EBV-associated synovial lymphoma in a chronically inflamed joint in rheumatoid arthritis receiving prolonged methotrexate treatment.

A patient with longstanding rheumatoid arthritis (RA) developed swelling in a chronically inflamed knee joint while receiving prolonged methotrexate treatment. Magnetic resonance imaging and positron-emission tomography showed soft tissue swelling with intense tracer uptake. Biopsy confirmed high-grade B-cell lymphoma. He developed complete remission with rituximab plus CEOP. The role of chronic inflammation and methotrexate in the pathogenesis of lymphoma in RA was discussed.

Papillomavirus-like particles induce acute activation of dendritic cells.

The role of viral structural proteins in the initiation of adaptive immune responses is poorly understood. To address this issue, we focused on the effect of noninfectious papillomavirus-like particles (VLPs) on dendritic cell (DC) activation. We found that murine bone marrow-derived dendritic cells (BMDCs) effectively bound and rapidly internalized bovine papillomavirus VLPS: Exposure to fully assembled VLPs of bovine papillomavirus, human papillomavirus (HPV)16 or HPV18, but not to predominately disordered HPV16 capsomers, induced acute phenotypic maturation of BMDCS: Structurally similar polyomavirus VLPs bound to the DC surface and were internalized, but failed to induce maturation. DCs that had incorporated HPV16 VLPs produced proinflammatory cytokines IL-6 and TNF-alpha; however, the release of these cytokines was delayed relative to LPS activation. Production of IL-12p70 by VLP-exposed DCs required the addition of syngeneic T cells or rIFN-gamma. Finally, BMDCs pulsed with HPV16 VLPs induced Th1-dominated primary T cell responses in vitro. Our data provide evidence that DCs respond to intact papillomavirus capsids and that they play a central role in VLP-induced immunity. These results offer a mechanistic explanation for the striking ability of papillomavirus VLP-based vaccines to induce potent T and B cell responses even in the absence of adjuvant.

Safety and immunogenicity trial in adult volunteers of a human papillomavirus 16 L1 virus-like particle vaccine.

BACKGROUND: Studies in animal models have shown that systemic immunization with a papillomavirus virus-like particle (VLP) vaccine composed of L1, a major structural viral protein, can confer protection against subsequent experimental challenge with the homologous virus. Here we report results of a double-blind, placebo-controlled, dose-escalation trial to evaluate the safety and immunogenicity of a human papillomavirus (HPV) type 16 (HPV16) L1 VLP vaccine in healthy adults. METHODS: Volunteers were given intramuscular injections with placebo or with 10- or 50-microg doses of HPV16 L1 VLP vaccine given without adjuvant or with alum or MF59 as adjuvants at 0, 1, and 4 months. All vaccine recipients were monitored for clinical signs and symptoms for 7 days after each inoculation. Immune responses were measured by an HPV16 L1 VLP-based enzyme-linked immunosorbent assay (ELISA) and by an HPV16 pseudovirion neutralization assay. The antibody titers were given as the reciprocals of the highest dilution showing positive reactivity in each assay. All statistical tests were two-sided. RESULTS: The prevaccination geometric mean ELISA titer for six seropositive individuals was 202 (range, 40--640). All vaccine formulations were well tolerated, and all subjects receiving vaccine seroconverted. Serum antibody responses at 1 month after the third injection were dose dependent in recipients of vaccine without adjuvant or with MF59 but were similar at both doses when alum was the adjuvant. With the higher dose, the geometric means of serum ELISA antibody titers (95% confidence intervals) to purified VLP 1 month after the third injection were as follows: 10,240 (1499 to 69 938) without adjuvant, 10,240 (1114 to 94 145) with MF59, and 2190 (838 to 5723) with alum. Responses of subjects within each group were similar. Neutralizing and ELISA antibody titers were highly correlated (Spearman correlation =.85), confirming that ELISA titers are valid proxies for neutralizing antibodies. CONCLUSIONS: The HPV16 L1 VLP vaccine is well tolerated and is highly immunogenic even without adjuvant, with the majority of the recipients achieving serum antibody titers that were approximately 40-fold higher than what is observed in natural infection.

Fibronectin and hyaluronan in bronchoalveolar lavage fluid from young patients with chronic obstructive pulmonary diseases.

Fibronectin (FN) and hyaluronan (HA) in bronchoalveolar lavage fluid (BALF) and FN released by alveolar macrophages (AM) were examined in 7 nonsmoking healthy control subjects, and 20 smoking patients with chronic obstructive pulmonary diseases (COPD). All patients and subjects were no more than 40 years old. According to the 95% confidence limits of HA and FN in BALF from nonsmoking healthy control subjects, the smoking patients were divided into two groups, those who had HA and FN levels within the limits for nonsmoking controls were classified as the first group (group 1) and those with levels above the control limits were classified as the second group (group 2). Our data showed that the concentrations of HA and FN in BALF and FN released by AM were significantly higher in group 2 than in group 1 patients. There were also significant differences between the two groups in pulmonary function measurements (DLco, FEV1, and FEV1/FVC) which were lower in group 2. HA levels in group 2 patients correlated directly with counts of inflammatory cells in BALF (BALF cells/ml, and numbers/ml of total cells, macrophages, neutrophils and lymphocytes), and the concentration of FN released by AM, and showed an inverse correlation with pulmonary function (DLco and FEV1/FVC). Our results suggest that the inflammatory repair response and fibrosis may play a role in the development of emphysema in young patients with COPD.

Rat placental hepatocyte growth factor/scatter factor: purification, characterization, and developmental regulation.

In view of significant species-specific sequence differences between human and rat placental hepatocyte growth factor (HGF)/scatter factor (SF), the rat placental HGF/SF (rpSF) was purified, and its properties compared with human placental HGF/SF (hpSF). Like hpSF, rpSF scattered Madin-Darby canine kidney cells at 1-2 ng/ml and is composed of two subunits of 60 kDa and 30 kDa. Higher amounts (> 50%) of uncleaved 90-kDa form was present in the HGF/SF preparations from both human and rat placentas. Rat placental SF reacts with antibodies raised against hpSF in rabbits and chickens. The SF activity when expressed per gram rat placental tissue rises rapidly up to 9 days and then levels off. When expressed per milligram tissue protein it also increases rapidly up to 9 days and then declines. The expression of HGF/SF mRNA during development parallels that of HGF/SF activity. The specific activity of HGF/SF receptor (c-met) mRNA also appears to peak at 6 days. These findings suggest that (i) in spite of significant (> 10%) sequence differences between rpSF and hpSF, they exhibit similar structural, biologic, and immunologic characteristics and (ii) HGF/SF and its receptor are expressed in high amounts on Day 6 and then decline in developing placenta.

Suprabasal change and subsequent formation of disulfide-stabilized homo- and hetero-dimers of keratins during esophageal epithelial differentiation.

Rabbit esophageal epithelium, a parakeratinized stratified epithelium, synthesizes as one of its major differentiation products a keratin pair consisting of a basic K4 (59 kDa) and an acidic K13 (41 kDa) keratin. Although immunohistochemical staining data suggest that in esophageal epithelia of some other species these two keratins are suprabasally located, antigenic masking of the epitopes in the basal cells has not been ruled out. Using several well-characterized monoclonal antibodies including AE8, which specifically recognizes K13, coupled with biochemical analysis of keratins of basal and suprabasal cells isolated from confluent rabbit esophageal epithelial culture, we have obtained direct evidence that K4 and K13 keratins are largely absent in the undifferentiated basal cells, but are present in large amounts in suprabasal cells. We also show that in the cornified cell layers that are formed during the terminal stage of esophageal epithelial differentiation, K4 and K13 keratins become disulfide-crosslinked to form three different dimers. Two of them (110 kDa and 100 kDa) are heterodimers and consist of equimolar amounts of K4 and K13; they presumably represent isomers crosslinked via different cysteine residues. The third dimer (90 kDa) was found to be a homodimer of the acidic K13 keratin. Trypsinization experiment established that at least some of the disulfide crosslinks in the K4/K13 heterodimer must involve cysteine residues residing in the trypsin-resistant rod domains of keratins. Air-oxidation of in vitro reconstituted filaments reproduced the two heterodimers, which most likely involve the crosslinking between type I and type II keratins of different coiled coils. The formation of these disulfide-crosslinked keratin dimers, instead of higher molecular mass oligomers or polymers as occurring in the epidermis and hair, may contribute to the formation of cornified cells with a physical stability and rigidity that are optimal for esophageal function. Our data also suggest that interactions involved in the formation of homodimers, thought to be metastable and unimportant during the initial step of filament assembly (i.e. tetramer formation), may actually play an important role in stabilizing a higher order structure in mature keratin filaments.

Effect of cations and temperature on kinetics of desmin assembly.

Smooth-muscle desmin, which was isolated from avian gizzard, was purified and used to form reconstituted intermediate filaments. Filament assembly was done in the presence of physiological cations, Ca2+, Mg2+, Na+, and Na+ plus Mg2+, and with non-physiological cations Cu2+ and Ni2+. Assembly was done at 2 degrees, 22 degrees and 37 degrees C, and was monitored by absorbance and by electron microscopy. Absorbance increased most rapidly during the first 2-5 min and then increased at a slower rate with the physiological cations, but decreased after that time with the non-physiological cations. For each physiological cation, absorbance increased with increasing temperature. This was particularly evident with Ca2+, which produced the lowest absorbance at 2 degrees C and the highest at 37 degrees C. When ionic strength was comparable, filament-forming buffers that contained bivalent cations were associated with higher absorbance values. Filament diameters were significantly smaller 60 min after assembly initiation than after 5 min. Average filament diameters, when formed in the presence of Cu2+ or Ni2+, were 10% greater than in the presence of the physiological cations and did not show a consistent tendency to decrease as time increased. These results demonstrate the importance, not only of the pH and ionic composition of the filament-forming buffer, but also of the temperature and duration of dialysis for reconstitution of desmin filaments.

Assembly of vimentin in vitro and its implications concerning the structure of intermediate filaments.

After dialysis against 10 mM-Tris-acetate (pH 8.5), vimentin that has been purified in the presence of urea is present in the form of tetrameric 2 to 3 nm X 48 nm rods known as protofilaments. These building blocks in turn polymerize into intermediate filaments (10 to 12 nm diameter) when they are dialyzed against a solution of physiological ionic strength and pH. By varying the ionic conditions under which polymerization takes place, we have identified two classes of assembly intermediates whose structures provide clues as to how an intermediate filament may be constructed. The structure of the first class, seen when assembly takes place at 10 to 20 mM-salt at pH 8.5, strongly suggests that one of the initial steps of filament assembly is the association of protofilaments into pairs with a half-unit axial stagger. Increasing the ionic strength of the assembly buffer leads to the emergence of short, full-width intermediate filaments at approximately 50 mM-salt at pH 8.5. In the presence of additional protofilaments, these short filaments elongate to many micrometers when the ionic strength and pH are further adjusted to physiological levels. The electron microscope images of the assembly intermediates suggest that vimentin-containing intermediate filaments are made up of eight protofilaments, assembled such that there is an approximately 22 nm axial stagger between neighboring protofilaments. We propose that this half-unit staggering of protofilaments is a fundamental feature of intermediate filament structure and assembly, and that it could account for the 20 to 22 nm axial repeat seen in all intermediate filaments examined so far.