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Biomedical subjects

K A Richard

Publications and source records attributed to K A Richard.

At least 19 recordsLinked to original sources
PubMed1994

Carbonyl-containing bisphosphonate esters as novel antiinflammatory and antiarthritic agents.

A study of the decomposition of the pyrazoline bisphosphonate ester 2 identified 3 as the sole bisphosphonate component. Evaluation in a delayed-type hypersensitivity granuloma model of chronic inflammation in mice (DTH-GRA) showed 3 to be a potent inhibitor of granuloma formation (sc, 10 mg/kg, 45%), but in a murine model of antigen-induced arthritis (AIA), no significant inhibition was observed. As a result, new ketonic bisphosphonate tetraethyl esters were synthesized from vinylidenebisphosphonic acid tetraethyl ester 4 and activated carbonyl compounds in 13-84% yield. 6 significantly inhibited the pathology of both the DTH-GRA (sc, 25 mg/kg, 45%) and AIA models (sc, 25 mg/kg, 55%). Other compounds in the series were not as potent. Our results show that bisphosphonate ester 6 can inhibit the chronic inflammatory response associated with cutaneous granuloma formation and erosive arthritis.

Animals↗
PubMed1993

Pyrazoline bisphosphonate esters as novel antiinflammatory and antiarthritic agents.

Vinylidenebisphosphonic acid tetraethyl ester (1) and diazo ketones 7a-1 in ether at 22 degrees C yield pyrazoline bisphosphonate tetraethyl esters 8a-1 in moderate to good yield. These compounds were evaluated in animal models of arthritis: rat adjuvant-induced polyarthritis (AIP) and murine antigen-induced arthritis (AIA) and a murine model of chronic inflammation, the delayed type hypersensitivity granuloma reaction (DTH-GRA). (5-Benzoyl-2,4-dihydro-3H-pyrazol-3-ylidene)-bisphosphonic acid tetraethyl ester (8a), and [5-(3-fluorobenzoyl)-2,4-dihydro-3H-pyraxol-3-ylidene]- bisphosphonic acid tetraethyl ester (8d) significantly inhibited the arthritis models, AIP (15 mg/kg) and AIA (25 mg/kg), as well as the DTH-GRA (25 mg/kg). Conversion of 8a to the corresponding bisphosphonic acid, 10a, resulted in loss of activity. Compounds with alkyl substituents on the pyrazoline nitrogen, 9a-d, were inactive in the DTH-GRA. These results show that 8a and 8d have novel antiinflammatory activity and are capable of inhibiting chronic arthritis and inflammation in animals. Such compounds might be useful in man for treating chronic tissue injury associated with arthropathies such as inflammatory joint disease as well as other chronic inflammatory diseases.

Animals↗
PubMed1993

The effect of an interleukin-1 receptor antagonist protein on type II collagen-induced arthritis and antigen-induced arthritis in mice.

OBJECTIVE: To investigate the anti-arthritic effect of recombinant human interleukin-1 receptor antagonist protein (IRAP) in two experimental models of arthritis. METHODS: Recombinant IRAP was administered daily to mice with type II collagen-induced arthritis (CIA) or with antigen-induced arthritis (AIA) provoked by methylated bovine serum albumin (mBSA). Disease incidence and severity were assessed by a clinical index and histologic features. Serum antibody to type II collagen, spleen cell proliferation to mBSA, and anti-IRAP antibodies were measured as indices of immune function. RESULTS: IRAP reduced the incidence and delayed the onset of CIA and suppressed the antibody response to type II collagen. In contrast, IRAP did not affect the pathogenesis of AIA and had no effect on either humoral or cellular immune responses to mBSA in arthritic mice. CONCLUSION: These observations suggest that interleukin-1 may play a prominent role in the development of some, but not all, forms of arthritis.

Animals↗
PubMed1993

The effects of several diphosphonates on murine bone marrow proliferation.

Six diphosphonates were examined for their ability to alter proliferative responses of mouse bone marrow cells to recombinant human M-CSF and recombinant murine GM-CSF. Risedronate ([2-(3-pyridinyl)-ethylidene] hydroxy bisphosphonic acid) added to in vitro cultures at 10 microM, suppressed the response to M-CSF by 58%, but had no significant effect on GM-CSF-induced proliferation. Ethane-1-hydroxy-1,1-bisphosphonic acid (EHDP), dichloromethylene bisphosphonic acid (Cl2MBP), 3-amino-1-hydroxy-propylidene-1,1-bisphosphonic acid (APD), (4-chlorophenyl)-thiomethylene bisphosphonic acid (tiludronate) and (cycloheptylamino)-methylene bisphosphonic acid (YM-175) had no significant effect. Treatment of mice for 5 or 14 days with 200 mg/kg/day p.o., Cl2MBP or 3 mg/kg/day p.o. of risedronate failed to inhibit M-CSF- or GM-CSF-induced proliferation by recovered bone marrow cells. Addition of Cl2MBP or risedronate in vitro to these cells did not reveal any change in sensitivity to CSFs as a result of exposure to diphosphonate in vivo.

Animals↗
PubMed1992

Site-specific chemical modification of interleukin-1 beta by acrylodan at cysteine 8 and lysine 103.

Acrylodan, which normally modifies cysteine residues, was employed to derivatize recombinant interleukin-1 beta (rIL-1 beta) under native conditions, using a reagent:protein ratio of 3:1. Two major covalent protein/acrylodan adducts were generated and subsequently purified by DEAE TSK 5PW ion exchange chromatography. Peptide mapping and mass spectrometry were used to locate the probe on the modified proteins. Both modified proteins carried one molecule of acrylodan each, one at Cys-8 and the other at Lys-103. Neither Cys-71 nor any of the other 13 lysine residues of rIL-1 beta was modified. Cysteine 71 is inaccessible to acrylodan, but the unusual specificity for Lys-103 could be caused by the location of that residue at the bottom of a hydrophobic pocket which might specifically bind the reagent. No double-labeled protein was detected, indicating that the introduction of the label at either site interferes with the labeling at the other. Both acrylodan-modified proteins exhibited bioactivity in the thymocyte proliferation assay at a level equivalent to that of the unmodified control protein (1.7 x 10(7) units/mg), which shows that the modification of either the Cys-8 or Lys-103 position with acrylodan does not interfere with the cellular bioactivities of the respective proteins. Furthermore, receptor binding assays yielded a Kd = 32.0 +/- 4.8 pM for the Lys-103-labeled protein, Kd = 69.5 +/- 12.7 pM for the unmodified protein, and Kd = 75.0 +/- 11.6 pM for the Cys-8-labeled protein. Thus, Cys-8 or Lys-103 modification of rIL-1 beta by acrylodan also does not interfere with the ability of the molecule to bind to its receptor. The slightly higher affinity of the Lys-103-labeled protein for the receptor suggests that the positive charge on this residue in the native molecule may interfere with IL-1 receptor binding. The two fluorescent labeled IL-1 proteins described herein should provide interesting probes for the study of IL-1/IL-1 receptor interactions.

2-Naphthylamine↗
PubMed1992

Chemical modification of interleukin-1 beta: biochemical characterization of a carbodiimide-catalyzed intramolecular cross-linked protein.

We have modified recombinant human Interleukin-1 beta using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide at pH 6.5, resulting in the formation of an internally cross-linked protein. The major product (30% yield) of the reaction (17 kD; pI = 6.2) was purified and fully characterized by peptide mapping using Endoproteinase Lys C. When digests were conducted under nondenaturing conditions, we found that the modified protein is different from the native protein. The native protein yielded 14 peptides after digestion, whereas only two large peptides and a tetrapeptide, Asn-Tyr-Pro-Lys, were released from the cross-linked protein (i.e., cleavage occurs only at residues Lys88 and Lys92). Using gel filtration, the two peptides were found to co-elute as a single species (15 kD), which represent a noncovalent complex of the amino terminal and C-terminal portions of the molecule. Further analysis of the modified protein by peptide mapping under denaturing conditions and by FAB MS analysis showed that Glu111 and Lys138 were internally cross-linked. The cross-linked protein had bioactivity (T-cell proliferation), fluorescence, and circular dichroism spectra similar to native IL-1 beta. In contrast, while having similar secondary structure, the digested cross-linked protein had less than 1% of T-cell proliferative activity of the undigested protein. These data show that the structural integrity surrounding and perhaps including the Asn-Tyr-Pro-Lys region may be crucial for the biological activity of rIL-1 beta and may be important for the binding of IL-1 to its receptor.

Amino Acid Sequence↗
PubMed1990

Induction of an acute erosive monarticular arthritis in mice by interleukin-1 and methylated bovine serum albumin.

We examined the effect of interleukin-1 (IL-1) administration on a mild and transient inflammatory response in the knees of mice injected intraarticularly with methylated bovine serum albumin (mBSA). Injection of mBSA on day 0 into nonsensitized mice caused a weak inflammatory response confined to the infrapatellar fat pads and involved infiltration by mononuclear cells, neutrophils, and eosinophils. The response developed between days 4 and 7 and resolved by day 28. No erosion of cartilage or subchondral bone was seen. In contrast, mBSA-treated mice injected with recombinant human IL-1 beta subcutaneously in the ipsilateral footpad on days 0-3 developed a severe monarticular arthritis in the antigen-injected knee. Pannus developed, extending over the articular surfaces, and extensive erosion of cartilage and subchondral bone occurred. Multinucleated giant cells, together with fibrin-like material, were observed at sites of active bone erosion and debris, and large numbers of neutrophils were seen in the joint space. These pathologic features represent a new arthritis model in which IL-1 profoundly augments a weak inflammatory response and induces acute erosive joint destruction, supporting the hypothesis that IL-1 is an important cytokine in the pathogenesis of arthritis.

Acute Disease↗
PubMed1989

Biotinylation of reactive amino groups in native recombinant human interleukin-1 beta.

Recombinant human interleukin-1 beta (rIL-1 beta) was chemically modified by a 10-fold molar excess (reagent:protein) of sulfosuccinimidyl 6-(biotinamido) hexanoate (sulfo-NHS-LC-biotin) or sulfosuccinimidobiotin (sulfo-NHS-biotin) under mild conditions. The primary product was purified in each case by cation exchange high performance liquid chromatography (HPLC) and digested with endoproteinase Lys C. Peptide mapping by C18 reverse phase HPLC permitted identification of three sites of biotinylation using both reagents; N-terminal alanine, lysine 93, and lysine 94. Few additional singly modified rIL-1 beta products were obtained under these conditions, despite the presence of 15 lysine residues in this protein. These data support the view that the N terminus as well as the trilysine sequence (residues 92-94) are readily susceptible to chemical modification and are exposed on the surface of the protein. Chromatography of intact biotinylated rIL-1 beta by C4 reverse phase HPLC resolved a protein modified exclusively at the N-terminal alanine from two proteins modified singly at either lysine 93 or lysine 94. In addition, a protein product modified at lysine 103 was also obtained when rIL-1 beta was similarly modified with sulfo-NHS-biotin. Since the only difference between the two biotinylation reagents relates to spacer length and its associated hydrophobicity, these data suggest that lysine 103 is not as accessible to surface modification reagents as are lysine 93, lysine 94, or alanine 1. Initial experiments indicate that none of the modifications described above decrease thymocyte proliferation by more than one order of magnitude. Therefore, these amino acid residues are not crucial for bioactivity, and we anticipate the use of these monobiotinylated proteins in structure/function analysis of IL-1 beta.

Amino Acid Sequence↗
PubMed1989

Soybean trypsin inhibitor. An IL-1-like protein?

Soybean trypsin inhibitor (SBTI) shares some structural homology with interleukin-1 (IL-1) and was tested for IL-1 bioactivity. Human T-cells proliferated maximally when stimulated with PMA and SBTI but failed to respond to either stimulus alone. This response was abrogated by neutralizing antibodies to IL-1 beta but not to IL-1 alpha. However, immunoblots showed no cross-reactivity between SBTI and anti-IL-1 antibodies. Furthermore, SBTI did not bind to IL-1 receptors on YT cells and did not activate a murine T-lymphoma or human T-hybridoma. Supernantants from monocytes stimulated with SBTI contained significant levels of IL-1 activity. The data show that SBTI has no direct IL-1 activity but can stimulate T-cells indirectly through an IL-1 dependent mechanism.

Humans↗
PubMed1989

Isolation and bioactivities of three IL-1 beta N-terminal variants.

Three distinct N-terminal variants of rhIL-1 beta can be generated by expression of the IL-1 beta gene in E. coli; the naturally occurring Ala1 species, Met0-Ala1 and des-Ala1 proteins. Since most studies with rhIL-1 beta have used a mixture of two or more variants, we have evaluated their individual bioactivities. The variants were resolved by cation exchange HPLC. Bioactivity measurement on murine thymocytes gave a potency order of Ala1 greater than des-Ala1 greater than Met0-IL-1 beta. Analysis using human T-cells co-stimulated with PMA showed a potency order of Ala1 greater than des-Ala1 greater than Met0-IL-1 beta. Thus changes in the N-terminal amino acid of IL-1 beta changes the activity of the protein. Since murine and human T-cells respond similarly, the interactions between the N-terminus of rhIL-1 beta and their receptors probably occur through comparable mechanisms.

Animals↗
PubMed1989

Local pathological responses to slow-release recombinant interleukin-1, interleukin-2 and gamma-interferon in the mouse and their relevance to chronic inflammatory disease.

1. The present study describes the pathological responses to local administration of recombinant cytokines in subcutaneously implanted slow-release ethylene vinyl acetate (EVA) co-polymer in mice. 2. EVA-recombinant human interleukin-1 beta (10(4) units) implants induced the formation of chronic granulomatous inflammatory tissue between 4 and 7 days after implantation, characterized by predominant macrophage infiltration, neovascularization and fibrosis which persisted up to 21 days after-implantation. EVA-recombinant human interleukin-1 alpha (10(4)-10(5) units) implants induced a qualitatively similar but less intense response. 3. In contrast, recombinant human interleukin-2 (10(2)-10(4) units) implants resulted in early lymphocytic vasculitis (4 days) and the development of a predominantly lymphoid lesion comprised of lymphoblasts and significant mononuclear cell proliferation by 7 days. 4. EVA-recombinant gamma-interferon (10(3)-10(4) units) implants failed to elicit a significant tissue response; with the exception of multinucleate giant cell formation the characteristics of these lesions closely resembled the mild fibrotic responses observed for EVA-bovine serum albumin (0.5-12.5 mg) implants. 5. These observations suggest that continuous endogenous local release of interleukin-1 or interleukin-2 in vivo is sufficient for the development of specific pathological features characterizing chronic immuno-inflammatory diseases.

Animals↗
PubMed1988

Resolution and biological properties of three N-terminal analogues of recombinant human interleukin-1 beta.

Recombinant human interleukin-1 beta (rhuIL-1 beta) was purified to apparent homogeneity using standard procedures. The protein was characterized by N-terminal sequence analysis and found to consist of three forms: Met (20%), Ala (75%), and des-Ala (5%). Utilizing TSK SP 5PW HPLC, each form was resolved and analyzed by 2-D acrylamide gel electrophoresis. The analogues were identical in molecular mass (17,500 d) but differed in pI (Met, pI = 6.7; Ala, pI = 6.8; des-Ala, pI = 6.8). Bioactivity measurements, using the C3H/HeJ thymocyte proliferation assay, showed that native Ala rhuIL-1 beta was 300-400% more active than des-Ala rhuIL-1 beta, and was 400-600% more active than Met rhuIL-1 beta protein.

Animals↗
PubMed1987

Cytokines involved in the augmentation of murine natural killer cell activity by pyrimidinones in vivo.

Therapeutically active pyrimidinones such as 2-amino-5-bromo-6-phenyl-4-pyrimidinone (ABPP, Bropirimine) are known to be potent immuno-modulators. This includes their ability to markedly augment murine natural killer (NK) cell activity as measured in ex vivo NK assays. We now report the use of a murine in vivo NK assay, based on the rate of NK-cell mediated clearance of radiolabeled tumor cells from the lungs, to directly demonstrate in vivo NK augmentation by several pyrimidinones, including ABPP. In order to evaluate the involvement of the cytokines interferon (IFN), interleukin-1 (IL-1), and interleukin-2 (IL-2) in ABPP-induced NK augmentation, we first showed that injection of purified IFN or IL-1, but not IL-2, resulted in significant enhancement of in vivo NK activity. However, the mixture of IFN, IL-1, and IL-2 synergistically enhanced NK activity more than with any of the cytokines alone. We then showed that ABPP induced significant serum levels of IFN and IL-1, but not IL-2. The induction of IL-1 by ABPP in vivo was further verified by demonstration of increased serum levels of the acute phase protein serum amyloid P in ABPP-treated mice. The possible induction of IL-2 by ABPP was further investigated by using cyclosporin A (CsA) to inhibit IL-2 production in vivo. No diminution of ABPP-induced NK augmentation was seen, however, in CsA treated mice. These results suggest a role for IFN and IL-1 in the augmentation of NK activity in vivo by ABPP, but no evidence for a role for IL-2 was found.

Animals↗
PubMed1985

Production of prostaglandin D2 by murine macrophage cell lines.

Several tumor-derived murine macrophage cell lines were evaluated in vitro as cloned prototypes of tissue macrophages for their ability to metabolize arachidonic acid. Unexpectedly, two cell lines, J774A.1 and WR19M.1, rapidly converted exogenous 14C-arachidonic acid (AA) to a single major prostaglandin metabolite. The compound, PGD2, was positively identified by TLC, HPLC, and GC-MS. The enzymatic formation of the PGD2 was shown by inhibition of its formation by indomethacin and reduced formation of 14C-PGD2 from 14C-PGH2 in boiled cells. When J774A.1 cells were prelabeled with 3H-AA, cultured for 24 hours, and stimulated with lipopolysaccharide (LPS), PGD2 was again the predominant product. No other tumor derived cell lines, including several other murine macrophage lines, produced significant amounts of PGD2. Elicited and activated murine peritoneal macrophages produced only small amounts of PGD2, but resident peritoneal macrophages produced modest amounts of PGD2. Exaggerated formation of PGD2 by J774A.1 and WR19M.1 cells may be a consequence of neoplastic transformation or the clonal expansion of a minor subpopulation of normal tissue macrophages.

Animals↗
PubMed1985

Mechanism of antitumor action of pyrimidinones in the treatment of B16 melanoma and P388 leukemia.

This study was undertaken in an attempt to understand the mechanism of antitumor action of pyrimidinones alone and in combination with cyclophosphamide (CY). Pyrimidinones such as 2-amino-5-bromo-6-(3-fluorophenyl)-4(3H)pyrimidinone (ABMFPP) were relatively nontoxic toward murine L1210 leukemia cell growth in vitro with the concentration of drug required for a 50% inhibition of cell growth being greater than 50 micrograms/ml. In contrast, ABMFPP showed anti-B16 melanoma activity in vivo which was sensitive to X-irradiation of the hosts. These results collectively suggest that pyrimidinones may act differently from conventional cytotoxic antitumor agents. Multiple i.p. injections of ABMFPP (125 mg/kg/injection) significantly augmented the cytotoxicity of both natural killer cells and macrophages in peritoneal exudates. The augmentation of both effector cell populations was delayed, but was more pronounced when animals received a dose of CY (100 mg/kg) prior to ABMFPP injections. The combination of CY and ABMFPP also showed a synergistic anti-P388 leukemia effect which appeared to be related to the initial reduction of the tumor burden by CY and the marked augmentation of the cytotoxicity of both natural killer cells and macrophages by ABMFPP. The antitumor activity of ABMFPP against B16 melanoma was almost completely eliminated when animals received a dose of 400 rads X-irradiation 5 days prior to tumor inoculation or a dose of 200 rads X-irradiation followed by several injections of anti-asialo monosialoganglioside antibody. The administration of anti-asialo monosialoganglioside alone also markedly reduced the anti-B16 melanoma activity of ABMFPP. The magnitude of reduction of the antitumor effect of ABMFPP by radiation and/or anti-asialo monosialoganglioside antibody directly correlated with the inhibition of the ABMFPP-mediated augmentation of immune responses. These results strongly suggest that the antitumor effect of ABMFPP alone or in combination with CY is at least in part mediated through its augmentation of natural killer cell and/or macrophage activities.

Animals↗