Zhi-Qing Hu

Mitogenic activity of (−)epigallocatechin gallate on B-cells and investigation of its structure-function relationship

(-)Epigallocatechin gallate (EGCg), the main constituent of green tea, strongly enhanced the direct plaque-forming cell (PFC) response to sheep red blood cells (SRBC) in vitro and showed strong mitogenic activity for mouse splenic B-cells but not for splenic T-cells and thymocytes. The enhancement of B-cell proliferation was not mediated by macrophages since their removal did not eliminate the activity. Among the derivatives of catechin examined, (+)catechin (C); (-)epicatechin (EC); (-)-epigallocatechin (EGC); (-)epicatechin gallate (ECg); (-)epigallocatechin gallate (EGCg); and theaflavin digallate (TF3), only the derivatives with the galloyl group (ECg, EGCg, and TF3) displayed significant enhancement of the spontaneous proliferation of B-cells. Structural analogs of the catechin and galloyl groups were also examined in the system. Gallic acid and tannic acid induced some enhancement, but rutin, pyrogallol and caffeine did not. The results indicate that the galloyl group on EGCg was responsible for enhancement. However, the basic conformations of the catechins are also important, because ECg, EGCg, TF3, gallic acid, and tannic acid had quite different potencies to induce B-cell proliferation.

Restoration of antibacterial activity of β-lactams by epigallocatechin gallate against β-lactamase-producing species depending on location of β-lactamase

The combined effects of (–)‐epigallocatechin gallate (EGCg) and β‐lactams were investigated against various β‐lactamase‐producing clinical isolates, including 21 Staphylococcus aureus, 6 Escherichia coli, 3 Klebsiella pneumoniae and 8 Serratia marcescens strains. Penicillin in combination with EGCg at 12.5μg mL−1 showed the most potent synergy against 100% penicillinase‐producing S. aureus. However, cefotaxime or imipenem in combination with higher concentration of EGCg (100 μg mL−1) only showed slight synergy against 2 of 17 Gram‐negative rods. Similar to the effect on the penicillinase from S. aureus, however, EGCg also directly inhibited the extracted β‐lactamases from the Gram‐negative rods, thereby protecting β‐lactams from inactivation. The different effects of the combinations on different β‐lactamase‐producing species were confirmed to be related to the cellular locations of β‐lactamases. In contrast to a 32.7% extracellular fraction of total β‐lactamase activity in a penicillinase‐producing S. aureus, the fractions were 0.6%, 0.6% and 1.2% in a TEM‐derived extended‐spectrum β‐lactamase‐producing E. coli, an inhibitor‐resistant β‐lactamase‐producing K. pneumoniae and an IMP‐producing S. marcescens, respectively. In conclusion, the combination of penicillin with EGCg showed potent synergy against penicillinase‐producing S. aureus in‐vitro. The combinations of β‐lactams and EGCg against β‐lactamase‐producing Gram‐negative rods do indicate a limitation owing to the cellular location of β‐lactamases.

Pharmacodynamic comparison of pantoprazole enantiomers: inhibition of acid-related lesions and acid secretion in rats and guinea-pigs

Pantoprazole is an irreversible proton pump inhibitor that is administered as a racemic mixture clinically. The effects of pantoprazole sodium (PAN·Na) enantiomers on acid‐related lesions were compared using models of pylorus ligation induced ulcer, histamine induced ulcer and reflux oesophagitis in rats and guinea‐pigs. Compared with (+)‐PAN·Na and (±)‐PAN·Na, (‐)‐PAN·Na showed much stronger inhibitory effects on pylorus ligation induced and histamine induced ulcers, but similar effects on reflux oesophagitis. The doses of (‐)‐PAN·Na, (+)‐PAN·Na and (±)‐PAN·Na required for 50% inhibition (ID50) of acid‐related lesions were 1.28, 5.03 and 3.40 mg kg−1 against pylorus ligation induced ulcer, 1.20, 4.28 and 3.15 mg kg−1 against histamine induced ulcer, and 2.92, 3.56 and 3.70 mg kg−1 against reflux oesophagitis, respectively. The inhibitory effects of PAN·Na enantiomers on basal gastric acid output were compared in rats with acute fistula. In contrast to inhibitory rates of 89.3% and 83.6% on gastric acid output by (‐)‐PAN·Na and (±)‐PAN·Na at 1.5 mg kg−1, (+)‐PAN·Na had an inhibitory rate of only 24.7% at the same dose. The above results indicate that (‐)‐PAN·Na is more potent than (+)‐PAN·Na at inhibiting acid‐related lesions owing to its stronger inhibition of acid secretion.

Enhancement of lymphocyte proliferation by mouse glandular kallikrein

Mouse glandular kallikrein (mGK) strongly enhanced the spontaneous and mitogen-induced proliferation of lymphocytes. Both blast formation and 3H-TdR incorporation were dose-dependently enhanced at the same time many cells were killed. The enhancing activity was independent of EGF, because EGF-binding proteins (mGK-9 in mGK-6,9 mixture and mGK-13), renal kallikrein (mGK-6) and human kallikrein all displayed the same enhancement. A serine proteinase inhibitor, diisopropyl fluorophosphate, could block the enhancement by mGK. The new function suggests that mGK is important in the immune system as a regulatory molecule.

Erythrocyte-dependent mitogenic activity of epigallocatechin gallate on mouse splenic B cells.

We previously reported that epigallocatechin gallate (EGCg), the main constituent of tea catechins, displays mitogenic effect on mouse splenic B cells. During research into the mechanism(s), it was found that the mitogenic activity of EGCg was dependent on the presence of red blood cells (RBC). When RBC in T cell-depleted spleen cells were removed, EGCg did not enhance the proliferation of B cells and even showed toxic effect at 25-50 micrograms/ml. When mouse, rabbit or sheep RBC as well as RBC-ghosts were added into the cultures, EGCg showed the mitogenic activity at a range of 1-50 micrograms/ml. Thereafter, we preincubated RBC with EGCg at 4 degrees C for various times and then washed the RBC to remove free EGCg in the suspensions. The EGCg-preincubated RBC also enhanced B cell proliferation. As short as ten minutes was sufficient for EGCg to bind to RBC membrane. These results indicate that EGCg first attached to the membrane of RBC and then stimulated B cell proliferation. The above results suggest an important immunoregulatory function of RBC.

Induction and identification of mast cells from long-term culture of mouse spleen cells without conditioned medium

A simple method is described for the preparation of large numbers of mast cells from mouse spleen cells in vitro. Mouse spleen cells were cultured with RPMI 1640 medium supplemented with 10% FCS and 2-ME. Half of the total volume of the medium was changed every 4-5 days. Mast cell numbers increased with the culture time and reached a peak between 16 and 20 days. Using this method, 2 x 10(6) mast cells could be induced from 1 x 10(7) nucleated normal spleen cells. T cells and supernatant derived from ConA-stimulated T cells were unnecessary for mast cell induction. Phenotype analysis by FACS showed that Thy1,2, L3T4, Ly-2, Ig, B220, Asialo GM1, and WGA receptors were all negative but functional IgE receptors were positive. The granules in the cells could be stained by alcian blue but not by safranin. There was 1.632 +/- 0.024 micrograms stored histamine in 1 x 10(6) of the cells. Histamine was released from the cells in an antigen-induced and IgE-mediated process. Compound 48/80 and A23187 induced degranulation of the cells, and the mast cells were able to respond to ConA.