Cytokines in murine lyme carditis: Th1 cytokine expression follows expression of proinflammatory cytokines in a susceptible mouse strain
The cardiac infiltrate seen in murine Lyme carditis is composed predominantly of macrophages, but small numbers of T cells are also present. To identify the cytokines present in cardiac lesions from susceptible mice, semiquantitative polymerase chain reaction was done on cardiac tissue from mice infected with Borrelia burgdorferi. The temporal expression of proinflammatory and T cell-derived cytokines was characterized in cardiac tissue at days 0, 3, 7, 14, 21, and 42 after infection with B. burgdorferi. Early in the course of infection, up-regulation of the proinflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha was detected. The Th1 cytokine interferon-gamma appeared after the expression of the proinflammatory cytokines and remained elevated throughout the study. Interleukin-4 was not detectable at any time in cardiac lesions. These data are the first to identify cytokines expressed at the lesional level in murine Lyme carditis and to demonstrate a Th1 pattern of cytokine expression in this lesion.
Multinucleated cells in pigmented villonodular synovitis and giant cell tumor of tendon sheath express features of osteoclasts
Pigmented villonodular synovitis (PVNS) and the histologically related lesion giant cell tumor of tendon sheath (GCTTS) are idiopathic, proliferative lesions that can induce osteolysis and formation of bone cysts. These lesions contain two predominant cell types: mononuclear polyhedral cells and multinucleated cells (MNCs). Previous studies demonstrated that the mononuclear cells exhibit phenotypic features consistent with derivation from a monocyte/macrophage lineage. The cell lineage of the MNCs and their relationship to osteoclasts are not known. To characterize the MNCs in these lesions and to establish the relationship of these MNCs to osteoclasts, histological sections from six cases of PVNS and two cases of GCTTS were studied. Mononuclear cells expressed CD14 and HLA-DR, in keeping with their relationship to cells of the monocyte/macrophage lineage. Characterization of the MNCs revealed features associated with an osteoclast phenotype. Seven of the eight specimens contained MNCs that were intensely tartrate-resistant acid phosphatase positive; approximately 5% of the mononuclear cells were tartrate-resistant acid phosphatase positive, and these tended to surround MNCs. MNCs in both lesions reacted strongly with the 23C6 monoclonal antibody that recognizes the alpha V beta 3 integrin (the vitronectin receptor), as did several mononuclear cells surrounding the MNCs. Most MNCs did not express CD14 or HLA-DR. Expression of receptors for calcitonin, a marker for osteoclasts, was detected on MNCs after incubation of sections with 125I-labeled salmon calcitonin and emulsion autoradiography. MNCs in four of six PVNS and two of two GCTTS samples demonstrated specific calcitonin binding. Expression of mRNA for calcitonin receptor was confirmed in all cases by reverse transcriptase polymerase chain reaction. These results demonstrate that MNCs in PVNS and GCTTS express phenotypic features of authentic osteoclasts and suggest that osteoclast-like multinucleated cells can arise in synovial soft tissues remote from bone.
A retrospective review was performed on 188 autopsied cases of rheumatoid arthritis at our institutions during 1958-1985, prior to the widespread use of methotrexate. Hepatic histology was reported in 182 cases. All available microscopic liver slides from cases in which the autopsy report described portal tract inflammation, fibrosis, cirrhosis, tumour, amyloid, vasculitis, or infections involving the liver were examined and graded by a hepatic pathologist blinded to the original diagnosis, along with a representative sample of cases with reports describing fatty change or no hepatic pathologic abnormalities. Ninety normal and abnormal cases were reviewed from the 182 for which hepatic histology was available. Fifteen cases of diffuse fibrosis were identified upon blinded review. Two cases were graded as severe fibrosis (grades 3 or 4 on a scale of 0-4) without an identifiable pathologic cause, in both of which the liver disease was suspected premortem (alcohol abuse and viral hepatitis). Although the incidence of fibrosis in this series is slightly higher than that previously described, serious fibrotic liver disease was rare. These results support the current practice of limiting pre-treatment liver biopsies prior to methotrexate therapy to patients with suspected liver disease.
OBJECTIVE: To evaluate the efficacy and tolerability of N-[4 hydroxyphenyl] retinamide (4-HPR), a synthetic retinoid, in the treatment of rheumatoid arthritis (RA).
METHODS: An uncontrolled, open clinical trial with synovial biopsy pre- and postmedication to evaluate the clinical effects of 4-HPR as well as its effects on metalloproteinase gene expression.
RESULTS: Twelve patients with severe, longstanding RA were enrolled in this study. Six patients withdrew before study completion, 2 because of drug toxicity, 2 because of a flare of RA, and 2 because of intercurrent medical problems. No patient met predetermined Paulus criteria treatment response, and there was no improvement in the laboratory parameters, except for a modest decrease in C-reactive protein. No decrease in messenger RNA for the metalloproteinases collagenase and stromelysin was seen in the 2 patients in whom paired synovial biopsies were obtained.
CONCLUSION: No beneficial clinical effect was observed with the retinoid 4-HPR in the treatment of severe, longstanding RA at the 300 mg/day dosage studied. The use of higher dosages is precluded by the observed toxicities. The effect of this drug in patients with early or mild disease was not studied. Although this particular retinoid was not effective in this pilot study, the use of other retinoids in RA should still be considered.
OBJECTIVE: To identify the cells that express transcription factor NF-kappa B subunits p50 and p65 in synovial tissue from patients with rheumatoid arthritis (RA) and to correlate the distribution of p50 and p65 with CD14 (macrophage lipopolysaccharide receptor) and members of the AP-1 transcription factor family, Jun and Fos.
METHODS: Immunohistochemistry was used to identify p50, p65, Jun and Fos in sections of synovial tissue from 13 patients with RA and 4 "normal" control subjects. Double staining for CD14 and each of the transcription factor subunits was performed.
RESULTS: Subunits p50 and p65 were present in the nuclei of synovial cells in all 13 RA patients, with expression varying from rare cells to more than half of all cells. In most cases, nuclear p50 and p65 were present in approximately one-third of synovial lining cells and in a variable proportion of cells scattered throughout the sublining region, including the endothelium. The distributions of p50 and p65 were similar. Jun and Fos were present in the nuclei of a large proportion of synovial lining cells with significantly less expression elsewhere. In each case the Jun/Fos distribution was clearly different from the p50/p65 distribution, although there was significant overlap in many cases. Cells expressing CD14 were mostly Jun/Fos negative and were predominantly p50/p65 positive. There was negligible staining for p50 or p65 in the 4 normal control synovium samples.
CONCLUSION: In most RA patients, the p50 and p65 subunits of NF-kappa B were present in the majority of CD14-positive cells within the lining and sublining regions and in a proportion of other cells throughout the synovium, including endothelial cells. NF-kappa B is likely to play an important role in the expression of macrophage-derived cytokines in rheumatoid synovium. Different but overlapping distributions of nuclear p50 and p65 versus Jun and Fos indicate separate or divergent mechanisms for the activation of NF-kappa B and the expression of AP-1 proteins in rheumatoid synovium.
OBJECTIVE: Therapeutic trials in rheumatoid arthritis with the monoclonal antibody Campath-1H have demonstrated recurrent clinical synovitis in some patients, despite profound depletion of circulating lymphocytes. This study was undertaken to examine the cellular infiltrates in synovial tissue at a time of persistent peripheral lymphopenia.
METHODS: Immunohistochemical staining of synovial tissue and peripheral blood lymphocyte phenotyping.
RESULTS: Synovial tissues from 2 patients with recurrent synovitis after Campath-1H therapy contained significant T lymphocytic infiltrates at a time when circulating T lymphocytes were markedly depleted.
CONCLUSION: These results demonstrate that peripheral blood analysis may not accurately reflect the synovial tissue response to monoclonal antibody therapy.
Early murine Lyme carditis has a macrophage predominance and is independent of major histocompatibility complex class II-CD4+ T cell interactions
To compare the role of macrophages and CD4+ T lymphocytes in early Lyme carditis, immunohistochemical techniques were used to analyze cardiac infiltrates in immunocompetent mice infected with Borrelia burgdorferi spirochetes. Macrophages predominated in the infiltrate during the first 4 weeks after infection. CD4+ and CD8+ lymphocytes each constituted < 5% of the infiltrate; B lymphocytes were rare. Infected mice deficient in class II major histocompatibility complex (MHC) antigen and depleted of CD4+ lymphocytes developed similar infiltrates, suggesting that class II MHC-CD4+ lymphocyte interactions do not play a critical role in disease initiation. Expression of mRNA encoding JE within areas of cardiac inflammation implicates this chemokine in the recruitment and activation of macrophages in this disease. These data demonstrate that early murine Lyme carditis requires neither class II antigen expression nor presentation of antigen to CD4+ T lymphocytes and suggest a direct response of macrophages to cardiac tissue invasion by B. burgdorferi.
Pigmented villonodular synovitis (PVNS) is an idiopathic proliferative synovial process composed of two predominant cell types: mononuclear histiocytic cells and giant cells. This lesion can be locally invasive and can result in bone cyst formation and late cartilage and bone loss. Because metalloproteinases have been implicated in the joint destruction occurring in inflammatory arthritis and in the ability of certain tumors to invade adjacent tissues, their presence in PVNS was determined. Synovial tissue samples were collected at surgical synovectomy from the knees of 10 patients with a prior histological diagnosis of PVNS. Pigmented villonodular synovitis synovium was examined for the presence of the metalloproteinases collagenase and stromelysin. Messenger RNA (mRNA) for collagenase and stromelysin was present in all patient samples, although in varying amounts. In situ hybridization studies on synovial tissue sections identified synovial lining cells as the predominant cells expressing these metalloproteinases. Occasional infiltrating mononuclear histiocytic cells also were producing metalloproteinase mRNA. Giant cells did not express mRNA for the metalloproteinases collagenase and stromelysin. These results suggest that collagenase and stromelysin may be among the mediators of cartilage and bone loss that can occur in PVNS.
In situ hybridization studies suggest a role for the basic region-leucine zipper protein hXBP-1 in exocrine gland and skeletal development during mouse embryogenesis
The spatial and temporal distribution of transcripts for the TRE/CRE-binding basic region-leucine zipper protein hXBP-1 was determined by in situ hybridization. Analysis of embryos from day 10.5 to 18.5 pc revealed high level expression of hXBP-1 RNA in two developing organ systems: 1) in bone and cartilage cells of the developing skeleton and toothbuds, and 2) in exocrine glands including the pancreas and the submandibular and salivary glands. High level expression was also found in whisker follicles and in selected cells in brown adipose tissue. In the developing skeleton, hXBP-1 RNA was expressed starting on day 11.5 pc in osteoblasts of newly formed intramembranous bone. Thereafter, hXBP-1 was expressed in both osteoblasts and preosteoblasts in bone formed directly by intramembranous formation as well as in bone formed during endochondral ossification. The most intense signal was observed in preosteoblasts and osteoblasts of newly forming bone. At day 11.5 pc low level hXBP-1 expression was also observed in matrix secreting chondroblasts of bones which are formed initially of cartilage, at the stage where they consist entirely of cartilage. Signal was also present in matrix producing chondroblasts of the mature zone of the growth region during endochondral ossification although at significantly lower level than in osteoblasts. hXBP-1 is thus the first transcription factor described, to our knowledge, whose level of expression is modulated during the osteoblast developmental sequence in vivo. The pattern of expression of hXBP-1 in the developing skeleton was found to be very similar to that of the genes encoding the tissue inhibitor of metalloproteinase and alkaline phosphatase throughout development. These observations suggest that hXBP-1 may play a role in regulating the expression of tissue specific genes (TIMP, osteonectin, osteopontin, osteocalcin) expressed in osteoblasts. It is intriguing that the promoter regions of several such genes contain potential hXBP-1 binding sites.
The class II (Ia) MHC molecules are cell surface proteins that regulate the activation of T cells. B lymphocyte expression of class II molecules has been shown to be influenced by a number of external stimuli. It has been previously demonstrated that treatment of these cells with IL-4 leads to an increase in class II gene transcription at 18 h as well as to an increase in steady state class II mRNA. It has also been previously demonstrated that LPS treatment of splenic B cells from athymic mice results in a decrease in steady state mRNA encoding the A alpha class II protein. This decrease persists for at least 18 h. Nuclear run-on transcription assays now demonstrate that although steady state mRNA levels for A alpha are decreased by LPS treatment of athymic mouse lymphocytes, LPS does not decrease A alpha gene transcription, but rather modestly activates transcription of this class II gene. LPS and IL-4 have been demonstrated to be synergistic stimuli for a number of genes. Costimulation of splenic lymphocytes from athymic mice with IL-4 plus LPS leads to activation of transcription, but the increase in transcription is no more than that seen with IL-4 stimulation alone. However, in costimulated lymphocytes, steady state A alpha-encoding mRNA levels are intermediate between the increased levels seen with IL-4 stimulation and the decreased levels seen with LPS stimulation. Therefore, LPS and IL-4 act nonsynergistically in class II gene transcription and the effects of LPS in decreasing steady state mRNA are most likely posttranscriptional. An IL-4-inducible and an LPS-inducible DNA-binding protein have been previously identified in splenic lymphocytes from athymic mice. Both nuclear binding proteins form complexes with the same DNA fragments from a control region of the A alpha gene. These nucleoprotein complexes comigrate under nondenaturing conditions and display identical patterns of binding with a panel of oligonucleotide competitors. Oligonucleotides representing protein binding sites of the IL-4 and LPS-induced DNA-binding proteins cross-compete for protein binding. Therefore, the binding proteins induced by LPS and IL-4 are likely related, and may function at different efficiencies as activators of A alpha gene transcription.
In situ hybridization studies of stromelysin and collagenase messenger RNA expression in rheumatoid synovium
Destructive joint changes in rheumatoid arthritis (RA) are thought to be mediated in part by the neutral proteinases collagenase and stromelysin. Collagenase messenger RNA (mRNA) has been previously localized to the synovial lining layer. In this study, synovial tissue from 8 patients with RA and 2 patients with osteoarthritis was examined for proteinase production by in situ hybridization. Stromelysin mRNA localized predominantly to the synovial lining layer cells. In serial sections, collagenase mRNA was shown to be localized to the same tissue areas as those producing stromelysin mRNA, and grain counts revealed a direct correlation between production of stromelysin mRNA and production of collagenase mRNA. All patients with RA were producing collagenase and stromelysin mRNA in detectable amounts. One of 2 osteoarthritis patients was producing these metalloproteinases, but in levels below those found in the RA patients. These data support the identity of the synovial lining cells as the major synovial cells producing collagenase and stromelysin in RA and provide new evidence for the coordinate production of collagenase and stromelysin in RA in vivo.
A lipopolysaccharide-induced DNA-binding protein for a class II gene in B cells is distinct from NF-kappa B
Class II (Ia) major histocompatibility complex molecules are cell surface proteins normally expressed by a limited subset of cells of the immune system. These molecules regulate the activation of T cells and are required for the presentation of antigens and the initiation of immune responses. The expression of Ia in B cells is determined by both the developmental stage of the B cell and by certain external stimuli. It has been demonstrated previously that treatment of B cells with lipopolysaccharide (LPS) results in increased surface expression of Ia protein. However, we have confirmed that LPS treatment results in a significant decrease in mRNA encoding the Ia proteins which persists for at least 18 h. Within the upstream regulatory region of A alpha k, an NF-kappa B-like binding site is present. We have identified an LPS-induced DNA-binding protein in extracts from athymic mice whose spleens consist predominantly of B cells. Binding activity is present in low levels in unstimulated spleen cells and is increased by LPS treatment. This protein binds to two sites in a regulatory region of the Ia A alpha k gene, one of which contains the NF-kappa B-like binding site. DNA fragments containing these sites cross-compete for protein binding. Analysis by DNase I footprinting identified a target binding sequence, named the LPS-responsive element. Although this target sequence contains an NF-kappa B-like binding site, competition with a mutant oligonucleotide demonstrated that bases critical for NF-kappa B binding are not required for binding of the LPS-inducible protein. Therefore, we hypothesized that this inducible protein represents a new mediator of LPS action, distinct from NF-kappa B, and may be one mechanism to account for the decrease in mRNA encoding the Ia proteins.
Aortitis as a feature of rheumatoid arthritis is considered rare. We have, however, identified 10 patients with aortitis from among 188 consecutive autopsy cases of rheumatoid arthritis. There were 5 men and 5 women with a mean duration of rheumatoid arthritis of 9.6 years. Nine were rheumatoid factor positive and had associated nodules. In addition to standard treatment regimens, 9 patients received corticosteroids. Although involvement of the thoracic aorta was most common, involvement of both the thoracic and abdominal aorta was present in 4 cases. Two patients had aneurysmal dilatation of the thoracic aorta and 1 of the abdominal aorta. Microscopic features of aortitis included necrosis of medial smooth muscle and elastica, with an inflammatory infiltrate comprising primarily lymphocytes and plasma cells. A panmural aortitis was seen in 3 cases. Rheumatoid granulomas were noted in the aortic wall in 5. The diagnosis of aortitis was not made until autopsy in any case. Aortitis was hemodynamically significant in 3 patients. Two had congestive heart failure secondary to thoracic aortitis and aortic valvulitis, and 1 had rupture of an abdominal aortic aneurysm at a site involved by aortitis. Seven patients had rheumatoid vasculitis with a mean of 10 organs involved. Six of these died of complications directly related to vasculitis, including 4 patients with coronary arteritis and associated myocardial infarction. Aortitis can be a feature of severe rheumatoid arthritis and is often associated with rheumatoid vasculitis. Hemodynamic compromise does occur and may be fatal.
The class II (Ia) major histocompatibility complex antigens are a family of integral membrane proteins whose expression is limited to certain cell types, predominantly B lymphocytes, macrophages, and thymic epithelial cells. In B cells, Ia expression is both developmentally regulated and responsive to external stimuli. The differentiation of early B stem cells to mature B lymphocytes is accompanied by the appearance of cell surface Ia antigens; the transition to plasma cells results in loss of class II gene expression. In Ia-expressing B cells, the T cell-derived lymphokine interleukin-4 (IL-4) increases such expression by an as yet undefined mechanism. Chloramphenicol acetyltransferase gene expression was cis-activated by a region of the Ia A alpha k gene in a B lymphoma line, but not in a myeloma line. A nuclear protein that bound to two sites within this region, upstream from previously described transcription elements, was found in normal spleen cells. This binding activity was also found in spleen extracts from athymic mice, which lack T lymphocytes, and in Ia-positive B lymphocyte tumor cell lines, demonstrating that it is a B cell protein. Further analysis showed the activity to be undetectable in an Ia-negative pre-B cell line and in three plasmacytoma cell lines that are Ia negative. IL-4 treatment of normal and athymic mouse spleen cells greatly increased the binding of this nuclear protein to these two sites, concomitant with increased MHC class II gene transcription. Thus, B cells contain a sequence-specific DNA-binding activity whose level is influenced both by IL-4 and by differentiation signals.
Tophi are rarely observed in patients without a prior history of gouty arthritis. We describe four patients whose initial manifestation of gout was tophaceous deposition in an unusual location, the finger pad. None of these patients had a history of acute gouty arthritis and none had tophi elsewhere. All four patients were postmenopausal women with decreased renal function; all were taking diuretics. We conclude that tophaceous gout without arthritis may be more common than previously recognized and that tophi may deposit in the finger pad. We recommend prompt aspiration and crystal analysis of white subcutaneous finger pad deposits in hyperuricemic patients even without a history of gouty arthritis.
Toxic epidermal necrolysis is a life-threatening disease, the pathogenesis of which remains largely unknown. Histologically, in addition to the characteristic epidermal alterations, there is a sparse mononuclear cell infiltrate in the dermis. The immunologic characteristics of this infiltrate are not well known. In a case of drug-induced toxic epidermal necrolysis with fatal outcome in a 48-year-old man, we demonstrated that the majority of the inflammatory cells were of helper/inducer T-lymphocyte subsets, having only a minority of cytotoxic/suppressor T-lymphocytes and rare cells with natural killer cell phenotype. The significance of these observations is discussed, with reference to the occurrence of lesions at epithelial sites bearing local networks of antigen-presenting cells (Langerhans' cells).
Nonenzymatic glycosylation of proteins of the erythrocyte membrane was determined by incubating erythrocyte ghosts with [3H]borohydride. The incorporation of tritium into protein provides a reliable assay of ketoamine linkages. The membrane proteins from 18 patients with diabetes incorporated twice as much radioactivity as membrane proteins from normal erythrocytes. After acid hydrolysis, amino acid analysis showed that the majority of radioactivity was localized to glucosyllysine. Autoradiograms showed that all of the major proteins of the erythrocyte membrane, separated by electrophoresis on sodium dodecyl sulfate gels, contained ketoamine linkages. No protein bands in either normal or diabetic erythrocytes showed significant preferential labeling. Erythrocyte membranes from three patients with hemolytic anemia showed reduced incorporation of tritium from [3H]-borohydride, indicating decreased nonenzymatic glycosylation. Two patients with diabetes and hemolytic anemia had incorporation of radioactivity similar to that of normal individuals. In these groups of patients the incorporation of tritium into erythrocyte membrane proteins correlated with levels of hemoglobin AIc. Thus the modification of membrane proteins like that of hemoglobin depends on blood glucose levels as well as erythrocyte age. These studies show that the enhanced nonenzymatic glycosylation of proteins in diabetics extends beyond hemoglobin to the proteins of the erythrocyte membrane and probably affects other proteins that have slow turnover and are exposed to high concentrations of glucose.
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A Virtual World Versus Face-to-Face Intervention Format to Promote Diabetes Self-Management Among African American Women: A Pilot Randomized Clinical Trial
BACKGROUND: Virtual world environments have the potential to increase access to diabetes self-management interventions and may lower cost.
OBJECTIVE: We tested the feasibility and comparative effectiveness of a virtual world versus a face-to-face diabetes self-management group intervention.
METHODS: We recruited African American women with type 2 diabetes to participate in an 8-week diabetes self-management program adapted from Power to Prevent, a behavior-change in-person group program for African Americans with diabetes or pre-diabetes. The program is social cognitive theory-guided, evidence-based, and culturally tailored. Participants were randomized to participate in the program via virtual world (Second Life) or face-to-face, both delivered by a single intervention team. Blinded assessors conducted in-person clinical (HbA1c), behavioral, and psychosocial measurements at baseline and 4-month follow-up. Pre-post differences within and between intervention groups were assessed using t tests and chi-square tests (two-sided and intention-to-treat analyses for all comparisons).
RESULTS: Participants (N = 89) were an average of 52 years old (SD 10), 60% had < /=high school, 82% had household incomes < US $30,000, and computer experience was variable. Overall session attendance was similar across the groups (6.8/8 sessions, P = .90). Compared to face-to-face, virtual world was slightly superior for total activity, light activity, and inactivity (P = .05, P = .07, and P = .025, respectively). HbA1c reduction was significant within face-to-face (-0.46, P = 02) but not within virtual world (-0.31, P = .19), although there were no significant between group differences in HbA1c (P = .52). In both groups, 14% fewer patients had post-intervention HbA1c > /=9% (virtual world P = .014; face-to-face P = .002), with no significant between group difference (P = .493). Compared to virtual world, face-to-face was marginally superior for reducing depression symptoms (P = .051). The virtual world intervention costs were US $1117 versus US $931 for face-to-face. CONCLUSIONS: It is feasible to deliver diabetes self-management interventions to inner city African American women via virtual worlds, and outcomes may be comparable to those of face-to-face interventions. Further effectiveness research is warranted.
TRIAL REGISTRATION: ClinicalTrials.gov NCT01340079; http://clinicaltrials.gov/show/NCT01340079 (Archived by WebCite at http://www.webcitation.org/6T2aSvmka).