Autoimmunity to the M(r) 32,000 subunit of replication protein A in breast cancer.

PURPOSE: We sought to identify autoantigens recognized by antibodies in breast cancer patient sera with potential diagnostic or prognostic significance. EXPERIMENTAL DESIGN: Serum from a female breast cancer patient exhibiting a high titer antinuclear antibody was used to screen a HeLa cDNA expression library, leading to the cloning of a cDNA for the M(r) 32,000 subunit of replication protein A (RPA32). RPA32 expression and localization were assayed in autologous tumor by monoclonal antibody staining. A specific ELISA using recombinant protein was used to screen sera from 801 breast cancer patients and 65 controls. RESULTS: A relationship between anti-replication protein A (RPA) antibodies and the ductal breast carcinoma of the proband was suggested by overexpression and aberrant localization of RPA32 in tumor cells as compared with surrounding normal ductal tissue and by the presence of anti-RPA32 antibodies before the diagnosis. The prevalence of anti-RPA32 antibodies was significantly higher (P < 0.01) among breast cancer patients (87 of 801 patients) than among noncancer controls (0 of 65 controls). Similarly, anti-RPA32 antibodies were present in 4 of 39 patients with intraductal in situ carcinoma. No associations were found between anti-RPA antibodies and survival, occurrence of a second tumor, metastases, or antibodies to p53. Reactivity to RPA32 also was detected in sera from 3 of 47 patients with other cancers. CONCLUSIONS: In view of the central role of RPA in DNA replication, recombination, and repair, we suggest that autoimmunity to RPA32 may reflect molecular changes involved in the process of tumorigenesis. The finding of antibodies to RPA32 before diagnosis and their prevalence in in situ carcinoma suggest that they are potentially useful markers of early disease.

Antinuclear antibodies as potential markers of lung cancer.

There are multiple case reports of antinuclear antibodies (ANAs) in patients with malignancies, yet to date there has not been a systematic survey of ANAs in lung cancer. We have previously reported that autoantibodies to collagen antigens resembling those found in the connective tissue diseases are consistently detected in the sera from lung cancer patients. In this work, we looked for the presence of ANAs in the sera from these same patients. Sera from 64 patients with lung cancer and 64 subjects without a history of cancer were retrospectively tested for reactivity on immunoblots of nuclear extracts of HeLa, small cell carcinoma, squamous cell carcinoma, adenocarcinoma, large cell carcinoma of the lung, and of normal lung cells. Associations were sought between the reactivities on immunoblots and lung cancer cell type, diagnosis, and progression-free survival by the method of classification and regression trees (CARTs). Cross-validated CART analyses indicated that reactivities to certain bands in immunoblots are associated with different types of lung cancer. Some of these autoantibodies were associated with a prolonged survival without disease progression. Our data suggest that autoimmunity is often a prominent feature of lung cancer and that molecular characterization of these antigens may lead to the discovery of proteins with diagnostic and prognostic value.

Periarticular osteoporosis in osteoarthritis of the knee.

OBJECTIVE: To test the hypothesis that bone mineral density (BMD) in periarticular subchondral regions of the knee joint is abnormal in patients with osteoarthritis (OA). METHODS: Sixty-two knees from patients with relatively mild OA (primarily radiographic grades 0, 1, 2) of the knee were compared with 62 knees from normal subjects matched for age, sex, race, and body side (right or left). BMD of the lumbar spine, distal femoral shaft, and several periarticular subchondral regions of the knee were determined by dual energy x-ray absorptiometry (DXA). To facilitate increased accuracy of BMD values, lateral DXA of the knees was also performed and used to provide a third dimension to the usual two-dimensional anterior-posterior g/cm2, producing measurements as g/cm3. Subchondral bone regions included both superficial and deep regions of the medial femoral, medial tibial, and lateral tibial compartments. RESULTS: BMD (g/cm3) was lower than normal in 6 subchondral bone regions of the knees (n=43) of white female patients with OA (average decrease -13.3%), significant in all 6 subchondral regions (p=0.001 to p=0.047). Two-dimensional BMD (g/cm2) was lower than normal in 6 subchondral regions of the knees (n=43) of white female patients with OA (average decrease -7.3%), significant in only 2 of 6 subchondral regions (p=0.011 to p=0.014). BMD (g/cm3) was lower than normal in 6 subchondral regions of the knees (n=19) of African American female patients with OA (average decrease -9.1%), significant in only one of the 6 subchondral regions (p=0.016). There was no significant difference in spinal BMD (L1-L4) or femoral shaft BMD between normal and OA for either racial group. About 13% of the OA patients had osteoporosis at the spine. CONCLUSION: A significant decrease in periarticular subchondral BMD is present in female patients with relatively mild OA of the knee whether or not they had osteoporosis based on a spine BMD measurement.

T cell receptor (V beta) bias in the response of rheumatoid arthritis synovial fluid T cells to connective tissue antigens.

T cell receptor (V beta) use in the response to type II collagen and cartilage proteoglycans was analysed in peripheral blood and synovial fluid T cells from RA patients. T cells from RA patients with an immune response to connective tissue antigens, and paired PB and SF samples were stimulated in vitro with type II collagen, high density aggrecan proteoglycans (PG), and the T cell mitogen concanavalin A. After short term culture, mRNA was extracted from cells and a reverse transcription-polymerase chain reaction was performed, using primers specific for eight TCR V beta determinants. Blood cells stimulated with ConA generated strong bands with virtually all the V beta primers tested, but the TCR (V beta) expression by SF T cells stimulated with mitogen was biased, suggesting a selection process during joint infiltration. The V beta phenotypes of cells responding to PG was restricted in individual RA patients, but the pattern of V beta use in the the RA population was not consistent. In contrast, the V beta phenotypes of SF cells responding to CII was highly biased in both individual patients and the RA population, with V beta 14, V beta 17, and V beta 8 phenotypes predominant. We conclude that the T cell response to connective tissue antigens is restricted compared with mitogen stimulation, with the highest degree of TCR bias seen in the response of SF T cells to stimulation with type II collagen.

Autoimmunity to collagen in human lung cancer.

Autoantibodies have been described in human cancer patients as well as in animal models of malignancy. The extracellular matrix and especially basement membranes act as barriers for tumor cell invasion. Collagen, particularly types I, III, and IV, are major constituents of the extracellular matrix. We tested the hypothesis that autoimmunity to collagen antigens is present in lung cancer. Sera from 67 patients with lung cancer and 50 reference subjects were tested for anticollagen antibodies by using purified human collagen types I-V and for antibodies binding human cartilage aggrecan proteoglycan. Antibody levels were determined by using ELISA. The relationship of serum levels of these antibodies to patient survival, histology, treatment response, disease stage, and pack years of smoking was examined by using multiple regression and discriminant function analyses. A subgroup of 45 patients in whom a smoking history was available was analyzed separately. Within 1 month of the initiation of therapy, mean serum levels of antibodies binding fibrillar collagen types I-III and V were significantly higher (P < 0.025) than were those in control sera (43.2% of patients positive for one or more anticollagen antibodies). Antibodies binding aggrecan proteoglycan were not different between patients and control sera. In the lung cancer patients, the levels of serum antibodies binding types IV and V collagens contributed to the variance of progression-free survival days, survival days, and the duration of favorable response in opposite directions. Histological cell type contributed to the variance in the level of serum antibody binding collagen types IV and V. Lower levels of antibody binding type IV and higher levels of antibody binding type V were associated with small cell carcinoma. The pack-years of smoking only contributed to the variance in the level of serum antibody binding type V collagen. We conclude that autoantibodies to fibrillar collagen antigens are present frequently in lung cancer patients, and their levels may be related to histological cell type and to the duration of the response to treatment.

Synovial thickening detected by MR imaging in osteoarthritis of the knee confirmed by biopsy as synovitis.

Previous studies have established the value of magnetic resonance imaging (MRI) in detecting articular changes characteristic of osteoarthritis (OA) of the knee. We have observed some MRI features in OA of the knee presumably indicating synovial thickening. To determine whether these MR features represent chronic synovial inflammation, we studied the knees of nine patients at the mild end of the spectrum of OA of relatively short duration (89%: < or = 4 yr), who were selected because MRI showed anatomical abnormalities compatible with synovial thickening. The painful knee was examined using conventional and weight-bearing radiographs, MRI, and arthroscopy. MR images suggestive of synovial thickening typically appeared in or near the intercondylar region of the knee, in the infrapatellar fat pad, or in the posterior joint margin. The site of an arthroscopic biopsy of the synovial membrane was guided by MRI to the area thought to represent synovial thickening for each patient knee. Pathological examination of these synovial membrane biopsies showed a mild chronic synovitis, and thus a correspondence with the synovial thickening detected by MRI. Our results suggest that MRI can be used to evaluate the extent of synovitis, observed as synovial thickening, in patients with early OA of the knee.

Factors affecting articular cartilage thickness in osteoarthritis and aging.

OBJECTIVE: To test the hypothesis that both aging and osteoarthritis (OA) contribute to the variance of human articular cartilage thickness of the knee and whether these contributions occur predominantly at weight bearing cartilage sites. METHODS: Thin, sagittal magnetic resonance images (MRI) of both knees were examined in 52 patients with idiopathic OA of the knee of short duration (87%: < or = 4 years) and 40 reference subjects of comparable age and sex distribution. Articular cartilage thickness was measured at the weight bearing and nonweight bearing femoral condylar, tibial plateau, and posterior patellar sites. Multiple regression analyses were performed to examine the influence of OA, age, sex, obesity (body mass index), and bone size on cartilage thickness. RESULTS: Age accounted for a significant linear decrease (p < 0.001) in both lateral and medial weight bearing femoral cartilage thickness of both knees, but failed to account for any significant variance in thickness at other cartilage sites. OA accounted for a significant decrease (p < 0.02) in the thickness of the femoral articular cartilage in the medial and lateral right knee compartments and in the lateral left knee compartment, but not in the medial left knee compartment. Significant cartilage thinning could be detected by MRI in patients with OA, even when the joint space was normal radiographically. CONCLUSION: The asymmetric decrease in the left knee may reflect the effect of mechanical factors. Our data show that articular cartilage thickness decreases at the femoral weight bearing sites both with age and as a consequence of OA and that these contributions can be distinguished from one another.

Modulation by beta-aminopropionitrile of vessel luminal narrowing and structural abnormalities in arterial wall collagen in a rabbit model of conventional balloon angioplasty versus laser balloon angioplasty.

This study was designed to assess the potential relationship between the late loss of angiographic luminal diameter and biochemical abnormalities of arterial wall collagen in rabbits subjected to angioplasty, and to test the hypothesis that beta-aminopropionitrile (beta APN), an inhibitor of lysyl oxidase, would inhibit such changes when administered orally for 1 mo after angioplasty. Endovascular injury was induced in rabbit iliac arteries by ipsilateral balloon angioplasty (BA) and by contralateral balloon angioplasty accompanied by exposure to continuous wave neodymium: yttrium aluminum garnet laser radiation (LBA). Computer measurement of angiographic luminal diameter demonstrated significant vessel narrowing at 1 and 6 mo after both procedures. By quantitative histology, the majority of the 1-mo loss in angiographic diameter could not be attributed to neointimal thickening. Analysis of collagen cross-linking by HPLC in collagen obtained from the LBA-injured segments of the arteries 1 mo after angioplasty revealed a significant increase, relative to values from uninjured arteries (P < 0.05), in the difunctional cross-link dihydroxylysinonorleucine (DHLNL). 6 mo after angioplasty, the content of hydroxypyridinium, the trifunctional maturational product of DHLNL, was significantly elevated in both BA- and LBA-treated arteries compared with values from uninjured arteries (P < 0.05). In animals administered beta APN, luminal narrowing at 1 mo, compared with controls, was attenuated (P < 0.01) and DHLNL content was decreased (P < 0.05) in arteries subjected to LBA, but not in arteries subjected to BA. The results suggest that lathyrogenic agents may be efficacious in favorably modulating LBA-induced alterations in vessel diameter and mural connective tissue.

MR features of osteoarthritis of the knee.

A group of patients with idiopathic osteoarthritis (OA) of the knee was surveyed using weight-bearing radiographs and MR imaging to compare the relative value of these methods in disease evaluation. Fifty-two patients with a clinical and radiological diagnosis of OA of the knee of relatively short duration (87%: < or = 4 yr) were compared to a reference group of 40 age- and sex-comparable subjects with no knee symptoms. All patients had a complete history, physical examination, standard anterior-posterior and lateral weight-bearing radiographs, T1-weighted, and FLASH MR images in both knees. The prevalence of MRI abnormalities was significantly greater in patients with OA of the knee in all radiographic grades (Kellgren and Lawrence) compared to the reference subjects. Significant differences were encountered for synovial thickening (OA, 73%; reference, 0%), synovial fluid (60%; 7%), meniscal degeneration (52%; 7%), osteophytes (67%; 12%), and subchondral bone involvement (65%; 7%), even in the patients at the mild end of the osteoarthritic spectrum, indicating the exquisite sensitivity of MRI compared with weight-bearing radiographs.

Dependence of proteoglycan induced arthritis in BALB/c mice on the development of autoantibodies to high density proteoglycans.

BALB/c mice were immunised with high or low density native human cartilage proteoglycans, or the respective core proteins obtained from chondroitin ABC lyase digestion. Mice injected with high density native proteoglycans developed arthritis whereas mice injected with low density proteoglycans or with core proteins did not. Analysis of the immune response by enzyme linked immunosorbent assay (ELISA) and western blot showed a stronger and more polyspecific response in animals injected with low density proteoglycans compared with mice with arthritis which had been injected with high density proteoglycans. Autoantibodies to mouse high density proteoglycans were only present in mice injected with native human high density proteoglycans, however. The data suggest that an arthritogenic epitope lies within the glycosaminoglycan rich region of the native proteoglycan molecule, which may induce an autoantibody response and subsequently arthritis in BALB/c mice.

Proteoglycans from osteoarthritic human articular cartilage influence type II collagen in vitro fibrillogenesis.

Collagen fibrils were formed in the presence of dermatan sulfate (DSPG) and high density (HDPG) proteoglycans isolated from human adult knee femoral articular cartilage. Eroded cartilage had a higher percentage of DSPGs in the extracted proteoglycans than normal cartilage (p = .018). The dermatan sulfate proteoglycans (DS-PGI and DS-PGII) were detected in normal and osteoarthritic cartilage. DSPGs compared to HDPG inhibited in vitro collagen fibrillogenesis producing a longer lag phase (p less than .05) and a slower rate of fibril formation (p less than .05). DSPGs from eroded osteoarthritic cartilage alone or in combination with HDPG produced a longer lag phase than DSPGs from normal cartilage alone or in combination with HDPG (p less than .05). The inhibition of fibrillogenesis by DSPGs suggests that collagen fibril formation in vivo may be abnormal due to the influence of molecular changes in proteoglycan as well as an increased proportion of DSPGs occurring in osteoarthritic cartilage. Abnormal fibril formation may produce a weakened cartilage matrix, thus contributing to an accelerated process of cartilage degeneration in osteoarthritis.

Articular cartilage defects of the knee: correlation between magnetic resonance imaging and gross pathology.

Magnetic resonance imaging (MRI) of the knee articular cartilage is possible owing to the contrast provided by different signal intensities of adjacent menisci and subchondral bone. The objective of this study was to determine the accuracy of MRI in quantitatively detecting thinning and focal defects of articular cartilage in vivo. High resolution MRI was performed followed by dissection of the knee within one hour of amputations above the knee of eight patients (62-89 years) with peripheral vascular disease. Articular cartilage was examined for erosions, surface irregularities, and appearance. Mean thicknesses of femoral and tibial articular cartilage sagittal sections from MRI were statistically indistinguishable from matched gross thicknesses. In those joints in which cartilage erosions, thinning, or irregularities were detected by MRI the same defects were apparent by gross examination. Cartilage that appeared normal by MRI had a normal gross appearance by gross examination. Thus high resolution MRI can accurately predict gross articular cartilage appearance and thickness, allowing an objective, quantitative, noninvasive assessment of eroded cartilage.

A human cartilage metalloproteinase with elastolytic activity.

A metalloproteinase with elastolytic properties found in human fetal and osteoarthritic cartilage could not be detected in normal adult cartilage. During extraction the enzyme appeared to be mostly associated with cartilage proteoglycans, from which it can be separated by ion exchange chromatography. This enzyme migrated during electrophoresis with an apparent molecular weight of 62,000 daltons and was found to be fully activated in the tissue under the study conditions. The enzyme showed a preference for substrates rich in non-polar amino acid residues and was capable of breaking down elastin and casein at neutral pH. The enzyme activity can be inhibited by chelating agents and specific affinity reagents, chloroketones, and is not inhibited by other proteinase inhibitors such as PMSF, aprotinin and alpha-1-antitrypsin. This enzyme may play a significant role in conditions demanding rapid cartilage matrix turnover and/or remodeling, such as normal embryonic development or osteoarthritis.

An animal model of pulmonary radiation fibrosis with biochemical, physiologic, immunologic, and morphologic observations.

An animal model of pulmonary radiation fibrosis was established, using male CBA/j mice. Both lungs of each mouse in one group (DL) were irradiated with two doses of 8.5 Gy each, separated by 30 days. A control group (CG) was sham-irradiated. There was a small but significant difference (P less than 0.03) in average breathing rate between DL and CG 27 weeks after the second irradiation which increased until the 34th week followed by a plateau. The accumulated hydroxyproline content of the irradiated mouse lung was 40% greater (P less than 0.02) than that of the sham-irradiated lung at 42 weeks and thereafter. Anticollagen antibodies assayed 52 weeks after irradiation by enzyme-linked immunosorbent assay were elevated by 49% in sera from the irradiated mice compared to sera from sham-irradiated mice. Mortality during the 52-week period following the second irradiation was low (13%) for both groups. Histological comparison of irradiated and control mouse lungs fixed under uniform inflation pressure indicated no significant differences. The model has unique features including an increase in collagen deposition, no acute changes attributable to radiation, a small but statistically significant abnormality in pulmonary function, an immunologic response to collagen, and low mortality.

Influence of antecedent lymphoid surgery on the odds of acquiring rheumatoid arthritis.

The occurrence of rheumatoid arthritis (RA) and the extent of antecedent lymphoid surgeries was examined using case control study methods. Two hundred sixty-four patients with definite or classic RA were considered and 283 patients with rheumatic diseases presumably of nonimmunologic origin were used as controls. The odds for developing RA were found to be significantly higher for patients with multiple lymphoid surgeries (tonsillectomy and adenoidectomy or tonsillectomy and adenoidectomy plus appendectomy) and exhibited a gradient, increasing with more extensive surgery. Significant rank correlations were found in patients with RA between the age at tonsillectomy and the rheumatoid factor (RF) titer. An earlier tonsillectomy correlated with lower titers of RF. A significant decrease of serum RF titer was also seen in patients with RA subjected to tonsillectomy, adenoidectomy and appendectomy. We conclude that antecedent removal of lymphoid tissue from the tonsils, adenoids and appendix constitutes a risk factor predisposing to RA. Moreover, this risk seems related to the quantity of lymphoid tissue removed.