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Heterophils are important mediators of innate resistance in poultry, especially in young birds that have not yet developed an acquired immune response. Invasion of the intestinal mucosa by Salmonella spp. initiates the recruitment of large numbers of heterophils to the lamina propria. Thus, the heterophilic response can control, but not eliminate, bacterial numbers in the bird until development of acquired immunity. Unfortunately, chicks and turkey poults are highly susceptible to Salmonella infections during the first 4 d posthatch due to the functional immaturity of both the innate and acquired immune systems. We have previously shown that the administration of Salmonella enteritidis (SE)-immune lymphokines (ILK) into either 18-d-old developing embryos or day-of-hatch chicks and poults conferred increased resistance to SE organ invasion. In this review, we present evidence that the protection induced by ILK is mediated by vigorous recruitment and activation of heterophils. These activated heterophils migrate rapidly to the site of bacterial invasion where they phagocytize and kill the SE. Specifically, in vitro studies demonstrate an enhancement of functional activities of the heterophils including chemotaxis, adherence, phagocytosis, and bacterial killing. In addition, during the activation process, membrane expression of adhesion molecules rapidly changes from L-selectins to beta2 integrins (CB11b/CD18) on the cells that become activated. These results further demonstrate the validity of preventive activation in poultry to induce the migration of large numbers of activated phagocytic cells to the site of infection by a pathogenic organism. Importantly, this immunopotentiation of the inflammatory response by ILK, as described here, induces the functional maturation of heterophils during the first 4 d posthatch.

Chicken heterophils activated in vivo following the intraperitoneal (i.p.) administration of Salmonella enteritidis-immune T lymphokines (SE-ILK) have been implicated in the protection against SE organ invasion. SE-ILK induces a heterophilia and directly (or indirectly) activates the granulocytes. The invasion of SE provides the secondary signal for directing activated heterophils to the site of bacterial invasion. We examined the mechanism of adherence within the avian heterophil system using an in vitro bovine serum albumin (BSA) matrix in which neutrophil adherence is primarily CD11/CD18 integrin mediated in mammalian systems. Activated heterophils displayed a four-fold increase in receptor-mediated adherence in vitro to BSA-coated slides as compared to control heterophils from PBS-injected birds. The increased adherence of activated heterophils can be partially blocked by either anti-alpha M (CD11b) or anti-beta 2 (CD18) antibodies in a dose dependent manner. Anti-alpha 3 (CD49c) antibody partially blocked adherence of both normal and activated cells. Fluorescence-activated cell scanning (FACS) analysis of the heterophils shows that both control and SE-ILK-activated heterophils collected at 4 h post injection with SE-ILK or PBS display similar amounts of integrin alpha 3 on their surface. This integrin is constitutively expressed and is responsible for the in vitro adherence of both groups. However, antibodies to the Mac-1 complex (CD11b/CD18) block only the adherence of SE-ILK-stimulated heterophils. Thus, the CD11b/CD18 heterodimer is apparently up regulated in response to the injected SE-ILK and plays a major role in the adherence of activated heterophils. Our studies in chickens parallel human and mouse studies showing the importance of the beta 2 integrins in adherence of activated cells.

We have previously reported that the prophylactic administration of factor(s) from T-cell supernatants derived from Salmonella enteritidis-immune chickens (ILK) have a favorable effect in controlling or eliminating salmonellosis in neonatal poultry. Experimentally, we have used the intraperitoneal injection as the standard method of administering ILK to neonatal poultry. However, this method is neither easy, practical, nor economical for the poultry industry. In the present study, we evaluated the effectiveness of oral (p.o.), intranasal (i.n.), and subcutaneous (s.c.) administration of ILK for ease of delivery, induction of protective resistance against Salmonella enteritidis (Se) organ invasion, and the ability to activate peripheral blood heterophils in day-old chickens. In the first experiments, delivery of ILK p.o., i.n., and s.c. significantly (P < 0.01) increased the resistance of day-old chickens to Se organ invasion. The level of protection was equivalent to that induced by the i.p. route. Administration of a comparable protein control (bovine serum albumin, BSA) by the 3 routes induced no protective effect against Se organ invasion. Likewise, a significant increase was found in the number of circulating heterophils within 4 h of administration of the ILK by all routes. In the 2nd experiment, the function of the heterophils from ILK-treated birds was compared with that of the control cells in adherence, chemotaxis, and phagocytosis assays. The heterophils from birds given ILK i.p., s.c., p.o., or i.n. had significantly (P < 0.01) increased functional activities when compared to the activities of the heterophils from the control birds. These studies indicate that the delivery of ILK either orally or parenterally, routes which can be used by the poultry industry, can confer protection to chickens against a localized enteric Se organ invasion by potentiating the systemic heterophilic innate response.

Peripheral blood mononuclear cells (PBMC) from five chronic schistosomiasis patients, three former patients, a SEA sensitized individual, and normal controls were tested in lymphoblastogenesis assays for their ability to proliferate in response to soluble egg antigen (SEA) and soluble worm antigen preparation (SWAP) from Schistosoma mansoni. Cells from all patients and the SEA sensitized individual gave significantly higher responses than the normal controls when stimulated with SEA and SWAP. However, the chronic patients' SEA responses were much lower than those of the former patients and the SEA sensitized individual. When cells from the same donors were tested in the in vitro granuloma assay, all produced significant granulomatous responses except the normal controls. Once it was established that all individuals in the study gave significant lymphoproliferative responses and granulomatous reactions, SEA was subjected to HPLC fractionation to identify immunogenic protein components of SEA. HPLC separation yielded 25 major fractions. SEA responses from the sensitized individual and former patients exhibited broad, unregulated responsiveness including fractions with neutral, less charged proteins while the chronic patients demonstrated a more restricted range of responsiveness. SEA-HPLC fractions 14, 21, and 22 contain the most immunodominant proteins based on cellular proliferation data from reactive individuals tested.

In order to circumvent the toxicity associated with high-dose interleukin 2 (IL2) administration and its rapid clearance from the circulation, a carrier system for IL2 is needed. Red blood cells (RBCs) coated with recombinant interleukin 2 (rIL2) provide a means of delivering IL2 into the system in a continuous low-dose manner which, in turn, maintains a low, potentially non-toxic, IL2 concentration. Murine RBCs coated with rIL2 (RBCs-rIL2) are able to induce cytotoxic activity in mouse spleen cells in vitro against malignant murine YAC-1 cells (53-62% cytotoxicity) using less than 4500 i.u./10(9) RBCs per mouse. Cytotoxicity (21-31%) becomes apparent upon cytotoxic testing of spleens cells stimulated in vivo. Using the murine Meth A sarcoma model, the effectiveness of this RBC-rIL2 vehicle is demonstrated in vivo by a 84% reduction in tumour size as compared with the soluble-rIL2-treated mice. Moreover, the RBC-rIL2 vehicle is able to induce tumoricidal cytotoxicity with very low rIL2 concentrations (about 10,000 i.u. of rIL2/mouse). These results indicate that rIL2 retains its biological activity when bound to the RBC and therefore could prove useful as a therapeutic delivery system for cancer treatment.

Mouse red blood cells (RBC) were incubated with recombinant human interleukin 2 (rIL 2) in protein-free media. Of the added cytokine, about 20% was bound to the cells. Dissociation of rIL 2 from RBC commenced when the carrier cells were transferred into fresh media. The release from the RBC vehicle was enhanced in the presence of soluble protein. When rIL 2-coated RBC (RBC-IL2) were incubated in protein-containing media for 24 h to release the majority of bound rIL 2, and then were subsequently incubated in fresh media with an IL 2-dependent cytotoxic T cell line (CTLL), residual bound rIL 2 was still released as demonstrated by CTLL proliferation. RBC were covalently coupled to specific monoclonal antibody (mAb) towards the lymphocyte cell-surface marker Thyl.2, coated with rIL 2, subsequently exposed to the target CTLL cells, then evaluated for CTLL/RBC rosette formation. The attachment of mAb to the RBCs surface did not markedly change the succeeding rIL 2 adsorption, and the bound rIL 2 did not impair antigen recognition by the mAb. At higher rIL 2 concentrations (1200-1500 i.u./10(8) cells), RBC-IL2 plus Thy1.2 mAb provided higher CTLL response than when RBC-IL2 plus non-specific mAb was used thus demonstrating enhancement by targeting. However, the targeting effects were not seen with lower rIL 2 concentrations (80-110 i.u./10(8) cells).

We have previously reported on Id/anti-Id-receptor interactions in clinical human schistosomiasis. These findings support a hypothesis that anti-SEA cross-reactive Id develop in some patients during the course of a chronic infection and participate in regulation of anti-SEA cellular immune responses. We report here on experiments that extend those observations to the regulation of granulomatous hypersensitivity measured by an in vitro granuloma model. T cells from chronic intestinal schistosomiasis patients were stimulated in vitro with anti-SEA Id and assayed in an autologous in vitro granuloma assay for modulation of granuloma formation. These anti-SEA Id-reactive T cells were capable of regulating autologous in vitro granuloma formation. Both CD4 and CD8 T cells could be activated to regulate granuloma formation. This regulatory activity, initiated with stimulatory anti-SEA idiotypic antibodies, was antigenically specific and was dependent on the presence of intact F(ab')2 Ig molecules. The ability to elicit this regulatory activity appears to be dose dependent and is more easily demonstrated in chronically infected intestinal patients or SEA-sensitized individuals. These data support the hypothesis that anti-SEA cross-reactive Id are important in regulating granulomatous hypersensitivity in chronic intestinal schistosomiasis patients and these cross-reactive Id appear to play a major role in cell-cell interactions that result in the regulation of anti-SEA cellular immune responses.

Improved methods have been developed for accurate determination of tricyclic antidepressants and their metabolites in body fluids by means of high-performance liquid chromatography. These facilitate pharmacokinetic studies, including individual variations in drug handling, comparisons of ordinary and sustained-release preparations, assessment of side effects, and the contribution of metabolites to clinical efficacy. Comparing concentrations in erythrocytes and plasma suggests that samples of whole blood might be used for determining concentrations of some tricyclic antidepressants.

To see if the clinical efficacy of amitriptyline and clomipramine is related to plasma level of the active drug or its main metabolite, or to the balance between them, plasma levels and clinical progress have been studied in 48 patients taking standard dosage. Dose for dose, plasma levels of clomipramine and desmethylclomipramine are usually higher than those of amitriptyline and its main metabolite nortriptyline, but no significant relationship between plasma levels and clinical improvement was found following clomipramine administration. By contrast, optimum responses to amitriptyline therapy were obtained at plasma levels of 50--100 micrograms/l for amitryiptyline itself and 25--75 micrograms/l for nortriptyline, but when their ratio exceeded 0--7 improvement declined. Steady states were reached after two to three weeks but a crude measure could be obtained from the plasma level after four days' treatment. Clomipramine and amitriptyline appeared to be equally effective, with clomipramine taking slightly longer to relieve depression.