Robert E. Ratzlaff

An arthropod defensin expressed by the hemocytes of the American dog tick, Dermacentor variabilis (Acari: Ixodidae)

Both soluble and cell-mediated components are involved in the innate immune response of arthropods. Injection of Borrelia burgdorferi, the Lyme disease agent, results in the secretion of defensin into the hemolymph of the ixodid tick, Dermacentor variabilis. The presence of the peptide is observed as early as 15 min post-challenge and remains present through 18 h post-challenge. As observed in insects and soft ticks, the transcript for defensin is detected as early as 1 h post-challenge in D. variabilis. RT-PCR resulted in an amplicon of 624 bp with a 225 bp region that translates to a 74 amino acid preprodefensin. The defensin encoding region was amplified, cloned and sequenced from the hemocytes. It appears as though defensin is stored in the granulocytes of the hemolymph and secreted into the hemolymph upon bacterial insult. The role of defensin as a contributing factor in determining vector competency is discussed.

Use of micrometers and calipers to measure various components of delayed-type hypersensitivity ear swelling reactions in mice.

The choice of the type of instrument to measure delayed-type hypersensitivity (DTH) in mice, as assayed by ear swelling reactions, influences the experimental results. When a caliper that applies little pressure to the ears is employed, DTH reactions in ears of mice sensitized to picryl chloride show an early onset at 2 h after challenge, comparable swelling at 4 h and a slow rise to a 24 h classical peak response thereafter. In contrast, 3 different micrometers that apply more pressure to the ears reveal a biphasic pattern of ear swelling reactions in mice immunized and challenged with picryl chloride. The early component of DTH measured by these micrometers peaks 2 h after challenge. Thereafter the measured ear thickness declines, and the onset of the classical delayed reaction is detected at 12 h after ear challenge. Yet another instrument, that in contrast to the caliper and micrometers mentioned above, applies all the pressure to only a very restricted area of the ear, fails to detect an early swelling reaction; the delayed reaction is first detected at 12 h after ear challenge and rises thereafter to a 24 h peak. The differences in outcome of the assays using the different instruments indicate that the early component or DTH reactions differs from the late component of DTH reactions in that the early swelling is easier to compress when pressure is applied by the instrument used for measurement. This is probably caused by the fact that the late reactions are due to a cellular infiltrate, whereas the early reactions are edematous in character, and are due to accumulation of plasma components.

Pathology and hematology of the Caribbean spiny lobster experimentally infected with Panulirus argus virus 1 (PaV1).

We examined the histopathological and hematological response of the Caribbean spiny lobster to experimentally induced infections with Panulirus argus Virus 1 (PaV1). The fixed phagocytes in the hepatopancreas were the primary sites of PaV1 infection in spiny lobsters. Fixed phagocytes were activated in early infections. However, as the disease progressed, the fixed phagocytes became infected and eventually lysed. Infected cells were subsequently observed in the hepatopancreas, gill, heart, hindgut, glial cells around the ventral nerves, and in the cuticular epidermis and foregut. In advanced infections, spongy connective tissues were heavily infected, as were glial cells around the optic nerves. The structure of the hepatopancreas was significantly altered as the disease progressed. The hemal sinuses among the hepatopancreatic tubules filled with massive amounts of cellular aggregates, including infected circulating hemocytes and spongy connective tissues. Atrophy of the hepatopancreatic tubules occurred in the late stage of viral infection. The virus caused significant decreases in total hemocyte counts and significantly altered several constituents in the hemolymph lysates of diseased lobsters, including: glucose, phosphorus, and triglycerides.

A role for platelet release of serotonin in the initiation of contact sensitivity.

Finding contact sensitivity (CS) responses that were fairly normal in ear swelling, and in serotonin (5-HT) dependence in mast-cell-deficient mice, led to experiments to determine whether platelets supplemented mast cells as a source of 5-HT in CS. Severe depletion of platelets, and consequently blood 5-HT, with antiplatelet antibody, strongly inhibited CS, especially in mast-cell-deficient mice, suggesting that platelets supplemented mast cells. Furthermore, human platelets sensitized in vitro with anti-(tri-nitro-phenyl) IgE, and transferred intravenously together with isolated late-acting effector T cells, provided CS initiation due to local 5-HT release. Similar, IgE-dependent in vitro release of 5-HT was C dependent. These findings establish the importance of antigen-specific platelet release of 5-HT in CS initiation.

Evidence of a neurogenic component during IgE-mediated inflammation in mouse skin

IgE-mediated inflammation was measured in mouse footpads that lacked sciatic innervation. Mice were passively sensitized with a monoclonal antibody, IgE anti-dinitrophenol, or were immunized for specific IgE production. Antigen-induced swelling in the denervated footpads was reduced 23-39% when compared to sham or untreated controls. Reduced IgE-mediated swelling responses were attributed to the loss of a mast cell-nerve interaction and not to blood vessel sensitivity to vasoamines. Furthermore, electrical stimulation of the distal segment of the sciatic nerve completely restored IgE-mediated inflammation. These data provide in vivo evidence that peripheral nerves participate in cutaneous IgE-mediated swelling reactions with the net effect of increasing inflammation.

Involvement ofan axonal reflex in IgE-mediated inflammation in mouse skin

A neurogenic component of IgE-mediated inflammation was demonstrated in mice by footpad denervation. Footpad swelling was reduced 26% following sciatic nerve transection, but unaffected by rhizotomy or spinal nerve transection. These data provide in vivo evidence that an axonal reflex is involved in IgE-mediated inflammation and completed distal to the cell bodies of the sensory neurons located in the lumbar spinal ganglia. Furthermore, depletion of neuropeptides with capsaicin also reduced IgE-mediated swelling by 26%, indicating that unmyelinated axons are involved in the neurogenic component of IgE-mediated inflammation.