Effects of a new advanced glycation inhibitor, LR-90, on mitigating arterial stiffening and improving arterial elasticity and compliance in a diabetic rat model: aortic impedance analysis.

BACKGROUND AND PURPOSE: We determined the effects of treatment with LR-90, an inhibitor of advanced glycation end products, on the mechanical properties of the arterial system in streptozotocin (STZ)-induced diabetic Sprague Dawley rats, using aortic impedance analysis, and further investigated the effects of LR-90 on the progression of aortic pathology. EXPERIMENTAL APPROACH: STZ-induced diabetic rats were treated with or without LR-90 (50 mg L(-1) in drinking water) for 8 weeks and compared with control groups. Arterial BP measurements, various metabolic parameters, aortic histopathology, collagen cross-linking, AGE accumulation, and RAGE protein expression in aortic tissue were determined. Pulsatile parameters were evaluated using a standard Fourier series expansion technique and impulse response function of the filtered aortic input impedance spectra. KEY RESULTS: LR-90 reduced glycated haemoglobin and triglycerides levels, although it had no effect on the glycaemic status. LR-90 did not affect arterial BP, but prevented the diabetes-induced increase in peripheral resistance and variations in aortic distensibility, as it reduced aortic characteristic impedance by 21%. LR-90 also prevented the elevation in wave reflection factor, as indicated by a 22.5% reduction and an associated increase of 23.5% in wave transit time, suggesting it prevents the augmentation of the systolic load of the left ventricle. Moreover, LR-90 inhibited collagen cross-linking and the accumulation of AGE and RAGE in the vasculature of diabetic rats. CONCLUSIONS AND IMPLICATIONS: Treatment with LR-90 may impart significant protection against diabetes-induced aortic stiffening and cardiac hypertrophy and provides an additional therapeutic option for treatment of AGE associated diabetic complications.

Characterization of a North American orf virus isolated from a goat with persistent, proliferative dermatitis.

The characterization of an orf virus (OV) isolated from skin lesions of a goat kid with severe, persistent, proliferative dermatitis, and designated orf virus-San Angelo 2000 (OV-SA00) strain, is described. The identity of OV-SA00 was confirmed by a combination of methods, including electron microscopy, amplification of specific fragments of viral DNA by polymerase chain reaction, restriction enzyme analysis of viral DNA and gene sequencing. Restriction endonuclease analyses of viral DNA and the protein profile studied by Western blot revealed differences between OV-SA00 strain and the profiles of other OV strains that have been published. The restriction enzyme profile of OV-SA00 was also different from the orf virus vaccine (OV-V) strain used to vaccinate this kid. Comparison of the nucleotide and deduced amino acid sequences indicated that OV-SA00 is closely related to OV-V strain, the Scottish OV strains orf11 and MRI Scab, and the human OV-CE/Shoe strain and more distant to bovine papular stomatitis virus (BPSV) reference strain and the pseudocowpox virus (PCPV)-MNV/Till strain. These results indicate that OV-SA00 is a strain of OV rather than a different parapoxvirus. Further studies are necessary to determine if the severity of orf-induced lesions in this goat kid was the result of individual host susceptibility factors.

Thioredoxin treatment increases digestibility and lowers allergenicity of milk.

BACKGROUND: By resisting digestion in the stomach, the major bovine milk allergen, beta-lactoglobulin, is believed to act as a transporter of vitamin A and retinol to the intestines. beta-Lactoglobulin has 2 intramolecular disulfide bonds that may be responsible for its allergic effects. OBJECTIVE: This study was carried out to assess the importance of disulfide bonds to the allergenicity and digestibility of beta-lactoglobulin. METHODS: beta-Lactoglobulin was subjected to reduction by the ubiquitous protein thioredoxin, which was itself reduced by the reduced form of nicotinamide adenine dinucleotide phosphate by means of nicotinamide adenine dinucleotide phosphate-thioredoxin reductase. Digestibility was measured with a simulated gastric fluid; results were analyzed by SDS-PAGE. Allergenicity was assessed with an inbred colony of high IgE-producing dogs sensitized to milk. RESULTS: As found for other proteins with intramolecular disulfide bonds, beta-lactoglobulin was reduced specifically by the thioredoxin system. After reduction of one or both of its disulfide bonds, beta-lactoglobulin became strikingly sensitive to pepsin and lost allergenicity as determined by skin test responses and gastrointestinal symptoms in the dog model. CONCLUSION: The results provide new evidence that thioredoxin can be applied to enhance digestibility and lower allergenicity of food proteins.

Analysis of the molecular basis of synovial rheumatoid factors in rheumatoid arthritis.

The objective of this study was to better understand the molecular basis of IgM rheumatoid factor in rheumatoid arthritis (RA). We recently generated 10 different monoclonal IgM RF (mRF) molecules isolated from the synovium of a single patient with RA. The heavy (H) and light chain (L) variable region (V) genes of these 10 mRFs were cloned and sequenced. Six mRFs used kappa light chains and 4 mRFs used lambda light chains. Of particular interest, 8 of 10 heavy chains used the JH4 joining region gene, and all five VH4 heavy chains used the DK4 diversity region gene with the JH4. Four of the VH4 clones used the same germline gene, likely representing a novel but closely related germline gene to VH4.18, and may be clonally related because of the extensive homology in their heavy chain sequence. Two VH4 clones shared the same light chain gene, VkappaIIIb kv325 (99% homology) and the same JK4 joining region gene, while three VH4 clones used two different light chain genes, an uncommon Vkappa4 and a Vlambda4 gene, respectively. In this RA patient, there was recurrent utilization of VH4-DK4-21/10-JH4 genes and a recurring association with gene elements Vkappa3 and Vlambda4. Recurring usage of Vkappa3 (kv325) and Vlambda4 (lv418) gene elements may result from a light chain editing process whereby immature autoreactive B cells encountering self-antigen attempt, and often succeed, in altering their specificities through secondary Ig light chain gene rearrangement. Moreover, the oligoclonality of these RFs suggest clonal relatedness secondary to an antigen-driven response.

Thioredoxin-linked mitigation of allergic responses to wheat.

Thioredoxin, a ubiquitous 12-kDa regulatory disulfide protein, was found to reduce disulfide bonds of allergens (convert S-S to 2 SH) and thereby mitigate the allergenicity of commercial wheat preparations. Allergenic strength was determined by skin tests with a canine model for food allergy. Statistically significant mitigation was observed with 15 of 16 wheat-sensitive animals. The allergenicity of the protein fractions extracted from wheat flour with the indicated solvent was also assessed: the gliadins (ethanol) were the strongest allergens, followed by glutenins (acetic acid), albumins (water), and globulins (salt water). Of the gliadins, the alpha and beta fractions were most potent, followed by the gamma and omega types. Thioredoxin mitigated the allergenicity associated with the major protein fractions-i.e, the gliadins (including the alpha, beta, and gamma types) and the glutenins-but gave less consistent results with the minor fractions, the albumins and globulins. In all cases, mitigation was specific to thioredoxin that had been reduced either enzymically by NADPH and NADP-thioredoxin reductase or chemically by dithiothreitol; reduced glutathione was without significant effect. As in previous studies, thioredoxin was particularly effective in the reduction of intramolecular (intrachain) disulfide bonds. The present results demonstrate that the reduction of these disulfide bonds is accompanied by a statistically significant decrease in allergenicity of the active proteins. This decrease occurs alongside the changes identified previously-i.e., increased susceptibility to proteolysis and heat, and altered biochemical activity. The findings open the door to the testing of the thioredoxin system in the production of hypoallergenic, more-digestible foods.

Polymerase chain reaction and restriction endonuclease digestion for selected members of the "Mycoplasma mycoides cluster" and Mycoplasma putrefaciens.

A specific diagnostic method using the polymerase chain reaction, together with restriction endonuclease digestion patterns, was developed for members of the "Mycoplasma mycoides cluster" that normally occur in the United States (i.e., Mycoplasma mycoides subsp. mycoides Large Colony and Mycoplasma capricolum subsp. capricolum in addition to "cluster" mycoplasma, bovine serogroup 7, and Mycoplasma putrefaciens. The digestion of "cluster" polymerase chain reaction DNA (1,225 bp) amplification products with restriction enzymes AseI and SspI gave mycoplasma species-specific patterns for all strains of M. mycoides subsp. mycoides Large Colony, M. capricolum subsp. capricolum, and bovine group 7 tested. Moreover, we found a nonspecific amplification product for M. putrefaciens that occurred with the oligonucleotide primers used for the "M. mycoides cluster" reaction. However, the restriction endonuclease digestion patterns observed with the restriction enzymes AluI, AseI, and SspI for M. putrefaciens were different than the digestion patterns obtained for the other "cluster" mycoplasmas. This report confirms the usefulness of polymerase chain reaction DNA amplification allied with restriction enzyme digestion profile analysis for the rapid and specific identification of mycoplasmas belonging to the "M. mycoides cluster" and M. putrefaciens.

The atopic dog: a model for food allergy.

The renewed interest in food allergy and its investigation has been hampered by the lack of an appropriate animal model with similar comparative aspects of form and function relative to humans. Therefore we have been characterizing an inbred colony of high immunoglobulin E-producing dogs that were immunized subcutaneously with food antigen extracts in alum and that developed clinical manifestations of food allergy after oral challenges with food antigen. These dogs had appreciably high IgE antibody titer to specific food antigens, as measured by an enzyme-labeled immunodot assay. Skin test results for the food antigens were consistently positive, as evidenced by a wheal-and-flare reaction. Gastroscopic food sensitivity was tested through an endoscope by injecting allergenic food extracts into the gastric mucosa after intravenous injection of Evans blue dye. Mucosal changes included swelling and erythema, some petechiae and blue patching, and in some instances generalized gastric erythema and hyperperistalsis. Examination of immediate-reaction biopsy specimens revealed edema and few inflammatory cells. Examination of late-reaction biopsy specimens revealed increased eosinophil and mononuclear cell infiltrations typical of late-phase allergic inflammatory responses. Direct mucosal challenge with food extracts confirmed the clinical and immunologic evidence of food allergy in these immunized dogs and suggests the usefulness of the atopic dog as a model for food allergy. This model might also be useful in detecting hidden food allergies in unexplained inflammatory gastrointestinal tract diseases.

Preferential utilization of a novel V lambda 3 gene in monoclonal rheumatoid factors derived from the synovial cells of rheumatoid arthritis patients.

OBJECTIVE: To further our understanding about the molecular genetics of rheumatoid factor (RF) in rheumatoid arthritis (RA). METHODS: The heavy and light chain variable region (V) genes of 5 new human monoclonal IgM RFs were cloned and sequenced using the polymerase chain reaction and the dideoxynucleotide termination method. RESULTS: The results reveal the recurrent usage in two RA patients of a novel V lambda 3 germline gene, designated Humlv3c93. Specifically, in 2 of 3 RFs (C93 and D53) from one patient, the light chains in the V lambda gene-encoded region were identical to each other and to the light chain of an RF (H4) from another patient. Serologically, the light chains of these 3 RFs were classified as members of the V lambda 3b sub-subgroup. Each of the RFs was encoded by a different VH gene. Both C93 and D53 bound specifically with human and rabbit IgG, whereas H4 was monospecific for rabbit IgG. CONCLUSION: Since the lv3c93 gene is not homologous to any reported V lambda sequence from natural autoantibodies, it is possible that lv3c93 may represent a disease-specific RF-related V lambda gene. Moreover, the amino acid sequence CSGGSCY in the third complementarity-determining regions of 2 of the RF heavy chains is encoded by the DLR2 gene segment and has been found previously in 2 other RA-derived RFs, and thus may play a significant role in antigen binding.

IgG3 reactive rheumatoid factor in rheumatoid arthritis: etiologic and pathogenic considerations.

Rheumatoid factor (RF) is a polyclonal autoantibody directed against the Fc portion of IgG. Although the role of RF in patients with rheumatoid arthritis (RA) is unclear, immune complexes that form between RF and IgG can activate the classical complement (C) pathway, leading to pathogenic outcomes involving inflammatory events and tissue damage. The specificity of serum RF and RF produced by rheumatoid synovial cells (RSC) is different. Serum RF has specificity for rabbit IgG and human IgG subclasses IgG1, 2, and 4, but binds poorly to IgG3. The affinity of serum RF for IgG Fc is low, having an association constant of 10(4)-10(5) M-1. RSC RF, however, has specificity for human IgG and high avidity for IgG3. Because of this greater specificity and avidity for IgG3, and because RSC RF may be pathogenically more important than serum RF, an important role for IgG3-reactive RF in RA may exist. Binding of RF to IgG may be dependent on the allotype and glycosylation of IgG. Infectious agents present in RA patients may directly or indirectly induce the production of certain RF. In this communication, we review and expand on several observations examining the role of IgG3-reactive RF in RA including: 1) binding differences between RF derived from RSC and serum; 2) glycosylation characteristics of IgG and its interaction with RF; 3) apparent allotype dependent binding of IgG3-reactive RF; and 4) possible relationship between infectious agents and the production of IgG3-reactive RF. Taken together, these observations suggest an important role for IgG3-reactive RF in better understanding the etiology and pathogenesis of RA.

Human monoclonal rheumatoid factor derived from rheumatoid synovial cells monospecific for rabbit IgG.

Rheumatoid factors (RF) in rheumatoid arthritis (RA) are polyclonal autoantibodies directed against antigenic epitopes located in the Fc portion of the IgG molecule. Hybridoma technology has overcome the difficulty of their polyclonality, so that monoclonal RF (mRF) can be examined for their individual binding specificities and genetics. We isolated a monoclonal IgM RF secreting hybridoma (designated H4) from the rheumatoid synovial cells (RSC) of a patient with RA. H4 bound specifically with rabbit IgG (RIgG) and had no human IgG (HIgG) reactivity. By direct binding ELISA and absorption experiments, 6% of the RIgG reactive RSC RF in this patient with RA was monospecific for RIgG. H4 was tested against RIgG F(ab')2 and pFc' fragments, and bound only to the pFc' fragment (CH3 domain). Moreover, H4 mRF had high avidity for RIgG in a capture ELISA. Total RNA was extracted and the variable region heavy (VH) and light (VL) chain cDNA were amplified using polymerase chain reaction technology. Sequence analysis of the IgM RF VH and VL chain genes indicated usage of the VH26 germline gene (VhIII gene family) and a new V lambda germline gene. Our results suggest preferential use of restricted germline genes in the formation of autoantibodies in human autoimmune diseases. The pathological significance of RIgG specific RF is still unclear. However, this finding suggests that all RSC RF production may not necessarily be induced by autologous IgG.

Allotypic dependency of the specificity and avidity of human monoclonal IgM rheumatoid factors derived from rheumatoid synovial cells.

OBJECTIVE: To better understand the genetic derivation and pathogenicity of rheumatoid factor (RF) molecules in rheumatoid arthritis (RA), we have focused our studies on rheumatoid synovial cells (RSC). METHODS: Five monoclonal human IgM rheumatoid factor (mRF)-secreting hybridomas were produced from the RSC of an RA patient. Fine subclass specificities and avidities of these RSC mRFs were compared with several paraprotein monoclonal IgM RFs using direct binding (reactivity) and competitive inhibition (specificity and avidity) enzyme-linked immunosorbent assays. RESULTS: The following observations were made: 1) RSC mRF had greater avidity for IgG than did paraprotein mRF; 2) 4 of the 5 RSC RF were highly avid for IgG3; and, 3) the avidity of RSC RF binding for IgG3 was highest for IgG molecules expressing the G3m(5) allotype. CONCLUSION: We conclude that RSC RF have different specificities and avidities than do paraprotein RF. This may suggest an antigen-driven process in RA synovium, with the production of higher-avidity IgG3m(5)-specific RSC RF, which could have special pathogenetic importance.

Molecular analysis of rheumatoid factors derived from rheumatoid synovium suggests an antigen-driven response in inflamed joints.

OBJECTIVE: Understanding the molecular genetic basis for rheumatoid factor (RF) production is necessary to a better understanding of the etiology and pathogenesis of rheumatoid arthritis (RA). We sought to define the genetic basis of RF in RA. METHODS: The heavy and light chain variable region genes encoding 4 human monoclonal RF were cloned and sequenced using the polymerase chain reaction and the dideoxynucleotide chain-termination method. RESULTS: The heavy and light chains of the C6 RF and the light chain of the G9 RF were encoded by 3 new RF-related Ig V-region genes. The heavy and light chains of D5 and G4 RFs were identical; most of their mutations caused amino acid substitutions. CONCLUSIONS: The RF-related Ig V-region gene repertoire is large and is still expanding. The data from D5 and G4 strongly suggest that these 2 RFs arise in an antigen-driven response in rheumatoid synovium. The presumed germline V genes for C6 may represent disease-specific RF-related V genes.

Allotype-dependent stimulation of peripheral blood and synovial lymphocytes by IgG3 in rheumatoid arthritis.

The immunopathologic process of rheumatoid arthritis (RA) is primarily expressed in the synovium where rheumatoid factor (RF) synthesis is concentrated. We hypothesized that RF synthesized by rheumatoid synovial cells (RSC) may be driven via a T cell-mediated immune response developed against IgG3 epitopes. To identify and characterize specific RSC RF epitopes and T cell antigens, two 28 amino acid peptides homologous with the C-terminus of IgG1 (P1) and IgG3 [G3m(5)] (P3) were synthesized and used in RF-binding studies and lymphocyte proliferation assays. Our results indicate that (i) the C-terminus of the CH3 domain contains epitopes for IgG3-reactive RSC RF; (ii) IgG3-reactive RSC RF binds primarily to IgG3 [G3m(5)]; (iii) P3 stimulated proliferation of T lymphocytes from both RA peripheral blood and RSC; and (iv) RF production was enhanced by P3 in selected RA cell cultures. These observations suggest that the C-terminus of IgG3 allotype G3m(5) may be important in T cell activation and RF production in RA.

Incidence of otitis media in CBA/J and CBA/CaJ mice.

The inbred CBA/J mouse has become a standard experimental animal for auditory study because of its lifelong good hearing. In a newly established mouse breeding colony that housed CBA/J and CBA/CaJ mice to reared as auditory subjects, otitis media frequently afflicted CBA/J mice, reaching an incidence of 90% in animals greater than 400 days of age. Otitis media was not found in CBA/CaJ mice. Three attempts to establish a colony that was free of otitis were unsuccessful. Although the primary pathogen was not clearly established, Pasteurella pneumotropica was isolated from infected bullae. Partial control of otitis media followed the introduction of tetracycline prophylaxis. The CBA/CaJ mice may be suitable replacements for CBA/J mice in studies that require inbred mice with good hearing, since their auditory thresholds did not differ significantly from those of otitis-free CBA/J mice.

Q fever vaccination of sheep: challenge of immunity in ewes.

Adult ewes (17 months of age) were vaccinated against Coxiella burnetii, using a formalin-inactivated whole cell (WC) phase I Henzerling strain vaccine or a chloroform methanol residue (CMR) vaccine. Nineteen pregnant ewes were placed in 3 categories [(i) unvaccinated, (ii) WC vaccine, and (iii) CMR vaccine] and were challenge exposed at approximately the 100th day of gestation with 210,000 plaque-forming units of C burnetii inoculated subcutaneously. Shedding of rickettsiae was measurably reduced, but was not prevented in vaccinated groups, as shown by inoculating ewes' placental tissues, amniotic fluid, and colostrum into mice, as well as by histopathologic lesions of placental tissues. The rickettsiae were shed in the placenta, amniotic fluid, or colostrum in 6 nonvaccinated ewes. In comparison, rickettsiae were detected in placental inoculations from 2 of 6 ewes in the WC vaccine group and 1 of 6 in the CMR group. In contrast to those in the vaccinated ewes, placentitis, high concentrations of rickettsiae in microscopic preparations, and weak lambs were typical for the nonvaccinated ewes.