Human rheumatoid factor crossidiotypes. I. WA and BLA are heat-labile conformational antigens requiring both heavy and light chains.

Evidence was obtained that both the WA and BLA crossidiotype (XId) groups are conformational antigens requiring both L and H chains and that with heat denaturation the antigens that define the XIds and antigen-binding activity are lost in parallel. In contrast, the primary structure-dependent crossreactive idiotype (CRI), PSL2, which is only weakly detected on native Wa and Bla monoclonal rheumatoid factors (mRFs), became prominently detected on the heated Wa and Bla mRFs. Heat denaturation may provide a simple method for distinguishing Ids determined by conformational antigen from primary structure-dependent Ids. In addition to heat denaturation, some acid conditions commonly used for preparation of RFs were also found to cause marked loss of Id antigen. The finding of PSL2-CRI on Bla mRF indicates that this Id is not unique to the WA XId.

Enhancement of glomerular immune complex deposition by a circulating polycation.

It is known that polycations bind to and neutralize glomerular polyanions. Here we examine the effect of the polycation polyethyleneimine (PEI) on glomerular deposition of preformed immune complexes. Bovine serum albumin (BSA)-anti-BSA immune complexes made in 40 times antigen excess were administered following intravenous injection of PEI. Glomerular localization of immune deposits was assessed by quantitative immunofluorescence and electron microscopy and compared to controls receiving diluent without PEI followed by the same dose in immune complexes. In rats receiving PEI, deposits were localized within the glomerular basement membrane (GBM) of all peripheral capillary walls and in the mesangium. In controls, deposits localized exclusively within the mesangium in smaller amounts than after PEI. Thus, neutralization of glomerular polyanion by a circulating polycation enhances the deposition and alters the distribution of immune complexes in glomeruli.

Human rheumatoid factor crossidiotypes. II. Primary structure-dependent crossreactive idiotype, PSL2-CRI, present on Wa monoclonal rheumatoid factors is present on Bla and other IgM kappa monoclonal autoantibodies.

The amino acid sequence of the L-CDR2 (complementarity-determining region) of Bla mRF (monoclonal rheumatoid factor) is identical to that of the Wa mRFs. The PSL2-CRI (crossreactive idiotype), as determined by anti-PSL2, which has been shown to be present on all Wa mRFs, is also present on the Bla mRF and other monoclonal autoantibodies. PSL2-CRI is, therefore, not unique to Wa mRFs and may be present on most IgM kappa monoclonal autoantibodies. Whether PSL2-CRI is a crossidiotype (XId) that is selectively present on autoantibodies or represents an allotypic marker for a V kappa III gene is undetermined.

Human rheumatoid factor cross-idiotypes. IV. Studies on WA XId-positive IgM without rheumatoid factor activity provide evidence that the WA XId is not unique to rheumatoid factors and is distinct from the 17.109 and G6 XIds.

The WA cross-idiotype (XId) is the major XId among human monoclonal rheumatoid factors (mRF) and is almost always associated with the light (L) chain XId, 17.109, and the heavy (H) chain XId, G6. A cell line, 35G6, was cloned that bears the WA XId, but shows no reactivity with immunoglobulin G (IgG) and is negative for the 17.109 and G6 XIds. The 35G6 L chain appears to be derived from the same VKIII-JKI genes as most WA mRFs L chains. In contrast to the WA mRFs H chains in which VH1 genes are used, the 35G6 IgM expresses a VH3 gene. Sequence comparisons with other WA XId-positive mRF suggested several common structural features that may be related to the WA XId and differences that may relate to lack of IgG reactivity. Cells similar to 35G6 have previously been described in pokeweed mitogen-stimulated cell lines of peripheral blood lymphocytes from normal individuals and patients with rheumatoid arthritis and type II mixed cryoglobulinemia. These observations were confirmed, and in addition, it was shown that the majority of WA XId-positive cells in these cultures were negative for the 17.109 and G6 XIds. The presence of the WA XId in the absence of IgG reactivity suggests that the WA XId is more directly associated with an antigen specificity other than IgG, and its association with RF activity may be incidental. It is postulated that these WA XId-positive RF-negative antibodies may serve a physiologic role as natural antibodies to a pervasive pathogen, and that IgG reactivity is a consequence of somatic diversification accompanying proliferation of the WA XId-positive RF-negative cell.

Predicting dental school performance based on prior dental experience and exposure.

PURPOSE: The purpose of this study was to evaluate whether the variables of students with prior dental assisting experience and students with a parent who is a dentist can be used as predictors of students' pre-clinical and clinical course performance in dental school. MATERIALS AND METHODS: The study population consisted of a cohort of 159 students in the Harvard School of Dental Medicine (HSDM) DMD graduation classes of 2001-2005. Data were collected via self-report using students' applications for admission to the HSDM DMD programme on which students provided information regarding whether they had prior dental assisting experience, including the type and duration of the experience and whether one or both of their parents were dentists. Data on the students' undergraduate science grade point average, Dental Admission Test academic average, Perceptual Ability Test (PAT) score, NBDE Part I and HSDM course grades (three pre-clinical and five clinical assessment categories) were collected from the Office of the Registrar. The pre-clinical categories included the first Oral Comprehensive Exam and the first two classes of the pre-clinical portion of the dental school, Treatment of Active Disease (TxAD) and Restorative Treatment (RTx). The clinical categories included the second Oral Comprehensive Exam and the cumulative grades received for the clinical procedures performed during the third and fourth years in the fields of Endodontics, Operative Dentistry, Periodontics and Prosthodontics. Descriptive and bivariate statistical analyses were performed and included in a multiple logistic regression model. RESULTS: The results revealed that for the variable of prior dental-assisting experience, no statistically significant differences were noted in the pre-clinical and clinical assessment categories. However, students who had any amount of assisting experience were 2.2 times more likely to earn a grade of honours in TxAD compared with students who did not have assisting experience (P = 0.05). Students with a parent who was a dentist performed better only in Operative Dentistry clinical assessment compared with students without a dentist parent (P < 0.05). CONCLUSIONS: Information on prior dental-assisting experience and having a parent who is a dentist have minimal merits for use as predictive agents based on these findings. Dental school admissions committees should continue to review a full spectrum of variables and ensure an applicant's true interest and motivation to pursue a career in dentistry.

Selective CCR2-targeted macrophage depletion ameliorates experimental mesangioproliferative glomerulonephritis.

The CCL2/CCR2 chemokine/receptor axis directs the chemotaxis of infiltrating monocytes/macrophages and T cells and plays a pivotal role in tissue damage and fibrosis in kidney diseases. The eradication of the activated leucocytes should diminish the production of inflammatory mediators, limit tissue damage and ameliorate disease. A recombinant fusion protein (OPL-CCL2-LPM) comprised of the human CCL2 (monocyte chemoattractant protein-1) chemokine fused to a truncated form of the enzymatically active A1 domain of Shigella dysenteriae holotoxin (SA1) has been developed. The CCL2 portion binds specifically to CCR2-bearing leucocytes and the fusion protein enters the cells, where the SA1 moiety inhibits protein synthesis resulting in cell death. The compound was tested in a model of anti-thymocyte serum (ATS)-induced mesangioproliferative glomerulonephritis (ATS-GN). Male rats were injected with ATS on day 0 and treated intravenously with vehicle, 50 or 100 microg/kg of OPL-CCL2-LPM Q2D from days 2, 4, 6 and 8. Urine and blood were collected on days 0, 5 and 9. Animals were sacrificed on day 9. No treatment-related effects on body weight or signs of clinical toxicity were observed. Urine protein levels were decreased in treated animals. At the highest dose, histopathological analyses of kidney sections revealed maximum reductions of 36, 31, 30 and 24% for macrophage count, glomerular lesions, alpha-smooth muscle actin and fibronectin respectively. These results indicate a significant protective effect of OPL-CCL2-LPM in this model of nephritis.

Nonsurgical closure of femoral pseudoaneurysms complicating cardiac catheterization and percutaneous transluminal coronary angioplasty.

OBJECTIVES: This study was performed to describe the initial experience and follow-up of ultrasound-guided compression of pseudoaneurysms in patients receiving systemic anticoagulant or antiplatelet therapy, or both, after recent cardiac catheterization or percutaneous transluminal coronary angioplasty. BACKGROUND: Femoral artery pseudoaneurysm formation after an interventional procedure is becoming more common as larger caliber catheters and prolonged anticoagulant and antiplatelet therapy are being used. Traditional treatment of this complication has been surgical repair. This study describes a new method of closing femoral pseudoaneurysms by using external compression guided by Doppler color flow imaging. METHODS: Fifteen patients, 3 undergoing cardiac catheterization and 12 undergoing coronary angioplasty, developed an expansile groin mass at the vascular access site diagnosed as a femoral artery pseudoaneurysm by Doppler ultrasound. Seven of the patients had undergone coronary stenting and were receiving postprocedural anticoagulant therapy. These patients underwent progressive graded mechanical (C-clamp) external compression guided by ultrasound. The mechanical compression was titrated to obliterate the vascular tracts to these aneurysms and maintain adequate flow in the femoral artery. RESULTS: After an average compression time of 30 min (range 10 to 120), these tracts remained closed. Follow-up ultrasound examination at 24 h or later confirmed continued closure in all. CONCLUSIONS: This study suggests that nonsurgical closure of femoral pseudoaneurysms is feasible. This technique may be valuable in managing vascular access-related complications after diagnostic and interventional procedures, even in patients requiring prolonged anticoagulant therapy.

Synthesis and evaluation of 2-pyridinone derivatives as HIV-1-specific reverse transcriptase inhibitors. 4. 3-[2-(Benzoxazol-2-yl)ethyl]-5-ethyl-6-methylpyridin-2(1H)-one and analogues.

A new series of potent specific 2-pyridinone reverse transcriptase (RT) inhibitors was developed based on the preliminary development lead 3-[(phthalmido)ethyl]-5-ethyl-6-methylpyridin-2(1H)-one (3), a non-nucleoside derivative which exhibited weak antiviral activity in cell culture against HIV-1 strain IIIB. One compound, 3-[(benzoxazol-2-yl)ethyl]-5-ethyl-6-methylpyridin-2(1H)-one (9,L-696,229), which was a highly selective antagonist of the RT enzyme (IC50 = 23 nM) and which inhibited the spread of HIV-1 IIIB infection by > 95% in MT4 human T-lymphoid cell culture (CIC95 = 50-100 nM), was selected for clinical evaluation as an antiviral agent.

Glomerular localization of platelet secretory proteins in mesangial proliferative lesions induced by habu snake venom.

Platelets have been implicated in mesangial cell proliferation in experimental and clinical glomerular disease. In this study, the temporal relationship between release of platelet secretory cationic proteins (PSCP) and progression of mesangial hyperplasia was examined in a model of mesangial proliferative glomerulonephritis induced by Habu snake venom (HSV). Intravenous injection of HSV (2 mg/kg body wt) led to capillary dilatation and ballooning into cysts filled with prominent platelet aggregates at 8 hr and 24 hr. At 48 hr, lesions were heterogeneous, some exclusively cystic, others exclusively nodular (comprised of confluent proliferative mesangial cells). Most lesions were mixed, showing features of cystic lesions containing clusters of proliferating cells. At 72 hr, all lesions were exclusively nodular. These lesions were associated with persistent localization of PSCP, as demonstrated by immunofluorescence microscopy. At 8 hr, PSCP were restricted primarily to platelets, became more intensified and diffuse at later time intervals, and by 72 hr was demonstrated in a homogeneous pattern interspaced throughout the nodular lesions. Studies utilizing antiserum to a specific platelet secretory protein, platelet factor 4 (PF4), showed an identical pattern of glomerular localization. Thus, before and during the proliferative phase of nodular formation, mesangial cells are exposed to a milieu replete with PSCP, some of which are presumably biologically active, suggesting a potential role for platelet-secreted proteins in mesangial hyperplasia in this model.

Expression of plasminogen activator-inhibitor-1 (PAI-1) during cellular remodeling in proliferative glomerulonephritis in the rat.

Pericellular proteolysis involves the plasminogen activator/plasmin system and plays an important role in cell remodeling involving cell migration and extracellular matrix turnover. Studies in this laboratory have previously characterized a model of proliferative glomerulonephritis induced by Habu snake venom (HSV) in the rat that involves cell migration, proliferation, and extracellular matrix accumulation. Because plasminogen activator-inhibitor-1 (PAI-1) has been used as a marker for cell migration as well as matrix accumulation, we were interested in examining the temporal and spatial expression and cellular sources of PAI-1 mRNA and translated protein over the course of HSV-induced proliferative glomerulonephritis. The results showed a highly localized and progressive expression of PAI-1 mRNA and translated protein by in situ hybridization and immunohistochemistry at the margins and periphery of glomerular lesions 8 and 24 hr after HSV. The expression of PAI-1 in glomerular lesions localized to the same sites as mesangial cell marker proteins, desmin and Thy-1.1, indicating that mesangial cells synthesize this important regulator proteolysis. Few cells expressed PAI-1 in the central aspects of glomerular lesions at later time intervals (48 and 72 hr) when cell proliferation and expression of extracellular matrix (fibronectin protein and mRNA) were maximal. Therefore, the expression of PAI-1 in this model was associated more with early events related to cell migration than with proliferation or extracellular matrix synthesis. These observations support the hypothesis that the plasminogen activator/plasmin system is involved in cell migration in early remodeling during glomerular disease.

Expression of embryonic fibronectin isoform EIIIA parallels alpha-smooth muscle actin in maturing and diseased kidney.

In this study we examined if an association exists between expression of an alternatively spliced "embryonic" fibronectin isoform EIIIA (Fn-EIIIA) and alpha-smooth muscle actin (alpha-SMA) in the maturing and adult rat kidney and in two unrelated models of glomerular disease, passive accelerated anti-glomerular basement membrane (GBM) nephritis and Habu venom (HV)-induced proliferative glomerulonephritis, using immunohistochemistry and in situ hybridization. Fn-EIIIA and alpha-SMA proteins were abundantly expressed in mesangium and in periglomerular and peritubular interstitium of 20-day embryonic and 7-day (D-7) postnatal kidneys in regions of tubule and glomerular development. Staining was markedly reduced in these structures in maturing juvenile (D-14) kidney and was largely lost in adult kidney. Expression of Fn-EIIIA and alpha-SMA was reinitiated in the mesangium and the periglomerular and peritubular interstitium in both models and was also observed in glomerular crescents in anti-GBM nephritis. Increased expression of Fn-EIIIA mRNA by in situ hybridization corresponded to the localization of protein staining. Dual labeling experiments verified co-localization of Fn-EIIIA and alpha-SMA, showing a strong correlation of staining between location and staining intensity during kidney development, maturation, and disease. Expression of EIIIA mRNA corresponded to protein expression in developing and diseased kidneys and was lost in adult kidney. These studies show a recapitulation of the co-expression of Fn-EIIIA and alpha-SMA in anti-GBM disease and suggest a functional link for these two proteins.

Differential expression of thrombospondin and cellular fibronectin during remodeling in proliferative glomerulonephritis.

Thrombospondin-1 (TSP-1) and an alternatively spliced fibronectin (Fn)-EIIIA isoform are adhesive proteins associated with embryogenesis and tissue remodeling. We compared, by immunohistochemistry and in situ hybridization, the course of TSP-1 and Fn-EIIIA expression in a model of glomerulonephritis induced by Habu snake venom (HV) and characterized by mesangial cell migration, proliferation, and extracellular matrix (ECM) synthesis. At 24 hr after HV, TSP-1 and Fn-EIIIA proteins localized in the central aspects of lesions associated with platelets and macrophages and at the margins of lesions coinciding with mesangial cell migration (determined by Thy-1 staining). Mesangial cells at this time expressed TSP-1 but not Fn-EIIIA mRNA. TSP-1 protein and mRNA peaked in lesions at 48 hr and were associated with cell proliferation (determined by PCNA, alpha-smooth muscle actin phenotype, and expression of beta-PDGF receptor mRNA). TSP-1 expression declined at 72 hr when expression of ECM synthesis peaked, as determined by increased expression of collagen Type IV, laminin, and TGF-beta1 protein and mRNA. Mesangial cell expression of Fn-EIIIA was first observed at 48 hr and was most abundant at 72 hr after HV. Therefore, platelet- and macrophage-derived Fn-EIIIA and TSP-1 in early lesions are associated with mesangial cell migration. Mesangial cell upregulation of TSP-1 is associated with migration and proliferation but not maximal ECM accumulation, whereas mesangial cell expression of Fn-EIIIA is associated with proliferation and ECM accumulation. These results suggest distinctive temporal and spatial roles for TSP-1 and Fn-EIIIA in remodeling during glomerular disease. (J Histochem Cytochem 47:533-543, 1999)

In situ hybridization in the study of the kidney and renal diseases.

In situ hybridization (ISH), combined with immunohistochemistry, has become a powerful tool in the investigation of temporal and spatial relationships between specific cellular sources of mRNA and ultimate localization of translated protein. This review provides a discussion on basic ISH methods and a comprehensive examination of the literature on applications of ISH to the study of nephrogenesis, normal kidney, and renal disease. The review covers literature examining expression of mRNA encoding growth factors, proteins involved in signal transduction and transcription, proteolysis, extracellular matrix and a variety of other products involved in regulating cell remodeling during nephrogenesis. Expression of mRNA encoding many of these products diminishes in normal adult renal tissue, only to be reactivated during renal disease. Such a recapitulation of the expression of mRNAs involved in nephrogenesis might indicate that similar cellular events are required for remodeling during renal disease. In addition, ISH has allowed for the investigation of interactions between cells intrinsic to the kidney and inflammatory cells (and their biologically active products) recruited from exogenous sources. Although diagnostic use of ISH is currently limited, the implications for the use of this methodology in future clinical applications is promising.

In situ hybridization in the study of remodeling in proliferative glomerulonephritis.

In situ hybridization combined with immunohistochemistry provides a powerful tool to study the temporal and spatial relationships between cellular sources of mRNA and localization of translated protein in normal biologic and pathologic processes. In this symposium, techniques in probe selection for the detection of mRNA in normal kidney and renal disease were discussed. Examples of the application of in situ hybridization in the study of renal disease were demonstrated using a model of proliferative glomerulonephritis induced by habu snake venom. This model follows an accelerated course of remodeling involving mesangial cell migration, proliferation, and extracellular matrix synthesis. The cellular sources and temporal expression of 2 adhesive proteins, fibronectin and thrombospondin, known to have a role in cell remodeling during embryogenesis and wound healing, were examined and compared to mesangial cell behaviors during the course of habu venom-induced glomerulonephritis. Mesangial cell migration in early lesions was associated with thrombospondin and fibronectin derived from platelets or macrophages. Thrombospondin mRNA and protein peaked at 48 hr after habu venom and were associated with mesangial cell proliferation; but thrombospondin mRNA and protein declined at 72 hr when expression of collagen type IV and laminin mRNA and protein peaked. Mesangial cell expression of fibronectin first appeared at 48 hr, and peaked at 72 hr after habu venom. Thus, mesangial cell migration was associated with exogenous fibronectin and thrombospondin derived from platelets or macrophages. Mesangial cell expression of thrombospondin was associated with migration and proliferation, whereas, expression of fibronectin was associated with proliferation and matrix synthesis. These results suggest distinctive temporal and spatial roles for thrombospondin and fibronectin in remodeling during glomerulonephritis and illustrate the utility of in situ hybridization and immunohistochemistry in the detection of cellular sources of translated proteins.

Amelioration of habu venom-induced glomerular lesions: potential role for platelet secretory proteins.

Platelets have been implicated as mediators of mesangial cell proliferation. Of interest is a potential role for platelet secretory proteins (some of which are known to be growth factors) in proliferative glomerular disease. This study examines the effect of sulindac, an inhibitor of platelet thromboxane A2 generation and platelet activation, on the development of glomerular cystic and proliferative lesions after injection of habu snake venom (HSV). To examine the association of platelet secretory proteins with glomerular lesions after HSV, antiserum against a pool of platelet secretory cationic proteins (PSCPs) was used, by immunofluorescence, as a marker of the secretory component of platelet activation in platelet-compromised and normal rats. Uninephrectomized rats received sulindac (60 mg/kg body weight) or vehicle daily before and after HSV (2 mg/kg body weight, IV). Glomerular cysts, proliferative nodules, and mixed lesions (cystic plus proliferative) were quantitated and PSCP localization was examined 48 hours after HSV. Sulindac substantially reduced the total number of glomerular lesions and preferentially reduced proliferative lesions when compared with controls. PSCPs localized in glomerular lesions in both groups and paralleled the severity of disease, but overall intensity of PSCP staining was less in sulindac-treated rats. Sulindac did not alter renal function before HSV, ruling out hemodynamic factors. The concomitant localization of PSCPs in glomerular lesions and amelioration by antiplatelet therapy supports a role for platelet secretory proteins in this model of proliferative glomerular disease.

Platelets in glomerular disease.

Platelets are viewed as inflammatory cells and play an important role in hemostasis and wound repair. During activation, platelets secrete a wide variety of products that have multiple effects on target cell behavior during tissue remodeling. Among these secretory products are chemotactins, growth factors and fibrogenic substances. This review examines evidence for a role of platelets in glomerular disease and discusses mechanisms of how platelet secretory products may alter permselectivity of the glomerular basement membrane, resulting in enhanced immune complex localization. Also, platelet secretory products may stimulate glomerular remodeling after injury through mechanisms involving cell migration, proliferation and extracellular matrix synthesis, ultimately distorting the normal glomerular architecture and function.

Temporal expression of autocrine growth factors corresponds to morphological features of mesangial proliferation in Habu snake venom-induced glomerulonephritis.

Habu snake venom induces an accelerated mesangial proliferative glomerulonephritis that follows a predictable course from early capillary aneurysms to micronodules comprised of confluent mesangial cells within 72 hours. We examined morphologically the course of mesangial cell proliferation and correlated it with the expression of messenger (m) RNA encoding two peptide growth factors, platelet-derived growth factor (PDGF) A and B chains and transforming growth factor-beta (TGF-beta). Rats were uninephrectomized and 24 hours later injected with Habu snake venom or saline. Kidney cortex and isolated glomeruli were obtained 24, 48, and 72 hours later for histological assessment, preparation and Northern analysis of mRNA, and immunohistochemical localization of PDGF using a polyclonal antibody that recognizes A and B chains. Maximal expression of PDGF B chain mRNA occurred at 24 hours and before the onset of mesangial cell proliferation; whereas maximal expression of PDGF A chain and TGF-beta mRNA occurred at 48 hours and during active mesangial cell proliferation. Expression of TGF-beta mRNA persisted at 72 hours at a time when PDGF A chain declined and PDGF B chain was not expressed compared to uninephrectomy and saline controls and at a time when mesangial cells within lesions reached confluence and proliferation subsided. PDGF protein localized in glomerular lesions associated with platelets at 24 and 48 hours and within mesangial cells at 48 and 72 hours. These results agree with the known roles of PDGF and TGF-beta as positive and negative modulators, respectively, of mesangial cell growth in vitro and suggest that a relative balance of the expression of these factors may operate in glomerular disease in vivo.