Biomedical subjects

T K Howcroft

Publications and source records attributed to T K Howcroft.

22 records · Page 2Linked to original sources

PubMed1991

Cytotoxic T lymphocyte (CTL)-mediated cytolysis proceeds in the absence of Na+/H+ antiport activity: regulation of cytosolic pH by the Na+/H+ antiport in a cloned CTL.

Cytotoxic T lymphocyte (CTL)-mediated cytolysis of specifically bound target cells (TC) is thought to be triggered by cross-linking the T-cell antigen receptor (TcR). Biochemical events associated with TcR cross-linking include increased intracellular calcium levels [Ca2+]i, hydrolysis of phosphatidylinositol (PI), and an increase in intracellular pH [pH]i. Whereas CTL-mediated cytolysis of some TC is calcium-dependent, and PI hydrolysis is speculated to trigger the CTL lethal hit via activation of PKC, little is known about changes in [pH]i relating to activation of the lethal hit stage. We report regulation of [pH]i in a cloned CTL by the electroneutral Na+/H+ antiport during activation with PMA and specific antigen-bearing TC. Furthermore, using 5-(N-methyl-N-isobutyl) amiloride (MIBA), a potent antiport inhibitor, we demonstrate that Na+/H+ exchange is not required for activation of CTL cytolytic activity.

Acid-Base Equilibrium↗

PubMed1988

Protein kinase C (PKC) inhibitors inhibit primary (1-0) but not secondary (1-1) or cloned cytotoxic T lymphocytes.

The isoquinolinesulfonamide PKC inhibitors H-7 and H-8 inhibit primary, in vivo generated cytotoxic T lymphocyte (CTL) activity by 50% at concentrations approximating their reported Ki values for PKC, 6 uM and 15 uM respectively. However, a greater than ten-fold higher concentration of H-7 (100 uM) is required to reduce secondary or clone 8F CTL-mediated cytotoxicity by 50%. At this concentration H-7 is also reported to inhibit calmodulin (CaM)-dependent enzymes. To distinguish between the effect of 100 uM H-7 on PKC versus CaM the napthalenesulfonamide CaM antagonist W-7 was investigated. W-7 inhibited primary, secondary and clone 8F CTL-mediated cytolysis by 50% near its reported Ki value for CaM-dependent kinase activity, 12 uM. We conclude that W-7 and 100 uM H-7 reduce cytolysis by inhibiting CaM-dependent reactions and not PKC. Thus, these findings indicate that primary killers require both PKC- and CaM-dependent activation pathways for lethal hit delivery, whereas highly lytic cultured CTL use only one pathway dependent upon CaM.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1987

Primary integumentary allograft reactivity in the American cockroach, Periplaneta americana.

Previous reports have failed to demonstrate integumentary allograft rejection in insects. We realized however, that these studies may not have fully appreciated the structure of the insect exoskeleton. Since the subcuticlar epidermal layer constitutes the only living tissue associated with insect integument, its destruction would indicate that the animal recognized and responded to the foreign tissue. Thus, we investigated allograft reactivity in the American cockroach, Periplaneta americana, by observing the fate of the epidermal portion of the integument. Each animal in a pair received a 3 X 4-mm integumentary allograft from its partner, as well as a 3 X 4-mm control autograft. The transplants were then examined histologically for signs of epidermal destruction at 0, 1, 3, 5, 7, and 10-70 days (in 10-day increments) posttransplantation. The results indicated that significant rejection of the allografts began by day 3, with peak reactivity occurring by day 7 when 92% of the grafts were scored as rejected. At later periods (greater than 20 days), the graft sites showed signs of repopulation by host epidermal cells. The allograft reaction was found to lag behind the xenograft reaction, which showed peak activity after only 1 day posttransplantation. Even so, allograft rejection in this insect occurred quite rapidly (as compared with some other invertebrates), and would appear to be due to a cytotoxic reaction against the epidermal layer.

Animals↗

PubMed1987

Demonstration of cell-mediated cytotoxicity to allogeneic and xenogeneic tissue in the American cockroach, Periplaneta americana, using a combination in vivo/in vitro assay.

Previous reports have claimed that insects lack the ability to reject integumentary allografts. However, when we followed the fate of the epidermal layer underneath the inert cuticle, we observed that the American cockroach not only rejected xenografts, but also demonstrated impressive reactivity against allografts. We have now developed a combination in vivo/in vitro assay that can quantitate the extent of allograft reactivity in the roach. Pieces of filter paper (3 X 4-mm) were implanted under the second and fourth tergites of donor animals. After 7 days, implants seeded with host hemocytes were removed, washed, and incubated in culture medium containing 3H-thymidine for 24 hr. Labeled grafts were reimplanted into paired animals to detect cytotoxicity as follows: grafts removed from under the second tergite were placed back into their original positions to serve as autograft controls; grafts removed from under the fourth tergite were reciprocally transferred between paired animals; and grafts were recovered after various time intervals and processed for scintillation counting. There was no significant difference in counts between allografts and autografts at day 1. Autografts sampled on days 3, 5, 7, and 10 had significantly higher counts than allografts, with peak reactivity occurring between days 3 and 5. This indicates that allogeneic cells were selectively destroyed, and confirms our recent data from conventional grafting studies that insects have the ability to react to allografts.

Animals↗