Primary local orbital amyloidosis: biochemical identification of the immunoglobulin light chain kappaIII subtype in a small formalin fixed, paraffin wax embedded tissue sample.

BACKGROUND: Amyloidosis refers to a heterogeneous group of disorders associated with the deposition of chemically distinct amyloid fibril proteins. Precise determination of chemical amyloid type has diagnostic, therapeutic, and prognostic relevance. Although immunohistochemical techniques are used routinely to determine the amyloid type, the results can be negative or inconclusive, so that biochemical characterisation is often required. The development and application of new biochemical microtechniques suitable for examination of extremely small tissue samples is essential for precise identification of the deposited amyloid proteins. AIMS: To investigate biochemically the amyloid proteins present in a formalin fixed paraffin wax embedded orbital tissue from a patient with localised orbital amyloidosis in whom immunohistochemistry was not helpful in the determination of amyloid type. METHODS: Extraction of amyloid proteins from fixed tissue and their identification was carried out by a recently developed microtechnique. An extremely small tissue sample was dewaxed and extracted with formic acid. The extracted material was analysed using electrophoresis, western blotting, and amino acid sequencing. RESULTS: Biochemical examination of the extracted proteins showed the presence of immunoglobulin (Ig) derived amyloid proteins, which were composed of the N-terminal fragments of the Ig light chain kappaIII subtype (AL-kappaIII) (16, 8, and 3 kDa). CONCLUSIONS: This is the first chemically proved AL case reported in association with primary localised orbital amyloidosis. The biochemical microtechnique used was useful in achieving a precise diagnosis of amyloid disease, in a case where the results of routine immunohistochemical examination of amyloid were inconclusive.

Immunophenotypic characterization of myelopoiesis in early and late myelodysplastic syndromes: use of CD44 as an aid in early diagnosis.

We investigated the possibility that myeloid cells from the bone marrow (BM) of myelodysplastic patients differ in their expression of CD44 antigen compared with expression of the antigen in normal controls. In addition, two triple-surface marker assays incorporating, respectively, CD44/CD33/CD66 and CD33/CD34/HLA-DR were used to evaluate the degree of myeloid maturation and assess the number of blasts in BM by flow cytometry. Patients with early-stage myelodysplastic syndrome (MDS; RA [FAB classification]) have significantly decreased expression of CD44 on gated myeloid cells. In contrast, patients with late-stage MDS (RAEB and RAEB-T [FAB classification]) showed an elevated expression of CD44 and an increased number of CD34 blasts compared with early-stage MDS patients and normal controls. Late-stage MDS patients also had an increase in the immature myeloid compartment (CD66 weak expression) compared with early-stage MDS patients and normal controls. We have already included this assay as part of our MDS evaluation protocol alongside BM morphology and cytogenetics.

Acute tumor lysis syndrome induced by high-dose corticosteroids in a patient with chronic lymphatic leukemia.

Acute tumor lysis syndrome (TLS) has been reported in hematological malignancies, such as aggressive non-Hodgkin's lymphoma, acute lymphoblastic leukemia, and rarely, in other malignancies (solid tumors) in association with the administration of cytotoxic therapy. We report a case of a patient with chronic lymphatic leukemia (CLL) who developed autoimmune hemolytic anemia treated by high dose corticosteroids and, following this treatment, developed acute tumor lysis syndrome. Only one similar case has been reported recently. Clinicians should be aware that corticosteroids alone may produce this potentially life-threatening complication.

The polymorphonuclear leukocyte--a new target for erythropoietin.

A previous study from our laboratory has shown that erythropoietin (EPO), beside its traditional role in erythropoiesis, acts as an alleviator of oxidative stress and inflammation in chronic hemodialysis (HD) patients, conferred in part by activated polymorphonuclear leukocytes (PMNLs). To substantiate this phenomenon, the existence of EPO receptors (EPO-Rs) on PMNL membrane was examined at the transcriptional and translational levels. mRNA for EPO-R was detected in PMNLs using specific primers directed towards the extracellular region of human EPO-R cDNA. The predicted 300-bp fragment was amplified by reverse transcriptase-polymerase chain reaction. Subcloning and sequence analysis revealed 100% homology of this fragment with human EPO-R. The receptor protein was detected on the surface of intact PMNLs using (125)I-EPO. The protein was further demonstrated by flow cytometric analysis using a fluorescent monoclonal anti-EPO-R. The percentage of PMNLs expressing EPO-R showed a strong correlation with the level of EPO in the serum, suggesting an upregulation of the receptor by the hormone. Taken together with our recent findings that EPO attenuates the oxidative stress and inflammation contributed by PMNLs in HD patients, the detection of functional EPO-R expression in PMNLs places these cells among the nonerythroid, EPO-responsive target populations.

Interaction between erythropoietin and peripheral polymorphonuclear leukocytes in hemodialysis patients.

The effect of erythropoietin (EPO) on the oxidative stress and inflammation caused by polymorphonuclear leukocytes (PMNLs) in chronic hemodialysis (HD) patients was investigated in vivo and in vitro. The studies were performed on isolated PMNLs from peripheral blood of healthy controls and HD patients before and following 6 weeks of EPO treatment. The oxidative stress was expressed by the rate of superoxide release from phorbol 12-myristate 13-acetate stimulated PMNLs, and the inflammatory state was evaluated by in vitro PMNL survival, in addition to white blood cell and PMNL counts of the enrolled subjects. Following 6 weeks of EPO treatment, in HD patients, the rate of superoxide release from PMNLs as well as WBC and PMNL counts fell significantly when compared with the pretreatment values. PMNLs from HD patients and healthy controls incubated in vitro with increasing amounts of EPO displayed a significant reduction in their rates of superoxide release and a significant improvement in survival. We have concluded that EPO interacts with PMNLs, attenuating their primed state in HD patients, thus reducing oxidative stress and the extent of inflammation. To the best of our knowledge, this attenuation of the primed state of PMNLs by EPO is a new finding.

Participation of peripheral polymorphonuclear leukocytes in the oxidative stress and inflammation in patients with essential hypertension.

Oxidative stress and inflammation have recently been linked to endothelial damage in essential hypertension (EH). Activated peripheral polymorphonuclear leukocytes (PMN) damage surrounding tissue by releasing reactive oxygen species (ROS) and proteolytic enzymes before self-necrosis. PMN necrosis further exacerbates inflammation and promotes chemotaxis and PMN recruitment. The number and properties of PMN from untreated EH patients is the focus of the present study. Oxidative stress was assessed by measuring the rate of superoxide anion release from separated, phorbol ester-stimulated PMN and the redox state of plasma glutathione. Inflammation was estimated indirectly by determining PMN number and their in vitro survival. PMN from EH patients (n = 37) released superoxide anion faster (P < .0001) than those of normotensives (NC, n = 37), 17.7 +/- 1.14 v 9.54 +/- 0.51 nmol/10 min/10(6) cells. The redox state of glutathione was twofold higher in EH plasma (P < .02) indicating systemic oxidative stress. PMN survival in vitro correlates linearly with the rate of superoxide release (r2 = 0.60, P < .02) and PMN count of EH patients, although in the normal range, were significantly higher (P < .0001), indicating necrosis and recruitment. Hypertensive plasma significantly reduced NC PMN viability, whereas normal plasma significantly increased EH PMN viability. What our studies show is that EH is accompanied by a primed state PMN that does not correlate with the levels of blood pressure. PMN priming in EH patients reflects an in vivo exposure to a constant stimulus ending in oxidative stress, increased self-necrosis, and cell recruitment. Oxidative stress and inflammation will result in endothelial damage and atherosclerosis in the long run.

Heparin induces apoptosis in human peripheral blood neutrophils.

Programmed cell death, by apoptosis, has been shown to play an important role in the regulation of haemopoiesis. Using trypan blue exclusion for distinguishing intact membranes, flow cytometry for detection of sub G1 peak and in situ terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL), this study shows that heparin induces apoptosis in vitro in human peripheral blood neutrophils. The known anti-proliferative effect of heparin in several in vitro cell systems has therefore to be interpreted in the light of apoptosis. In addition, apoptosis may help explain the anti-inflammatory effects resulting from the interaction between vessel wall heparan sulphate and chemoattracted peripheral blood neutrophils.

Neural-targeted gene therapy for rodent and primate hemiparkinsonism.

Expression of the rate-limiting enzyme for catecholamine biosynthesis, tyrosine hydroxylase (TH), via retroviral and plasmid expression vectors improved the efficacy of conditionally immortalized nigral neural cells in ameliorating rodent and nonhuman primate models of Parkinson's disease through neural transplantation. No improvement in rotational behavior occurred when sham transplants or nondopaminergic transplants were performed. Transplantation of the temperature-sensitive immortalized parental nigral neural line with a TH expression vector resulted in improvement for at least 2 months. Improvement was accompanied by HPLC evidence of increased L-DOPA production and immunocytochemical evidence of TH in the transfected cells increased over that of the parental line. No tumor formation was detected. These results suggest that: (1) temperature-sensitive immortalized neural cells may be genetically engineered successfully to improve their efficacy for the treatment of parkinsonism; and (2) a change in L-DOPA production, as opposed to growth factor production or other factors, is likely to account for the observed improvement, since the parental and derived lines differ by a single gene.

The possible role of fibronectin in multiple myeloma.

Fibronectin (FN) has an active role in the immune response, interacting with a number of different cells and components. It has been implicated in the formation of cryoprecipitates in rheumatic diseases and is present in tissues where under pathological conditions immune complexes are deposited. Under physiological conditions of pH and ionic strength both heavy and light chain of all multiple myeloma and normal IgG show affinity to FN. FN binds to both B and T cells and is shown to inhibit thrombin and collagen-induced platelet aggregation. We have found elevated levels of FN in the plasma of multiple myeloma patients tested compared to a group of normal subjects. Even though the level of FN did not correlate with the level of the paraprotein, our findings raise the possibility that FN might be implicated in some of the clinical symptoms of multiple myeloma.

Transplantation of human neuroblastoma cells, catecholaminergic and non-catecholaminergic: effects on rotational behavior in Parkinson's rat model.

Cultured human catecholaminergic and non-catecholaminergic donor cells were used in neural transplantation experiments in a rat model of Parkinson's disease. Using two different human catecholaminergic neuroblastoma cell lines, one control non-catecholaminergic neuroblastoma cell line, and one sham control (tissue culture medium), transplants were made into the striatum using a modified Ungerstedt hemiparkinsonian rat model. Significant decreases in apomorphine-induced rotational behavior were produced by two of three catecholaminergic cell lines. Grafted cells staining positively for tyrosine hydroxylase (TH) and catecholamine fluorescence indicated viable catecholamine activity in the two cell lines which produced reductions in rotational behavior. Catecholamine fluorescence was not detected in either of the two controls. These data suggest a link between catecholamine secretion by transplanted cells and motor improvement using a rat rotational behavior model.

High plasma fibronectin levels in multiple myeloma patients: possible mechanisms and clinical implications.

Fibronectin (FN) has been implicated in the formation of cryoprecipitates in rheumatic diseases and is present in tissues where, under pathological conditions, immune complexes are frequently deposited. We found elevated levels of FN in the plasma of 92% of multiple myeloma patients tested compared with a group of normal subjects, although the level of FN did not correlate with the level of the paraprotein. We then characterized the interacting fragments on both molecules and found that under physiological conditions of pH and ionic strength both heavy and light chain of all multiple myeloma and normal IgG showed affinity to FN; the active fragment on FN was found to be the aminoterminal heparin-binding domain. These findings raise the possibility that FN might be implicated in some of the clinical symptoms of multiple myeloma.

Four different mutations in codon 28 of alpha spectrin are associated with structurally and functionally abnormal spectrin alpha I/74 in hereditary elliptocytosis.

Hereditary elliptocytosis (HE) Sp alpha I/74 is a disorder associated with defective spectrin (Sp) heterodimer self-association and an abnormal tryptic cleavage of the 80-kD alpha I domain of Sp resulting in increased amounts of a 74-kD peptide. The molecular basis of this disorder is heterogeneous and mutations in codons 28, 46, 48, and 49 (codons 22, 40, 42, and 43 in the previous nomenclature which did not include the six NH2-terminal amino acids) have been reported. In this study we present data on seven unrelated HE Sp alpha I/74 kindred from diverse racial backgrounds in whom we identified four different mutations all occurring in exon 2 of alpha Sp at codon 28. Utilizing the polymerase chain reaction we established a CGT----CTT; Arg----Leu 28 mutation in one kindred of Arab/Druze origin. In two unrelated white kindred of English/European origin the substitution is CGT----AGT; Arg----Ser 28 and in two apparently unrelated white kindred from New Zealand, the mutation is CGT----TGT; Arg----Cys 28. Finally, in one American black kindred and in a black kindred from Ghana the mutation involves CGT----CAT; Arg----His 28. Allele specific oligonucleotide hybridization confirmed that the probands are heterozygous for the respective mutant alleles. All four point mutations abolished an Aha II restriction enzyme site which allowed verification of linkage of the mutation with HE Sp alpha I/74. Our results imply that codon 28 of alpha Sp is a "hot spot" for mutations and also indicate that Arg 28 is critical for the conformational stability and functional self association of Sp heterodimers.

Malignant tumor masquerading as eosinophilic gastroenteritis.

A 49-year-old patient presented with urticaria, vomiting, diarrhea and peripheral eosinophilia. A histological diagnosis of eosinophilic gastroenteritis was made. Within 3 weeks of admission a highly papillary adenocarcinoma of the right ovary was diagnosed. The gastrointestinal symptoms and the eosinophilia disappeared after partial resection of the tumor and chemotherapy. A possible relationship between cancer, eosinophilia and eosinophilic gastroenteritis is discussed.

Prenatal diagnosis of congenital methemoglobinemia with mental retardation.

A case of congenital enzymopenic methemoglobinemia associated with severe mental retardation is described. The deficiency of cytochrome b5 reductase activity in the erythrocytes and the leukocytes of the propositus is demonstrated by kinetic measurement and by disc gel electrophoresis. Analysis of cultured amniotic fluid cells during a second pregnancy of the mother revealed an almost complete deficiency of the enzyme. The absence of cytochrome b5 reductase activity in the blood from the aborted fetus confirmed the prenatal diagnosis. The data presented support the opinion that in pregnancies at risk for the severe form of congenital enzymopenic methemoglobinemia, prenatal diagnosis is warranted.

Serum immunoglobulin-lipid complexes in plasma cell dyscrasia.

Immunoglobulin-lipid complexes (SILC) are found in the serum of all patients with monoclonal gammopathy. Fractionation by density gradient ultracentrifugation, Sephadex G-200 chromatography and Sepharose protein A affinity, followed by extensive immunoassaying for immunoglobulins and lipoproteins, does not confirm the accepted assumption that SILC are immune complexes between monoclonal immunoglobulins and lipoproteins. Lipid extraction of monoclonal fractions isolated on protein A columns followed by thin-layer chromatography shows a lipid pattern characteristic of the one found in cellular membranes. It is proposed that SILC are hydrophobic complexes between immunoglobulins and lipids, as described in membranes of B lymphocytes.

Glucose-6-phosphate isomerase deficiency-Nahariya: extreme in vitro and in vivo lability of the mutant enzyme.

A glucose-6-phosphate isomerase deficiency is described in an Arab boy suffering from chronic hemolytic anemia. The patient was probably true homozygous for the defect. The residual enzyme activity in his red blood cells (RBC) was approximately 30% of normal. The most striking enzyme abnormality observed was an extreme heat lability: upon incubation at 45 C, greater than 90% of activity was lost within 15 min. Furthermore, an increased affinity for the substrate glucose-6-phosphate was shown. The lability of the enzyme was also shown to exist in vivo by separating the patient's RBC into four fractions of different cell age by centrifugation on a discontinuous density gradient. This in vivo lability of the enzyme is believed to be the main cause of the hemolytic diathesis. Remarkably, the residual activity of the enzyme in the RBC of obligate heterozygotes was comparable to that in the patient. However, their enzyme activity was only slightly more labile than that in normal RBC and consequently no signs of hemolysis were noticed.

Spontaneous immunoglobulin changes in human plasma-cell dyscrasia.

A series of spontaneous changes affecting the nature of the immunoglobulin secretion of plasma cels is described in a patient initially diagnosed as IgG lambda benign monoclonal gammopathy. After several years a slight increase in the amount of serum monoclonal immunoglobulin occurred; shortly thereafter an aggressive form of multiple myeloma was diagnosed. Unexpectedly a rapid spontaneous decrease of the monoclonal immunoglobulin, accompanied by the appearance in the serum of increasing quantities of a complex containing intact lambda light chains, then occurred. Concomitantly a fragment of the corresponding free light chain was was detected in the urine. A parallel is drawn between the facts observed in this patient and in an animal model recently proposed to explain the different types of structural immunoglobulin abnormalities in multiple myeloma.