New, third class of amyloid fibril protein.

An unusual protein AR was isolated from the amyloid fibril preparation derived from a patient with primary amyloidosis. Protein AR was unique in its antigenicity, and revealed no structural identity with any known amyloid proteins or with immunoglobulin chains or fragments. Thus a new third class of amyloid fibril proteins besides the immunoglobulin light-chain variable region fragments and the nonimmunoglobulin protein AS, has been characterized. A component antigenically related to protein AR was found in the serum of the patient.

Amyloid-related serum protein SAA in endotoxin-induced amyloidosis of the mink.

The concentration of the amyloid AA-related serum protein (SAA) was markedly increased after endotoxin injections in mink, mouse, rabbit, and man. It was particularly studied during the development of endotoxin-induced amyloidosis of the mink. Protein SAA was markedly elevated in all mink 24 h after the first endotoxin injection but had fallen to relatively low levels before the onset of amyloidosis at about 4 wk. These results are consistent with SAA being a circulating precursor of the amyloid fibril protein, AA. However, both proteins may be derived by proteolysis from a common precursor.

Antibodies reacting with cytoplasm of subthalamic and caudate nuclei neurons in chorea and acute rheumatic fever.

46% of sera from 30 children with rheumatic chorea showed IgG antibody reacting with neuronal cytoplasm of human caudate and subthalamic nuclei. The antibody was also detected in 14% of 50 children with active rheumatic carditis. 55 normal control sera, as well as 148 sera from a broad variety of other disease states showed a low prevalence (1.8-4.0%) of positive reactions. In rheumatic chorea the presence of anti-neuronal antibody appeared to correlate with severity and duration of clinical attacks. Antibody reacting with neuronal cytoplasm was completely removed by absorption with Group A streptococcal membranes or with isolated human neurons from caudate nucleus. Partial absorption of antibody was also recorded using Group A cell wall preparations but not with Group A carbohydrate. No absorption of positive reactions was seen with streptococcal Group D membranes or cell walls. In rheumatic chorea, anti-neuronal antibody appeared to represent cross-reaction with antigens shared by Group A streptococcal membranes.

A serum component related to nonimmunoglobulin amyloid protein AS, a possible precursor of the fibrils.

A nonimmunoglobulin protein with the molecular weight of 9,145 (protein AS) has been shown to be a principal component of the amyloid fibrils in different clinical types of amyloidosis. A protein component, antigenically closely related to protein AS, was detected in human sera. The protein AS-related component (protein ASC) was found in the sera of many groups of patients, including 48 out of 55 patients with various clinical types of amyloidosis. No structural relationship of protein ASC to the plasma component of amyloid was found. Protein ASC was also present with high frequency in the serum of diseases known to be frequently complicated by amyloidosis. In some cases, ASC was found in the sera of patients 2-3 yr before the diagnosis of amyloidosis was established. Protein ASC was also frequently found in hypogammaglobulinemia. Among normal individuals, protein ASC was seldom detected in the serum by our techniques, but there was a noticeable increase with age and during pregnancy. Moreover, a more sensitive technique, immunoelectro-osmophoresis, revealed protein ASC in a higher number of sera from both patients and normal controls. Thus protein ASC was suggested to be a normal serum constituent, usually present only in minor quantities. Under certain conditions, protein ASC increases considerably in serum, and may in such instances act as a precursor for the deposition of amyloid fibrils in the tissues.

Localization of T and B cells and alpha fetoprotein in hepatic biopsies from patients with liver disease.

Peripheral blood and hepatic tissue T- and B-lymphocyte distributions, serum alpha fetoprotein (AFP) concentrations, and hepatic AFP were studied in 46 patients undergoing diagnostic percutaneous liver biopsy. The patients included 26 with alcoholic liver disease, 13 with nonalcoholic hepatitis or cirrhosis, and 7 with either normal histology or minor nonspecific changes. Serum AFP was determined by radioimmunoassay and hepatic tissue AFP by indirect immunofluorescence. Peripheral blood T lymphocytes were identified by the sheep red-cell rosette technique; and B lymphocytes by fluoresceinated anti-immunoglobulin antisera and IgG aggregates. Tissue identification of T lymphocytes was accomplished using an extensively absorbed rabbit antihuman thymocyte antiserum and indirect immunofluorescence; tissue B lymphocytes were identified using pepsin F (ab')2 fragments of rabbit IgG antibodies to human immunoglobulins. T lymphocytes predominanted in hepatic lymphoid infiltrates from patients with alcoholic liver disease (91+/-4%), whereas in patients with chronic active or chronic persistant hepatitis, viral hepatitis, or cryoptogenic cirrhosis proportions of T and B lymphocytic infiltrates were similar (50+/-15%). Hepatic tissue AFP was detected in 9 of 18 patients with alcoholic hepatitis; serum AFP concentration was increased in only 1 of these 9 patients. Tissue AFP was not observed in the remaining biopsy material nor were serum AFP concentrations increased. Peripheral blood T-cell numbers were significantly decreased in patients with alcoholic liver disease (P less than 0.01) and in nonalcoholic hepatitis or cirrhosis (P less than 0.025). A close relationship between peripheral blood T-lymphocytopenia and hepatic T-cell infiltrates was observed in patients with alcoholic liver disease; this relationship was less apparent in patients with nonalcoholic hepatitis or cirrhosis.

Tissue T and B cell infiltration of primary and metastatic cancer.

Immunofluorescent techniques were utilized to identify the types of infiltrating lymphocytes adjacent to human malignant tumors arising from a wide range of anatomic sites. 24 of 29 primary tumors and 5 of 8 metastatic lesions showed varying degrees of lymphocytic infiltration. T cells predominated in the infiltrates in primary tumors (mean 80%, range 50-100%) and this pattern was evident regardless of anatomic site or the presence or absence of metastatic spread. By contrast, B cells predominated at the margins of three of five tumor metastases. Mononuclear cells bearing the Fc receptor were not a prominent component of the infiltrates associated with either primary tumors or metastases, but tumor cell binding of fluoresceinated IgG aggregates was observed in 12 of 29 primary tumors. A significant reduction in peripheral blood T cell numbers occurred in a third of the patients studied. This decrease was not clearly related either to the extent of local tumor T cell infiltration or to the presence of disseminated disease. These preliminary findings provide a descriptive analysis of the local and systemic distributions of immunocompetent cells in cancer.

Antibodies to human caudate nucleus neurons in Huntington's chorea.

Antibodies reacting with neuronal cytoplasmic antigens present in normal human caudate and subthalamic nuclei were detected in 37 of 80 probands afflicted with Huntington's disease (HD). IgG antibodies were detected by immunofluorescence using frozen sections of unfixed normal human and rat brain. Specificity of IgG binding was confirmed using pepsin F(ab')2 fragments of IgG isolated from positive sera. In vitro complement fixation of IgG antibody was detected in 22 of 31 sera tested. Neuronal cytoplasmic antigens reacting with positive HD sera were diminished after trypsin or RNAase treatment of tissue sections but were not removed by DNAase, neuraminidase, EDTA, or dithiothreitol treatment. Antibody staining of neurons could be removed after absorption with isolated caudate nucleus neurons or by using perchloroacetic acid extracts of caudate nucleus. Prevalence of antibody reacting with neuronal cytoplasm was 3% in 60 normal controls and 6% among a wide variety of patients with diverse neurological disorders. However, one-third of 33 patients with Parkinson's disease showed presence of antineuronal antibody. Among patients with HD, a significant association was noted between duration of clinical disease greater than 7 yr and titers of antibody of 1:2 or greater (P less than 0.001). When 115 family members of HD probands were tested, 30% of unaffected spouses showed presence of antineuronal antibody. 23.2% of first-degree relatives at risk for developing HD was also positive (P less than 0.001). 10.5% of second-degree relatives showed presence of antineuronal antibody. These data may support an environmental or infectious factor somehow involved in the ultimate expression of HD.

Autoantibodies to the HLA-B27 sequence cross-react with the hypothetical peptide from the arthritis-associated Shigella plasmid.

We previously reported elevated serum antibody levels to a peptide representing the HLA-B27 polymorphic region (B27 peptide) in HLA-B27(+) ankylosing spondylitis (AS) patients. A plasmid (pHS-2) isolated from arthritogenic Shigella flexneri strains had been shown to encode an amino acid sequence homologous to HLA-B27. Rabbit antibody to this sequence (pHS-2 peptide) strongly cross-reacted with B27 peptide and, to a much lesser extent, with Klebsiella nitrogenase peptide. Serum antibody levels to pHS-2 peptide were studied in 160 spondylarthropathy patients. 12 of 115 (10.4%) AS patients, 2 of 45 (4.4%) patients with Reiter's syndrome or reactive arthritis as well as 6 of 147 (4.1%) normal controls were shown to have elevated anti-pHS-2 peptide antibodies. Antibody levels to B27 and pHS-2 peptides were significantly correlated in 134 HLA-B27(+) patients (r = 0.333, P less than 0.001). 13 of 15 affinity-purified anti-B27 peptide antibodies from patients strongly cross-reacted with pHS-2 peptide, whereas only 3 weakly cross-reacted to nitrogenase peptide. Leucine appeared to be a critical residue for this cross-reaction. AS patients' anti-B27 peptide antibodies reacted with HLA-B27 transfected L cells. These results may suggest that pHS-2 peptide more efficiently "mimics" B27 peptide than does nitrogenase peptide. Involvement of pHS-2 in pathogenesis of spondylarthropathy through molecular mimicry mechanisms requires further study.

Amyloid A in systemic amyloidosis associated with cancer.

Amyloid fibrils from two cases of cancer-associated, systemic amyloidosis with renal cell carcinoma and mesothelioma as the respective underlying disorders were studied. The immunochemical studies suggested strongly that amyloid A comprised a principal fibril component in both cases of cancer-associated amyloidosis. This was definitively proven by amino acid sequence analyses, which revealed structural homology between a purified subcomponent of the amyloid fibrils from both of the two cases of cancer-associated amyloidosis and previously sequenced amyloid A proteins. The chemical composition of the amyloid fibrils from systemic amyloidosis associated with cancer thus corresponded to that seen in amyloidosis reactive to inflammatory diseases and Hodgkin's disease. Amyloid proteins of immunoglobulin light chain type, which are found associated with myelomatosis, macroglobulinemia, and idiopathic (primary) amyloidosis, were not found in the two amyloid preparations. Renal cell carcinoma appears to be an effective stimulator of amyloid formation, while only one case of amyloidosis associated with mesothelioma has been reported previously.

T-lymphocyte subpopulations in peripheral blood and tissues of cancer patients.

Patients with cancer often show impaired immune functions; however, the basis of this suppression is still not understood. In several experimental systems, human T-cells with receptors for Fc of immunoglobulin G may function as suppressors, and those with receptors for Fc of immunoglobulin M may function as helpers. Peripheral blood as well as tumor tissue infiltrates were examined for proportions and numbers of T gamma, T mu, or Ia-positive T-cells. Forty-five untreated patients with solid tumors and 24 patients with lymphomas were studied. An increase in the percentage of peripheral blood T gamma cells (p < 0.001) and a decrease in T mu cells (p < 0.0005) were recorded in all tumor patients when compared with 30 normal controls. Percentages and absolute numbers of peripheral blood Ia-positive T-cells were decreased (p < 0.001 and < 0.00001) in solid-tumor patients; by contrast, the proportion of peripheral blood Ia-positive T-cells was elevated (p < 0.005) in lymphoma subjects. Studies of cancer tissues from 46 untreated patients using immunofluorescence and mouse hybridoma antibody specific for T-cells showed that tumor lymphocytic infiltrates were composed mainly of T-cells. Double staining with fluorescein-conjugated specific anti-T gamma and Ia-positive T-cells within solid-tumor lymphoid infiltrates. A comparison of peripheral blood and tumor lymphocyte T-cell profiles revealed that, in some patients, low proportions of Ia-positive T-cells in blood were paralleled by a high percentage of such cells in tumor lymphoid infiltrates.

Expression of serum amyloid A genes in mink during induction of inflammation and amyloidosis.

Serum amyloid A (SAA) is an acute phase protein and the precursor of amyloid protein A (AA) in deposits of secondary amyloidosis. Several isotypes exist in mink, but previous studies suggest that mink AA is derived from only one. To assess the effect of repeated episodes of inflammation and induction of amyloidosis, qualitative and quantitative changes in hepatic and extrahepatic SAA mRNA were studied. Young female mink received subcutaneous lipopolysaccharide injections for amyloid induction. Studies were performed using RNA probes and oligonucleotide probes specific for each of two SAA mRNA species. Northern blot hybridization showed that hepatic SAA1 and SAA2 mRNA levels increased dramatically after inflammatory stimulation, and were subsequently maintained at elevated levels, showing considerable interindividual variation, but only a slight decrease during repeated inflammatory stimuli and the early stages of amyloid deposition. No preferential accumulation of mRNA specifying a particular isotype was found during the experiment. Differential expression of mink SAA mRNA during repeated inflammatory stimulation does not seem to explain why only SAA2-derived AA is found in amyloid deposits. Extrahepatic SAA mRNA seemed to be independently regulated and may thus represent another, yet not characterized, SAA isotype.

Filter function and immune complex trapping in splenic ellipsoids.

The role of splenic ellipsoids in the trapping of particulate material and immune complexes was investigated in mink (Mustela vison). The ellipsoids were prominent, with typical features such as a permeable endothelium and a discontinuous basement membrane surrounded by a sheath of macrophages and reticular cells. Ellipsoidal trapping of circulating particles was demonstrated 10 min after intracardiac injection of colloidal carbon and fluorescent microspheres. Preformed peroxidase-antiperoxidase immune complexes were detected in ellipsoids 10 min and also 1 h after intracardiac injection. Erythrocytes were frequently observed in the ellipsoidal sheath, and many phagocytized fragments of erythrocytes were found in the ellipsoidal macrophages. It was concluded that mink ellipsoids are effective blood filters with a role in retention of circulating particulate material, and that mammalian splenic ellipsoids also have the ability to trap immune complexes.

Evaluation of the VIDAS Listeria (LIS) immunoassay for the detection of Listeria in foods using demi-Fraser and Fraser enrichment broths, as modification of AOAC Official Method 999.06 (AOAC Official Method 2004.06).

A multilaboratory study was conducted to compare the VIDAS LIS immunoassay with the standard cultural methods for the detection of Listeria in foods using an enrichment modification of AOAC Official Method 999.06. The modified enrichment protocol was implemented to harmonize the VIDAS LIS assay with the VIDAS LMO2 assay. Five food types--brie cheese, vanilla ice cream, frozen green beans, frozen raw tilapia fish, and cooked roast beef--at 3 inoculation levels, were analyzed by each method. A total of 15 laboratories representing government and industry participated. In this study, 1206 test portions were tested, of which 1170 were used in the statistical analysis. There were 433 positive by the VIDAS LIS assay and 396 positive by the standard culture methods. A Chi-square analysis of each of the 5 food types, at the 3 inoculation levels tested, was performed. The resulting average Chi square analysis, 0.42, indicated that, overall, there are no statistical differences between the VIDAS LIS assay and the standard methods at the 5% level of significance.

Peripheral neuropathy in systemic lupus erythematosus.

We studied 33 consecutive patients with systemic lupus erythematosus (SLE) for neuropathy by employing the Neuropathy Symptom Score (NSS), Neurological Disability Score (NDS), EMG and nerve conduction velocity (NCV) studies, and determinations of vibration thresholds (VT). Polyneuropathy defined as NCV abnormalities of two or more nerves occurred in seven patients (21%). Neuropathic symptoms showed a poor correlation with NCV and VT, while clinical neuropathic signs, VT, and NCV correlated with each other in most instances. When reporting frequencies of neuropathy in SLE, NCV studies should be used as a basis. NSS, NDS, and VT give additional quantifiable information and can be useful in the follow-up of patients and for evaluating the response to therapy.

The Norwegian multicenter study.

The largest multicenter, double-blind trial comparing piroxicam with naproxen in the treatment of osteoarthritis has recently been conducted in Norway. More than 2,000 patients were enrolled in the 12-week study. Both drugs showed similar efficacy, and serious gastrointestinal side effects occurred in less than 1 percent of the patients taking either agent. These findings are similar to results with other nonsteroidal anti-inflammatory drugs. This study indicates that in the treatment of osteoarthritis, piroxicam is as effective as naproxen and poses no greater risk of serious gastrointestinal side effects.

Characterization of renal tissue lymphocytes in patients with interstitial nephritis.

Five patients with interstitial nephritis who presented with a variety of clinical profiles were studied with particular emphasis on documentation of the cellular types of potentially immunocompetent lymphocytes and mononuclear cells present within interstitial renal infiltrates. Immunohistologic studies coupled with conventional light and electron microscopic observations indicated that most mononuclear cells making up renal interstitial infiltrates were T cells. Some chronic inflammatory cell foci within renal interstitium were characterized by clusters of Ia antigen-positive T cells considered to be markers for activated lymphocytes. B cells were present in very small proportions (5 percent or less). The profile of immunocompetent cells present in lesions of interstitial nephritis suggests a major role for cell-mediated immunity in this disorder. Increase in tissue lymphocytes of the T gamma subclass with receptors for the Fc portion of immunoglobulin G (IgG) also suggests local activation of intrinsic suppressor cell mechanisms.

A Danish kindred with familial amyloid cardiomyopathy revisited: identification of a mutant transthyretin-methionine111 variant in serum from patients and carriers.

PURPOSE: In familial amyloid cardiomyopathy of Danish origin, the amyloid microfibrils contain a mutant transthyretin (TTR) with a methionine-for-leucine substitution at the molecular position 111. We studied the possible occurrence of this variant TTR-Met111 in serum from afflicted as well as nonafflicted family members and their offspring, in order to define its possible role as predictor of the disease and to describe its mode of inheritance. PATIENTS AND METHODS: Stored, frozen serum samples obtained from 1959 through 1960 from 36 of 40 living members of the kindred were analyzed. The method employed to detect the abnormal TTR was based on the electrophoretic separation of fragments produced by cyanogen bromide cleavage at the two methionine sites. RESULTS: All sera from family members with amyloid cardiomyopathy contained the variant transthyretin TTR-Met111 as did sera from half of their offspring. In contrast, nonafflicted family members and their offspring were seronegative for TTR-Met111. Three cousins from the second generation died between 1980 and 1986 of amyloid cardiomyopathy. The presence of variant TTR-Met111 preceded their deaths by 20 to 26 years. CONCLUSIONS: The occurrence in serum of the mutant transthyretin TTR-Met111 is linked to the occurrence of amyloid cardiomyopathy in patients and their offspring, while unafflicted branches of the family are negative for the variant protein. That the occurrence in serum of TTR-Met111 precedes the onset of clinical amyloid cardiomyopathy by several decades makes the variant TTR a marker for the disease. The distribution of afflicted family members and seropositivity for the variant TTR shows an autosomal dominant mode of inheritance. CLINICAL SIGNIFICANCE: The results make possible early detection of potential patients and provide tools for genetic counseling. Cardiac transplantation may provide a new therapeutic option.

Tissue distribution of lymphocytes in rheumatic heart valves as defined by monoclonal anti-T cell antibodies.

Fresh cardiac valvular tissues and atrial appendages removed from 106 Indian patients with rheumatic heart disease at the time of corrective cardiac surgery were examined to determine the characteristics of valvular interstitial lymphocytic infiltrates using conventional histologic staining along with indirect immunofluorescent techniques. Precise identification of the phenotypic profiles of inflammatory mononuclear cells was attempted using anti-IgG, anti-Ia, and monoclonal mouse hybridoma reagents identifying T cells (OKT3) as well as T cell subsets (OKT4 helper/inducer and OKT8 suppressor/cytotoxic cells). A similar group of 21 patients undergoing cardiac valvular resection in Albuquerque was studied. The mean age of Indian patients providing valve tissues was 27.7, whereas in those in Albuquerque, it was 52 years. Twenty-five percent of rheumatic heart valves in Indian patients showed significant interstitial lymphoid infiltrates, and one third of the rheumatic valves from patients in Albuquerque showed similar mononuclear cell collections. Lymphoid infiltrates contained a predominance of T cells (70 to 80 percent) and only occasional B cells. Most of the T cells were OKT4-positive, with only a minor representation of suppressor/cytotoxic OKT8-positive T cells. In many instances, OKT4-positive helper T cell collections were closely juxtaposed to fibroblasts and collagen fibrils. These findings suggest that the chronic rheumatic scarring process may involve helper/inducer T cells as an ancillary factor in the indolent contracture and fibrosis of deformed cardiac valvular structures. Attempts to demonstrate residual streptococcal antigens by indirect immunofluorescence using a wide panel of heterologous rabbit F(ab')2 reagents with specificity for group A streptococcal membranes, cell wall mucopeptide, or group A carbohydrate gave negative results.

Direct PCR of washed blood cells.

We report a simple and rapid method for direct DNA amplification of washed blood cells by PCR. Small samples (2-100 microliters) of blood were washed, the cells resuspended in a buffer and used directly for PCR after boiling. Amplification of a specific DNA sequence of the human transthyretin gene, directed by the primers, was successfully performed. The method gives comparable results to amplifications made by purified DNA from blood.

Ankylosing spondylitis. Current drug treatment.

The administration of drugs constitutes an important component of the therapeutic programme in ankylosing spondylitis (AS). The main objective of initiating such therapy is to reduce pain, stiffness and discomfort. There are at present 3 groups of drugs available for the management of AS. The first group is represented by drugs thought to influence the disease process itself. In this group, sulfasalazine is the only drug which is controlled trials has been shown to suppress disease activity in AS. We recommend the use of sulfasalazine in patients with high disease activity, with peripheral arthritis and in those with AS of short duration. The second group of drugs includes nonsteroidal anti-inflammatory drugs (NSAIDs), which suppress inflammation without influencing the disease process. These drugs should be administered selectively during periods of high disease activity. Moreover, 1 drug should be used in appropriate dosage before it is assumed to be inefficient. High doses of NSAIDs may be prescribed before bedtime in patients suffering from severe pain and stiffness during the night. The toxicity profile of NSAIDs includes gastrointestinal and renal side effects. The third group comprises analgesics and muscle relaxants. Such drugs should be used rather frequently in patients with longstanding AS refractory to treatment with NSAIDs. Peripheral arthritis and enthesopathy are generally managed by local injections of corticosteroids, while AS complicated by psoriasis or inflammatory bowel disease is treated as primary AS. AS occurring in juveniles is best treated with aspirin and an NSAID, although careful observation is necessary for the development of Reye's syndrome (with aspirin) and gastric irritation (with NSAIDs). When patients with AS undergo surgery, the possibility of silent gastrointestinal bleeding due to the use of NSAIDs and salicylates should not be ignored. Patients treated with oral corticosteroids should receive a bolus injection of soluble corticosteroid prior to surgical intervention. NSAIDs may be administered pre- and postoperatively to relieve stiffness induced by immobility. Rapid treatment of intervening infections and use of NSAIDs is recommended in AS complicated by renal amyloidosis. During pregnancy and lactation, ibuprofen may be the preferred drug in AS.