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Amyloid A (AA) amyloidosis is a fatal disease caused by extracellular deposition of fibrils derived from serum AA (SAA). AA amyloid fibril formation has previously been modeled in macrophage cultures using highly amyloidogenic mouse SAA1.1, but attempts to do the same with human SAA invariably failed. Our objective was to define conditions that support human SAA-derived amyloid formation in peripheral blood mononuclear cell (PBMC) cultures. Two conditions were found to be critical - omission of fetal calf serum and use of StemPro34, a lipid-enriched medium formulated for hematopoietic progenitor cells. Cultures maintained in serum-free StemPro34 and provided with recombinant human SAA1 in the complete absence of amyloid-enhancing factor exhibited amyloid deposition within 7 d. Amyloid co-localized with cell clusters that characteristically included cells of fibrocytic/dendritic morphology as well as macrophages. These cells formed networks that appeared to serve as scaffolding within and upon which amyloid accumulated. Cells in amyloid-forming cultures demonstrated increased adherence, survival and expression of extracellular matrix components. Of the three human SAA1 isoforms, SAA1.3 showed the most extensive amyloid deposition, consistent with it being the most prevalent isoform in Japanese patients with AA amyloidosis. Attesting to the reproducibility and general applicability of this model, amyloid formation has been documented in cultures established from eight PBMC donors.

Systemic reactive amyloid A (AA) amyloidosis is one of the critical complications associated with rheumatoid arthritis (RA). Recently, there are several useful reports of anti-tumor necrosis factor therapy for RA-related systemic reactive AA amyloidosis patients. However, the time-kinetic transition between effective anti-inflammatory therapies and regression of AA amyloid deposits remains uncertain. Here, we report a RA patient with systemic reactive AA amyloidosis who was successfully treated with prednisolone and etanercept, showing marked regression of gastroduodenal mucosal amyloid deposits within only 4 months. This is the first case report of RA-related systemic reactive AA amyloidosis histopathologically demonstrating rapid regression of amyloid deposits on gastroduodenal mucosa after adequate suppression of the underlying inflammatory condition.

A 60-year-old woman was found to have proteinuria and a lung nodule. The surgically resected left upper lobe contained a nodule, in which the adenocarcinoma was surrounded by a heavy deposition of amyloid. Subsequent renal and gastric biopsies demonstrated amyloid deposition with Aλ immunoreactivity. She was treated with 2 courses of VAD (vincristine, doxorubicin and dexamethasone), resulting in the disappearance of Bence Jones proteinuria. Her nephrotic syndrome has been improving during the subsequent 3 years. The rare lung nodule consisting of adenocarcinoma and amyloid deposition was a diagnostic clue in this primary systemic AL amyloidosis patient.

We report 3 patients with nephrotic syndrome ascribed to primary systemic AL amyloidosis that were successfully treated with VAD (vincristine, doxorubicin and dexamethasone) alone. M-protein in serum disappeared soon after VAD, and nephrotic syndrome gradually improved in parallel with a decrease in daily protein excretion in urine. Long-term follow-up of these patients showed neither relapse of nephrotic syndrome nor reappearance of M-protein. High-dose melphalan followed by autologous stem cell support is a standard therapy for primary systemic AL amyloidosis, but in high-risk cases for this treatment, such as elderly patients and those with multiple organ involvement, VAD might be a therapeutic option.

Objective: Intensive chemotherapy targeting plasma cell dyscrasia has been recently employed for the treatment of primary systemic AL amyloidosis. We prospectively studied the clinical usefulness of cyclic VAD (vincristine, doxorubicin and dexamethasone) in patients with primary systemic AL amyloidosis who were ineligible for high-dose melphalan with autologous stem cell support., Patients and Methods: Eight patients (mean age, 60.4+/-8.8 years) were treated with cyclic VAD until the disappearance of M-protein from both serum and urine. Of these, seven showed nephrotic syndrome before the start of VAD irrespective of a decrease in creatinine clearance. Serial follow-up studies after VAD evaluated hematological status and organ function., Results: Four patients (50%) showed a marked decrease in abnormal plasma cells in the bone marrow and normalized kappa/lambda ratios of serum free light chain in conjunction with disappearance of M-protein after 1 to 3 courses of VAD. There were no serious adverse events, and nephrotic syndrome gradually improved with no hematological relapse in the follow-up period of 3 to 5 years. The remaining 4 patients showed worsening of congestive heart failure and/or systemic edema ascribable to dexamethasone, resulting in cessation of cyclic VAD before disappearance of M-protein. All of these patients died of multiple organ failure or required permanent hemodialysis within 1 year after the start of cyclic VAD., Conclusion: Cyclic VAD is a potent therapeutic option in primary systemic AL amyloidosis, but in patients with renal or cardiac dysfunction careful management for adverse events, especially body fluid retention, is necessary.

We report a patient with myeloma-associated systemic AL amyloidosis who showed chronic polyarthralgia as the main symptom. The clinical picture was similar to that of rheumatoid arthritis with regard to symmetrical swelling with tenderness in multiple joints, but inflammatory reactions were almost normal and autoantibodies were negative. He was diagnosed as having systemic AL amyloidosis based on deposition of kappa-light chain-immunoreactive amyloid in biopsied tissue and Bence Jones protein in serum and urine. Magnetic resonance imaging and bone scintigraphy suggested this disease as the cause of the polyarthralgia. Systemic AL amyloidosis may be important in the differential diagnosis of chronic polyarthralgia.

Objective: Primary systemic AL amyloidosis arises from immunoglobulin light chains produced by plasma cell dyscrasia. To prospectively investigate the production of M-protein and plasma cells in bone marrow before and after chemotherapy, we performed flow cytometry and analysis of serum free light chains (FLCs)., Patients and Methods: Fifty-nine patients with primary systemic AL amyloidosis (mean age, 59.9+/-8.8 years) were enrolled in this study, and of these 31 were serially studied before and after chemotherapy. Complete hematological remission was defined as normalization of the FLC kappa/lambda ratio., Results: MPC-1(-)CD45(-) (p<0.05) and MPC-1(+)CD45(-)CD49e(-) (p<0.005) were significantly higher, and MPC-1(-)-CD45(+) (p<0.05), MPC-1(+)CD45(+)CD49e(-) (p<0.0001) and MPC-1(+)CD45(+)CD49e(+) (p<0.0005) were significantly lower in the patients with AL amyloidosis than in controls. There was a significantly positive correlation between the serum predominant FLC/serum creatinine ratio and MPC-1(+)CD45(-)CD49e(-) (p<0.05). After chemotherapies, such as high-dose melphalan with autologous stem cell support, 20 of 31 patients with AL amyloidosis achieved complete hematological remission. There were no significant differences in any subtype of plasma cells before treatment between the remission and non-remission groups, but in the former group MPC-1(+)CD45(-)CD49e(-) and MPC-1(-)CD45(+) were significantly decreased and increased after chemotherapy compared with before, respectively., Conclusion: Abnormal plasma cells in the bone marrow, particularly the MPC-1(+)CD45(-)CD49e(-) subset, may be important as a follow-up marker before and after chemotherapy in primary systemic AL amyloidosis. These cells maintain low levels as long as no relapse occurs.

A patient with primary systemic AL amyloidosis achieved partial hematological response after 2 courses of VAD (vincristine, doxorubicin and dexamethasone) and subsequent high-dose melphalan followed by autologous peripheral blood stem cell transplantation despite involvement of multiple organs, including the heart. In this patient natriuretic peptides and free light chains in serum were useful as markers of cardiac involvement and therapeutic effects, respectively. When amyloidosis-related dysfunction is seen in multiple organs, this intensive chemotherapy might be a possible therapeutic option, although several modifications in the regimen and careful management are necessary.

Objective: Immunoglobulin-related free light chains (FLCs) in serum have recently become quantitatively detectable using the nephelometric assay in plasma cell disorders, including multiple myeloma and AL amyloidosis. To investigate whether FLCs are useful as a diagnostic and therapeutic marker in Japanese patients with primary systemic AL amyloidosis, we determined these values in serum before and after chemotherapy., Patients and Methods: The serum FLC analysis was carried out in 25 patients with primary systemic AL amyloidosis (mean age, 60.1+/-8.4 years). All of the patients were shown to have either ALkappa- or ALlambda-immunoreactive amyloid deposits on biopsied tissues. Thirteen patients were treated with VAD (vincristine, doxorubicin and dexamethasone) alone (n=6) or VAD and subsequent high-dose melphalan followed by autologous stem cell support (n=7), and serum FLCs were serially determined before and after the chemotherapy., Results: Before chemotherapy the amyloidogenic FLC was elevated in serum with or without abnormal e/e ratios in 24 patients, including 5 with undetectable M-protein in both serum and urine on immunofixation. After chemotherapy the amyloidogenic FLC in serum was significantly decreased irrespective of high-dose melphalan (p<0.05), and all the patients with normalized kappa/lambda ratios showed a good prognosis., Conclusions: With respect to sensitivity and quantification serum FLCs will be a key marker for diagnosis and therapeutic effects in primary systemic AL amyloidosis. The prognosis of patients with this disease may be improved if the kappa/lambda ratio in serum can be normalized by intensive chemotherapy.

This report concerns two patients with severe protein losing enteropathy and refractory diarrhea due to AA amyloidosis who were successfully treated with corticosteroid and octreotide. In these patients, biopsied tissues from the gastrointestinal (GI) tract showed extensive deposition of AA amyloid, which was caused by rheumatoid arthritis in one case and was of unidentified etiology in the other. Both patients manifested severe diarrhea unresponsive to conventional treatment with hypoproteinemia, and protein leakage from the small intestine to the ascending colon was confirmed by 99mTc-diethylene triamine pentaacetic acid human serum albumin (HSA-D) scintigraphy. Soon after starting a long-acting somatostatin analogue, octreotide, with co-administration of oral prednisolone, their general status improved in parallel with a rapid decrease in the volume of watery diarrhea and an increase in serum levels of albumin and IgG. Also on 99mTc-HSA-D scintigraphy protein leakage from the GI tract was apparently decreased in both patients. Combination therapy with a somatostatin analogue and corticosteroid may be effective for protein losing enteropathy with intractable diarrhea ascribable to GI amyloidosis. Because of the lack of specific therapies in this serious clinical situation, the described therapy should actively be considered as a therapeutic option not only in AA amyloidosis, but also in other types of systemic amyloidosis.

To investigate whether there is a correlation between subtypes of plasma cells in the bone marrow and the production of M-protein, flow cytometry and serum free light chain (FLC) analyses were carried out in 17 patients with primary systemic AL amyloidosis (mean age, 59.9+/-8.8 years) and controls with M-protein (MGUS controls, n=6) and without it (negative controls, n=9). The patients showed a significantly higher value in the serum predominant FLC:serum creatinine ratio (43.8+/-63.2) and CD38++ CD19- CD56+ subpopulation (monoclonal plasma cells) (2.57+/-5.35%) than either the negative (p<0.0005 and p<0.001, respectively) or MGUS controls (p<0.05). With respect to maturation of plasma cells in the bone marrow, the intermediate (MPC-1+ CD45- CD49e-) and mature (MPC-1+ CD45+ CD49e-) subtypes were significantly higher (49.2+/-23.2%, p<0.005) and lower (27.6+/-21.3%, p<0.005) in the patients than in the negative controls, respectively. The serum predominant FLC:serum creatinine ratio was elevated in parallel with an increase in CD38++ CD19- CD56+ and MPC-1+ CD45- CD49e- cells and a decrease in mature subtypes (MPC-1+ CD45+ CD49e- and MPC-1+ CD45+ CD49e+ cells), There was a significantly positive correlation between the serum predominant FLC:serum creatinine ratio and either CD38++ CD19- CD56+ (r=0.510, p<0.05) or MPC-1+ CD45- CD49e- cells (r=0.481, p<0.05). In primary AL amyloidosis M-protein is probably produced by increased monoclonal plasma cells in the bone marrow, particularly by the intermediate subpopulation with a phenotype of MPC-1+ CD45- CD49e-.

Patients with AL amyloidosis were treated with VAD (vincristine, doxorubicin and dexamethasone) with or without high-dose melphalan followed by auto-PBSCT according to eligibility criteria based on disease severity, and prospectively investigated the therapeutic benefits and complications. Thirty-one patients were enrolled in this study. VAD and subsequent high-dose melphalan with auto-PBSCT were performed only in patients who met all of the eligibility criteria. Among patients ineligible for this treatment, VAD alone was performed in those with satisfactory general status. Eleven patients met the eligibility criteria, and of these, 7 were treated with VAD and subsequent high-dose melphalan with auto-PBSCT. Seven patients received VAD alone, and the remaining 17 were treated with the supportive therapy. Among the 14 patients treated with chemotherapy, 9 (5 of the 7 treated with VAD and high-dose melphalan, and 4 of the 7 treated with VAD alone) showed complete hematological response with apparent improvement of amyloidosis-related clinical symptoms. Serious complications of chemotherapy were cytomegalovirus infection and pneumocystis carinii pneumonia seen in 1 and 2 patients, respectively. These chemotherapies may be effective for reduction of M-protein and are also useful in improving of amyloidosis-induced organ dysfunction. In patients who cannot tolerate high-dose melphalan, VAD alone is a potent therapeutic option, although there are possible harmful effects on the heart and peripheral nerve.

This report concerns a patient with IgM AL amyloidosis due to a B cell lymphoproliferative disorder who was successfully treated with VAD and subsequent high-dose melphalan followed by autologous stem cell support. After this chemotherapeutic regimen, the patient showed complete hematological remission and improvement in nephrotic syndrome. These findings suggest that high-dose melphalan may also be effective for lymphoplasmacytoid cells producing monoclonal IgM which are phenotypically distinct from plasma cells. Myeloablative therapies, such as high-dose melphalan, should definitely be considered as a treatment option for AL amyloidosis, irrespective of the type of precursor immunoglobulin.

Objective: To detect amyloid deposits in the early phase of illness, we investigated the usefulness of abdominal fat aspiration biopsy along with genotyping of serum amyloid A (SAA) in patients with rheumatoid arthritis (RA)., Patients and Methods: We performed abdominal fat aspiration biopsy with phenol Congo red staining and determined inflammatory markers, including CRP and SAA, in 217 patients with an RA history longer than 5 years (mean age, 64.1 +/- 10.6 years). Genotypes of SAA1 and 2 were investigated in 127 patients with RA by a polymerase chain reaction-restriction fragment length polymorphism analysis., Results: In the abdominal fat aspiration biopsy 17 patients (7.8%) demonstrated amyloid deposits, which were confirmed as AA type by immunostaining on biopsied tissues from other organs, and nine of them showed no clinical symptoms ascribable to amyloidosis. RA patients with amyloidosis showed significantly higher serum levels of CRP (p < 0.05) and SAA (p < 0.0001) than those without amyloidosis. In the genotyping, amyloid deposition was significantly correlated with the frequency of SAA1.3 (p < 0.005 vs. 1.1, p < 0.05 vs. 1.5). Comparison of inflammatory markers between the number of SAA1.3 alleles showed that the SAA/CRP ratio and SAA concentration were higher in the 1.3 homozygote than in the others (p < 0.05). Two patients demonstrated amyloid deposits at the second abdominal fat biopsy one year after the first, and their SAA1 genotypes were 1.3/1.5 and 1.3/1.3., Conclusion: In RA patients confirmed as having SAA1.3, serial examinations with abdominal fat aspiration biopsy might contribute greatly to the early detection of amyloidosis during the long-term follow-up.

Objective: Systemic reactive AA amyloidosis is an intractable complication in patients with a long history of rheumatoid arthritis (RA). To help to more easily and reliably detect the presence of this form of amyloidosis in patients with RA and start intensive treatment as early as possible, we examined the sensitivity and usefulness of abdominal fat aspiration biopsy with phenol Congo red staining in the diagnosis of AA amyloidosis., Patients and Methods: Ten patients were diagnosed with systemic reactive AA amyloidosis secondary to RA (all women; mean age, 70.2 +/- 6.4 years; mean disease duration of RA, 20.3 +/- 11.2 years) based on histopathological examinations of biopsied specimens mainly from the gastroduodenal mucosa. Abdominal fat aspiration biopsy was performed in these patients, and the specimens were treated with both classical alkaline and phenol Congo red staining., Results: Phenol Congo red staining revealed amyloid deposits in all 10 patients, while conventional alkaline Congo red staining showed a positive result in 7 patients. In the patients with a positive result with alkaline Congo red staining, reactivity of one grade or two higher was demonstrated by the phenol Congo red method., Conclusion: Phenol Congo red staining is superior to the classical alkaline Congo red staining with respect to the detection of AA-amyloid deposits in biopsied abdominal fat tissue specimens. In addition to easy access and procedural safety, abdominal fat aspiration biopsy might contribute reliably to the diagnosis of systemic reactive AA amyloidosis secondary to RA when phenol Congo red staining is employed.