to the practicing physician. Bladder cancer is a common, costly, and potentially dangerous disease with the opportunity for significant technological inroads in the area of applied biomarkers. An appreciation of basic epidemiological considerations informs our consideration of the state of the art and identifies specific strategies amenable to further innovation.

Monoclonal antibody therapy for hairy cell leukemia.

The use of MoAb therapy for the treatment of HCL offers great promise and potential for improving progression-free survival. Rituximab has activity in the setting of previously treated HCL and the ability to eradicate MRD after 2-CdA given as frontline therapy. Alemtuzumab, epratuzumab, Hu-Max-CD20, and other candidate MoAb's should be studied in HCL. Appropriate pharmacologic investigation, use of antigen modulation, and assessments of soluble antigen levels should be considered with future clinical trial s of MoAb's in HCL to optimize therapeutic strategies.

Dangerous small B-cell clones.

The detection of a monoclonal immunoglobulin in serum or urine usually raises concerns about the size of the underlying B-cell-derived clone and possible systemic effects caused by its expansion. However, a small clone can synthesize a very toxic protein, producing devastating systemic damage and protean clinical presentations. The resulting "monoclonal component-related diseases," although difficult to diagnose, may be progressive and even fatal. The monoclonal protein can aggregate and deposit systemically as occurs in light-chain amyloidosis, monoclonal immunoglobulin deposition disease, crystal-storing histiocytosis, and monoclonal cryoglobulinemia. Alternatively, some monoclonal proteins possess antibody activity toward autogenous antigens and cause chronic cold agglutinin disease, mixed cryoglobulinemia, and peripheral neuropathies. Other humoral mediators may contribute to neuropathy in variant disorders such as the POEMS (polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes) syndrome. The clone synthesizing the noxious monoclonal proteins is often small, and sensitive techniques may be required to detect these immunoglobulins. A delay in diagnosis can allow irreversible organ damage and dramatically shorten survival. Prompt recognition of suggestive signs and symptoms should trigger a thorough diagnostic approach to reach the correct diagnosis quickly, because this is the key to effective therapy. Although the treatment of these conditions is not optimal, significant advances have been made, improving the duration and quality of life.

[Radiation exposure in (90)Y-Zevalin therapy: results of a prospective multicentre trial]. / Strahlenexposition bei der (90)Y-Zevalin-Therapie: Ergebnisse einer prospektiven multizentrischen Studie.

UNLABELLED: AIM of this study was the assessment of the radiation exposure from preparation and application of (90)Y-Zevalin, the measurement of the dose rate at the patient, the exposure of family members as well as the determination of the activity concentration in urine of patients. METHODS: Overall data from 31 therapeutic administrations carried out in four institutions were evaluated. During preparation and application of (90)Y-Zevalin the finger exposures of radiochemists, technicians, and physicians were measured. The dose rate of the patient was measured immediately after radioimmunotherapy. In patients treated in a nuclear medicine therapy unit, urine was collected over a two day period and the corresponding activity was determined. Family members of outpatients were asked to wear a dosimeter over a seven day period. RESULTS: During the preparation we found a maximum skin dose of 6 mSv at the average, and during application of 3 mSv, respectively. After administration of (90)Y the dose rate was 0.4 +/- 0.1 microSv/h at 2 m distance. Urine measurements yielded a cumulated 24 h excretion of 3.9 +/- 1.4% and 4.4 +/- 1.4% within 48 h, respectively, that is equivalent to 43 +/- 18 and 50 +/- 20 MBq of (90)Y, respectively. Family members received a radiation exposure of 40 +/- 14 microSv over seven days. CONCLUSION: During preparation and application of (90)Y-Zevalin appropriate radiation shielding is necessary. For family members as well as nursing staff no additional special radiation protection measures beyond those being common for other nuclear medicine procedures are necessary.

Fanconi's syndrome induced by a monoclonal Vkappa3 light chain in Waldenstrom's macroglobulinemia.

Fanconi's syndrome (FS) is a disorder of sodium-dependent proximal tubule reabsorption, which may complicate plasma cell disorders producing a free monoclonal light chain (LC). FS often occurs in the setting of smoldering myeloma and features cytoplasmic crystalline inclusions of monoclonal kappa LC in proximal tubular cells and malignant plasma cells. Although the clinical and pathological presentation may vary, including lack of crystal formation, monoclonal kappa LCs that underlie FS show a striking genetic and biochemical homogeneity: they almost always belong to the Vkappa1 subgroup of variability and originate from 2 germline genes, O2/O12 or O8/O18. Their variable domain sequences present unusual hydrophobic residues, responsible for the resistance to proteolysis, which leads to LC accumulation in the endocytic compartment of proximal tubule cells. We report a patient with slowly progressive Waldenstrom's macroglobulinemia and full-blown FS with accumulation of a monoclonal kappa LC within proximal tubules, but no detectable crystalline organization. This LC, which belonged to the unusual Vkappa3 subgroup and derived from the L2/L16 germline gene, showed no common substitution with previously described FS kappaI LC and was sensitive to trypsin digestion. These data show that molecular and biochemical characteristics of kappa LCs in patients with FS are more heterogeneous than initially suspected. Mechanisms other than resistance of LCs to endosomal proteolysis probably are involved in the pathogenesis of FS-associated plasma cell dyscrasias.

Insights into mechanisms responsible for mesangial alterations associated with fibrogenic glomerulopathic light chains.

Our previous studies have shown that human mesangial cells (HMCs) incubated with fibrogenic glomerulopathic monoclonal light chains (G-LCs) obtained from the urine of patients with light chain deposition disease produce increased extracellular matrix (ECM) when compared with HMCs not exposed to fibrogenic LCs. This overproduction of ECM proteins is regulated by transforming growth factor-beta (TGF-beta); blocking TGF-beta normalizes the production of ECM proteins. All ECM proteins, after synthesis, have to go through the secretory pathway in the endoplasmic reticulum (ER) and Golgi complex for final maturation and secretion. Blocking the secretory pathway may reduce the accumulation of ECM proteins. We tested the effect of tunicamycin, a specific inhibitor of N-linked glycosylation in the ER which inhibited glycosylation and brefeldin A, an inhibitor of vesicle transport between the endoplasmic reticulum and the Golgi complex, on ECM protein production, both resulting in subsequent upregulation of glucose-regulated protein 78. Overproduction of fibronectin and tenascin by HMCs was normalized by tunicamycin and brefeldin A. Similarly, when HMCs were exposed to exogenous TGF-beta, the increase in fibronectin was reversed by tunicamycin and brefeldin A. Exogenous platelet-derived growth factor-beta (PDGF-beta) did not induce fibronectin overproduction but significantly stimulated proliferation of HMCs. In summary, this study further supports the notion that fibrogenic G-LCs promote the accumulation of ECM proteins, through the actions of TGF-beta. Importantly, the data indicate that altering protein trafficking in the ER results in impairment of secretion of proteins into the ECM. Furthermore, the data also reveal that PDGF-beta and TGF-beta act independently and that PDGF-beta activation by itself cannot increase ECM proteins directly, but only by increasing the number of HMCs.

A comparison of the biodistribution and biokinetics of (99m)Tc-anti-CD66 mAb BW 250/183 and (99m)Tc-anti-CD45 mAb YTH 24.5 with regard to suitability for myeloablative radioimmunotherapy.

Radioimmunotherapy (RIT) with radiolabelled monoclonal antibodies (mAbs) is an effective method of achieving myeloablation in leukaemia patients prior to stem cell transplantation (SCT). We wished to compare the approaches of specific binding to leukaemic blasts and non-specific binding to benign red marrow cells, which results in a myeloablative "cross-fire" effect. Therefore, we prospectively evaluated the biodistribution and biokinetics of the anti-CD45 mAb YTH 24.5 and the anti-CD66 mAb BW 250/183 with regard to their suitability for myeloablative RIT. The red marrow selective anti-CD66 mAb BW 250/183 (IgG1) binds to normal granulopoietic cells. In contrast, the anti-CD45 mAb YTH 24.5 (IgG2b) binds to 85-90% of acute leukaemic blasts and almost all haematopoietic white cells. Patients with leukaemic blast infiltration of the marrow <25% and assigned for RIT and SCT were included. Twelve patients (eight male, four female; median age 46+/-7 years) with AML (5), CML (5) or ALL (2) were examined. Both mAbs were labelled with technetium-99m. Within 48 h, 906+/-209 MBq (99m)Tc-anti-CD66 mAb and 760+/-331 MBq (99m)Tc-anti-CD45 mAb were injected consecutively. Scintigraphic and urinary measurements were performed 1, 2, 4 and 24 h after injection. Serum activities were evaluated 2, 5, 10, 15, 30 and 60 min and 2, 4 and 24 h after injection. Compared with the anti-CD45 mAb, the anti-CD66 mAb showed an approximately fourfold higher accumulation in the red marrow, a 2.5-fold lower accumulation in the liver and similar accumulation in the kidneys. The serum activity (% of the injected dose) initially decreased faster for the anti-CD45 mAb but was similar for the two mAbs 24 h after injection: 3.3%+/-1.2% (anti-CD66 mAb) and 2.4%+/-1.1% (anti-CD45 mAb). The cumulated urinary excretion was 17%+/-6.6% (anti-CD66 mAb) and 27.3%+/-7.9% (anti-CD45 mAb) 24 h after application. In these patients with low tumour load, the anti-CD66 mAb BW 250/183 showed more favourable properties in terms of biodistribution and pharmacokinetics. Thus, it appears superior to anti-CD45 mAb YTH 24.5 in selectively increasing the marrow dose and avoiding extramedullary organ toxicity.

Role of uCyt+ in the detection and surveillance of urothelial carcinoma.

OBJECTIVES: To test the clinical value and role of uCyt+ as a noninvasive tool for the detection and surveillance of urothelial carcinoma. METHODS: Included in this prospective study were 235 patients (mean age 71.5 years, range 32 to 86). Of these, 98 patients had signs and symptoms suggestive of bladder cancer and 137 patients were being followed up after complete transurethral resection of superficial urothelial cancer (UC). All patients underwent urinary cytology and the uCyt+ test performed on ThinPrep (thin layer). All underwent subsequent cystoscopy and evaluation of any suspicious lesion by biopsy. RESULTS: A total of 102 patients had histologically proven UC. In the group of patients with signs and symptoms suspicious of UC, the sensitivity of cytology increased from 5% for G1 to 84.6% for G3 tumors; for uCyt+, it was 85% for G1, 100% for G2, and 92.3% for G3 tumors. Combining cytology and uCyt+, the sensitivity was 85% for G1 and 100% for G2 and G3. In the group of follow-up patients, the sensitivity of cytology increased from 4.3% for G1 to 94.4% for G3 tumors; for uCyt+, it was 78.2% for G1, 70% for G2, and 94.4% for G3 tumors. Combining both tests, the sensitivity was 78.2% for G1, 90% for G2, and 100% for G3. CONCLUSIONS: The uCyt+ is a valid test in the detection of UC of all grades and stages. It improves the sensitivity of cytology in low-grade tumors. The two tests combined may be a highly sensitive method to detect UC early in detection and surveillance.