Sunlmar~We have used site-directed mutagenesis to change amino acid residues in the heavy chain of the pathogenic R4A anti-double-stranded DNA (dsDNA) antibody and have looked for resultant alterations in DNA binding and in pathogenicity. The data demonstrate that single amino acid substitutions in both complementarity determining and framework regions alter antigen binding. Changes in only a few amino acids entirely ablate DNA specificity or cause a 10-fold increase in relative binding. In vivo studies in mice of the pathogenicity of the mutated antibodies show that a single amino acid substitution leading to a loss of dsDNA binding leads also to a loss of glomerular sequestration. Amino acid substitutions that increase relative affinity for dsDNA cause a change in localization of immunoglobulin deposition from glomeruli to renal tubules. These studies demonstrate that small numbers of amino acid substitutions can dramatically alter antigen binding and pathogenicity, and that the pathogenicity of anti-DNA antibodies does not strictly correlate with affinity for DNA.

Anti-double-stranded (ds) DNA antibodies are not only an important diagnostic marker for SLE, but also play an important role in tissue injury. Microbial antigen may be a stimulus for the production of these antibodies. We isolated 99D.7E, an IgG2b monoclonal antibody from a nonautoimmune BALB/c mouse that is cross-reactive with both ds-DNA and phosphorylcholine, the dominant hapten on the pneumococcal cell wall. While partially protective against a bacterial challenge, 99D.7E is also pathogenic to the kidney. To identify those molecular motifs that confer on anti-PC antibodies the potential for autoreactivity, we created a panel of 99D.7E mutants with single amino acid substitutions in the heavy chain, and examined the changes in antigen binding and renal deposition. Our results support the hypothesis that charge and affinity for dsDNA are not adequate predictors of the pathogenicity of anti-DNA antibodies. Differential renal damage from anti-dsDNA antibodies may be due to differences in fine specificity, rather than differential affinity for dsDNA. Importantly, high affinity IgG antibodies cross-reactive with bacterial and self antigen exist and can display pathogenic potential, suggesting that defects in peripheral regulation of B cells, activated by foreign antigen but cross-reactive with self antigen, might lead to autoimmune disorders. ( J. Clin. Invest. 1996. 97:2251-2259.)

Cassia oil (CO) from different parts of Cinnamomum cassia have different active components. Very few pharmacological properties of cassia leaf oil have been reported. This study investigated and compared effects of cassia leaf oil and cinnamaldehyde on lipopolysaccharide (LPS)-activated J774A.1 cells. Volatile compositions in cassia leaf oil were identified by gas chromatography-mass spectrometry (MS)/MS. Effects of CO and cinnamaldehyde on LPS-activated J774A.1 cells were investigated by determining nitric oxide (NO) production using Griess reaction assay; expression of pro-inflammatory cytokines, enzymes involve in inflammatory mediators; antiinflammatory cytokines, and iron exporter ferroportin1 (Fpn1) using reverse transcription-polymerase chain reaction; and production of tumor necrosis factor (TNF-α) and interleukin (IL)-10 using ELISA. The main component of CO was cinnamaldehyde. Both oils at 1-20 μg/ml markedly inhibited NO production in LPS-activated J774A.1 cells with IC50 value of 6.1 ± 0.25 and 9.97 ± 0.35 μg/ml, respectively. They similarly inhibited mRNA expression of pro-inflammatory cytokines and chemokines. These mediators included TNF-α, IL-1β, IL-6, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1α in LPS-activated cells. They also significantly decreased expression of inducible enzymes inducible nitric oxide synthase, cyclooxygenase-2, microsomal prostaglandin-E synthase-1. In the opposite way, they increased mRNA expression and the production of antiinflammatory cytokines IL-10 and transforming growth factor-β. In addition, they promoted the expression of Fpn1. These results demonstrated that inhibitory effects of cassia leaf oil from C. cassia mainly came from cinnamaldehyde. This compound not only inhibited inflammatory mediators but also activated antiinflammatory mediators in LPS-activated J774A.1 cells. It may also have an effect on iron regulatory proteins in activated macrophages.

Context: Doxorubicin is a chemotherapy agent used in non-Hodgkin's lymphoma but side effects limit its use. Citral is a mixture of neral and geranial found in essential oils of lemon grass. Objectives: We evaluated the activity of citral, doxorubicin, and combination on cytotoxicity, apoptosis, and anti-proliferative effects using human lymphoma Ramos cells. Materials and methods: Cells were treated with doxorubicin alone or in combination with citral (10, 20, and 40 mM). Cytotoxic and apoptosis studies were done after 24 and 18 h incubations, respectively. Cytotoxic effects of citral on normal human peripheral blood mononuclear cells (PBMCs) were also investigated for its safety. Changes in the expression of BCL-2 family genes were analyzed by quantitative RT-PCR. Results: Citral had cytotoxicity on cells with an IC 50 value of 77.19 ± 4.95 mM. Citral at concentrations of 10, 20, and 40 mM additively increased the cytotoxic and apoptotic effects of doxorubicin, leading to decreased IC 50 (mM) of the drug from 2.50 ± 0.01 to 2.16 ± 0.03, 1.90 ± 0.04, and 1.23 ± 0.04, respectively. Enhanced cytotoxicity was not observed in normal human PBMCs. Citral (40 mM) in combination with doxorubicin (1.5 mM) increased the expression of pro-apoptotic protein BAK but significantly decreased the expression of antiapoptotic protein BCL-XL to 5.26-fold compared with doxorubicin-treated cells. It did not change the anti-proliferative activity of drug. Discussion and conclusion: Citral potentiated cytotoxicity of doxorubicin by increasing apoptotic effects. We conclude that citral may have beneficial effects in patients with B cell lymphoma treated with chemotherapy.

Context: Glycosmis parva Craib (Rutaceae) is reported to have cytotoxic and anti-inflammatory activities by decreasing COX-2 expression. Objective: To investigate the effect of G. parva on human colorectal cancer cells expressing COX-2, HT-29 cells. Materials and methods: HT-29 cells were treated with ethyl acetate extract from the leaves of G. parva (GPE 6.25-100 mg/ml) for 24-72 h. Cell viability was evaluated by the resuzurin reduction assay. An apoptotic study was performed using annexinV/FITC-PI staining. The cellcycle pattern was investigated by PI staining. The expression of BCL-2 family genes was analyzed by quantitative RT-PCR and expression of cyclins and COX-2 were done by RT-PCR.Results: GPE at 6.25-100 mg/ml reduced HT-29 cell viability with IC 50 values of 69.49, 55.89, and 48.94 mg/ml at 24, 48, and 72 h, respectively. HT-29 apoptosis was induced by 18.23% at 100 mg/ml. Cells in S phase decreased by 5.22% and 13.28% at 50 and 100 mg/ml, respectively, causing G0/G1 (10.6% at 50 mg/ml) and G2/M (15.67% at 100 mg/ml) accumulation. GPE at 50 mg/ml downregulated cyclin A (11.46%), cyclin E (17.98%), BCL-2 (0.32-fold), and COX-2 (29.06%) expression with an increased BAK expression (1.79-fold). Discussion and conclusion: GPE reduced HT-29 cell viability, inhibited cell proliferation, induced apoptosis, and arrested the cell cycle. Underlying mechanisms may involve decreases in COX-2, cyclin A, and cyclin E expression in addition to changes in BCL-2 family gene expression. Fundamental knowledge of GPE anticancer effects found in this study could lead to future use of this compound for colorectal cancer treatment.