Preventive Anti-CD2 Treatment does not Impair Parasite Control in a Murine Toxoplasmosis Model.

Targeting human CD2 with the monoclonal antibody (mAb) CB.219 reduces intestinal inflammation in a colitis model where T cells carry human CD2. Here, we asked whether this mAb has adverse effects on infection control. Mice expressing human CD2 on T cells (huCD2tg) were orally infected with Toxoplasma (T.) gondii and treated with the human CD2-specific mAb CB.219 in a preventive setting. The intestinal T. gondii loads in CB.219 treated mice did not differ from the control group. Histologically, huCD2tg mice showed moderate ileal inflammation that did not change with CB.219 treatment. In the ileum, CB.219 treatment reduced the protein levels of interferon-γ, transforming growth factor ß and interleukin-6, whereas interleukin-18 mRNA was slightly increased. The infiltration of neutrophils, macrophages, and T cells into the ileum was unaffected by CB.219 treatment. However, CB.219 treatment decreased the numbers of forkhead box P3(+) regulatory T cells (Treg) in ileum and liver of huCD2tg mice. This was confirmed in vitro using human peripheral blood mononuclear cells. Taken together, targeting CD2(+) T cells by the human CD2 mAb CB.219 does not prevent beneficial immune reactions necessary for pathogen control. Further experiments will address gut specificity, underlying mechanisms, and general applicability of CB.219 treatment.

In vivo proliferation of rat lamina propria T lymphocytes: general hyporesponsiveness but increased importance of the CD2 and CD28 pathways.

Lamina propria T lymphocytes (LPL-T) have a low proliferative potential in vitro. We asked whether LPL-T are also hyporesponsive in vivo and whether this is specific for the alphabeta T cell receptor (TCR). Mitogenic mAb directed at the alphabeta TCR, CD2, CD28, or control mAbs plus IL-2 were injected into rats. Proliferation and/or apoptosis were detected by double staining using 5-bromo-2'-deoxyuridine/TUNEL and the alphabeta TCR. LPL-T were hyporesponsive to various stimuli compared to other T cells. The strongest proliferation was found upon CD2/CD28 stimulation (LPL-T: 281 +/- 6%; spleen: 642 +/- 31%). LPL-T proliferation was only detectable at 24 h while proliferation in other compartments also occurred later. Hyporesponsiveness was not caused by enhanced T cell apoptosis upon alphabeta TCR stimulation. In conclusion, stimulation of LPL-T results in much shorter and weaker in vivo proliferation than in other lymphoid organs. Overall, CD2/CD28 costimulation is the strongest T cell stimulus in vivo.

Anaphylaxis to raw potato.

BACKGROUND: Potato allergy has been described rarely, generally in relation to the Oral Allergy Syndrome (OAS). Adults with seasonal allergic rhinitis have been reported in whom peeling of raw potatoes causes oculonasal symptoms, wheezing, and contact urticaria. Skin testing with fresh fruits and vegetables has been recommended in cases of OAS, although the sensitivity of commercial potato extract is reportedly equal to that of fresh potato. CASE REPORT: This report describes a 4-year-old with raw potato-induced anaphylaxis. He rapidly developed urticaria, angioedema, respiratory distress, vomiting and diarrhea after biting into a raw potato that was being used for painting in preschool. Review of systems is significant for viral-induced wheezing, but no symptoms suggestive of seasonal allergic rhinitis were evident. His mother has a history of seasonal allergic rhinitis and contact urticaria with raw potato. Skin testing to commercial potato extract was negative and skin testing to fresh potato by the prick + prick method was markedly positive. Skin testing to birch tree was negative. An open challenge to a small amount of cooked potato was negative. Food challenge to raw potato was not considered indicated in this case of immediate anaphylaxis to a single food. CONCLUSIONS: This patient had clinical and skin test reactivity to raw and uncooked potato in the absence of OAS. The patient will be followed for the development of seasonal allergic rhinitis.

Prevention of asthma morbidity: recent advances.

Asthma prevalence has risen substantially in recent decades and is an increasing cause of disability for American children. Concern about the rise in morbidity has led to treatment guidelines and a growing body of clinical research. Recent trials continue to support the role of inhaled corticosteroids as the most effective therapy to control airway inflammation associated with persistent asthma. Growth suppression due to inhaled corticosteroids has also been well documented, although the long-term effects and relative potencies of different agents require further study. Other anti-inflammatory agents such as cromolyn and the new class of leukotriene receptor antagonists have demonstrated benefit in milder patients. Leukotriene receptor antagonists and long-acting beta2-agonists may allow for reduction of inhaled steroid doses. Control of environmental allergens and irritants is essential. New evidence suggests an increasingly important role for allergen immunotherapy.

Resolution of childhood peanut allergy.

BACKGROUND: Peanut allergy creates great fear in many families because it is one of the leading causes of fatal and near-fatal food-induced allergies. Earlier reports suggested that peanut allergy was life-long, but a recent study described resolution of peanut allergy in some children. OBJECTIVE: Tolerance to peanut allergy in childhood was studied. Examination of the natural history of childhood peanut allergy was explored. METHODS: A retrospective review of all children with peanut allergy seen at the Children's Hospital of Philadelphia in a 3-year period (n = 293). Children with histories of peanut allergy were challenged at the mean age [3.8 years; range 1.5 to 8 year] which was 1.8 years [range: 0.5 to 6.8 years], following their last known clinical reaction. Food allergy or tolerance was confirmed by open challenges. RESULTS: Thirty-three patients with histories of peanut allergy and a positive skin test to peanut underwent oral challenges. Not one patient (n = 5) with a history of peanut anaphylaxis developed tolerance to peanuts. In comparison, 9 of 17 patients with history of urticaria upon ingestion to peanuts developed tolerance. Also, 4 of 10 patients with flaring of their atopic dermatitis upon ingestion to peanuts developed tolerance. The 14 patients with a negative challenge to peanut had a significantly smaller wheal and flare reaction than the 19 patients with positive challenges. Tolerance to peanut was documented by a positive challenge reverting to a negative challenge in one patient. Oral challenge of 13 additional patients with positive skin tests and histories of only refusing to eat peanut resulted in 5 (39%) positive challenges. CONCLUSION: A selected group of peanut-allergic children, who do not have a history anaphylaxis to peanut, may develop tolerance to peanuts.

Routine and influenza vaccination rates in children with asthma.

BACKGROUND: Children with asthma may be at increased risk for low immunization rates given that they have recurrent illnesses that often result in acute care visits to their pediatrician, visits to the emergency room, admissions to the hospital, and visits to subspecialists, where immunizations are not routinely administered. OBJECTIVES: To assess immunization rates for routine and influenza vaccines in children with asthma and assess factors that may contribute to delay. METHODS: We conducted a cross-sectional survey of 117 children aged 6 to 48 months with onset of asthma within the first 15 months of life. Subjects were recruited from an allergy and immunology clinic at an urban, tertiary care center. Those judged to have immunization delay did not have the required 4 DTP, 3 OPV, and 1 MMR vaccine by age 24 months (4:3:1 series). Receipt of influenza vaccine was determined for eligible children with moderate to severe asthma. RESULTS: Seventy-four (80%) children had up-to-date immunizations at age 24 months. Those with delay had fewer visits to a subspecialist than those who were up-to-date (1 versus 2 visits P = .010). Twenty-two (25%) of 87 eligible subjects received influenza vaccine. Recipients were more likely to have been hospitalized than nonrecipients (77% versus 49%, P = .022). CONCLUSIONS: Though the majority of young children with asthma were up-to-date for routine immunizations, only 25% of children with moderate to severe asthma received influenza vaccine. Greater efforts must be made by pediatricians and asthma subspecialists to ensure that children with moderate to severe asthma are immunized against influenza virus.

Latex allergy in pediatric emergency department personnel.

Allergy to natural latex proteins has been recently recognized as a dangerous entity among health care professionals. Cutaneous symptoms related to latex glove use vary from the redness and scaling of contact dermatitis to urticaria. In addition, anaphylactic reactions have been reported. We report the spectrum of reactions to latex glove use in 93 members of our emergency department (ED) staff during a one-month study period. In addition, we attempt to correlate these symptoms with serologic evidence of atopy and latex allergy. Eighty-four of these subjects underwent total serum immunoglobulin E and latex-specific radioallergosorbent test (RAST) testing. Fifty-four percent of subjects reported symptoms relating to latex glove use, categorized as either contact dermatitis (48.4%) or urticaria (5.4%). Of the urticaria group, two subjects reported additional symptoms related to latex glove use such as rhinitis, conjunctivitis, or sneezing. All three groups of subjects (asymptomatic, contact dermatitis, and urticaria) were alike with respect to age, sex, and race. The urticaria group reported a higher incidence of environmental allergies (chi 2, P = 0.02). Serum total immunoglobulin E levels and latex-specific RAST results did not differ among the three groups. The one subject with a positive latex-specific RAST reported urticarial and nasoocular symptoms when exposed to latex gloves. Seventeen percent of symptomatic subjects reported decreased use of latex gloves because of these symptoms. It was concluded that many members of our pediatric ED staff exhibit a sensitivity to latex antigens. Clinical symptoms, rather than serologic testing, must be used to identify latex-sensitive individuals in this setting. Recommendations are offered to assist in the identification and management of hospital personnel who exhibit allergy to latex-containing products.

A comparison of electrochemiluminescence and flow cytometry for the detection of natural latex-specific human immunoglobulin E.

In vitro correlates of type 1 hypersensitivity to natural latex (NL) proteins continue to be limited by both sensitivity and specificity. Methods which have detection limits in the picogram range, namely, radioallergosorbent assays (RAST) and enzyme-linked immunosorbent assays (ELISA), are inadequate for the identification of NL hypersensitivity in certain at-risk groups, such as health care workers. A flow cytometry assay (FCA), previously shown to be comparable to RAST and ELISA in the identification of NL-sensitized pediatric patients with spina bifida, was compared with electrochemiluminescence (ECL) in the evaluation of pediatric patients with spina bifida and NL-sensitized adult health care workers. As with RAST and ELISA, ECL is capable of detecting picogram amounts of specific analyte. The ECL assay detected NL-specific immunoglobulin E (NL-IgE) in three of six health care workers with strong histories of NL hypersensitivity. All six patients were negative by FCA. Further, 2 of 11 spina bifida patients found to be NL-IgE negative by FCA were NL-IgE positive by ECL. These findings suggest that in sensitivity the ECL assay is an improvement over the FCA for the identification of NL-sensitive individuals.

Latex hypersensitivity in children: clinical presentation and detection of latex-specific immunoglobulin E.

OBJECTIVE: To better understand the clinical characteristics, diagnosis, and possible prevention of immediate hypersensitivity reactions to latex in a hospitalized, pediatric patient population. METHODS: We performed a retrospective case analysis of the first 35 patients with latex allergy evaluated by our service over a 2-year period at our institution. Characteristics of patients and clinical reactions were analyzed and the presence of latex-specific immunoglobulin E was assessed using in vitro methods. In a limited group of patients, the success of strict environmental control and premedication with steroids and antihistamines was evaluated for the prevention of latex allergic reactions. RESULTS: The majority of our patients had life-threatening reactions. In previous reports, most pediatric patients underwent reactions in the perioperative period and belonged to two well-recognized "high-risk" patient groups (spina bifida and genitourinary malformations). In our series, 21 patients (60%) had reactions outside of the operating room setting, and 14 patients (40%) had primary diagnoses outside of the previously recognized "high-risk" groups. Many patients had a history of multiple surgical procedures, and a history of a surgical procedure in the first year of life was very common. A pre-existing clinical history of latex allergy was present in only 18 of the 35 patients, and a severe or life-threatening allergic reaction was the presenting feature of latex allergy in 11 of the 35 patients. Using in vitro assays, we were able to detect latex-specific immunoglobulin E in the sera of all but two of our patients. Latex gloves and latex-containing intravenous sets were common triggers for reactions. When exposure to latex occurs systemically, as through an intravenous line, premedication with steroids and antihistamines may fail to protect against anaphylaxis. CONCLUSIONS: Our experience indicates that the incidence of latex hypersensitivity in children is increasing, that the circumstances (patient profile, hospital location, route of exposure) in which life-threatening reactions may occur are more broad than previously reported, and that a better understanding of both environmental sources of latex antigens and host responses to latex exposure are needed for improved prevention of serious reactions.

Physician-targeted program on inhaled therapy for childhood asthma.

BACKGROUND: Inhaled medications are the mainstay of asthma therapy, but significant deficiencies exist in the knowledge and skills of physicians regarding use of metered-dose inhalers (MDI) and spacer devices. OBJECTIVE: We developed, implemented, and evaluated the effects of a physician-targeted educational program on inhaled therapy in a group of pediatric residents in our institution. METHODS: Patient-directed instruction sheets on aerosol therapy were developed on the basis of literature review and expert guidelines. These served to establish a consistent foundation for the educational curriculum. The program was delivered through one-on-two teaching sessions (45 minutes). Residents were provided with a summary of theoretical and practical information and with devices for practice (a placebo MDI, InspirEase and AeroChamber holding chambers, and the AeroChamber device with mask). Each session included review of an educational monograph, demonstration of proper technique, and practice with the different devices. The program was evaluated by a randomized-control design. Assessment of practical skills included number of correct steps for the use of MDI (maximum score, 7), InspirEase (maximum, 7) and AeroChamber (maximum, 6). Theoretical knowledge was assessed with 25 multiple-choice questions. RESULTS: Pretest scores in the experimental group (n = 24) were 3.7 of 7, 1.9 of 7, and 0.3 of 6 steps correct for MDI, InspirEase, and AeroChamber devices, respectively, and 13 of 25 for the theoretical knowledge assessment. The control group (n = 26) had similar pretest scores. After the program the experimental group significantly improved in all parameters: 6.3 of 7, 5.9 of 7, and 4.5 of 6 steps correct for MDI, InspirEase, and AeroChamber devices, respectively, and 18 of 25 questions correct (p < 0.01 for all parameters). CONCLUSIONS: Implementation of a simple educational program among pediatric residents can significantly increase their skills in the use of inhalational therapy.

What do pediatricians in training know about the correct use of inhalers and spacer devices?

Most patients with asthma in the United States are cared for by nonspecialist physicians. Because inhaled medications are the mainstay of asthma therapy and their successful use requires both practical skills and theoretic knowledge, we wondered how much nonspecialist physicians know about the use of metered-dose inhalers and spacer devices. Fifty pediatricians in training were interviewed individually. Practical knowledge was assessed by asking each to demonstrate correct use of a placebo inhaler and a spacer device (Inspirease [Key Pharmaceuticals, Inc., Miami, Fla.] and Aerochamber with mask [Monaghan Medical Corp., Plattsburgh, N.Y.]). Of the seven recommended steps for use of metered-dose inhalers, the residents demonstrated an average of 3.8 steps correctly. The most common errors included not shaking the metered-dose inhaler before use (18% of residents correct) and insufficient breath holding (28% correct). In testing spacer use, the most common errors included not shaking the canister (16% correct) and incorrect number of activations and inhalations (12% correct). Many residents were not familiar with correct assembly of the spacer (48% correct). Theoretic knowledge of metered-dose inhaler and spacer use was evaluated by a written questionnaire. The most common deficiencies in theoretic knowledge related to the purpose of slow inspiration and breath holding. Most of the participants had been treating children with asthma and had prescribed metered-dose inhalers (45 of 50, 90%) and spacer devices (76%) in the past.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometric measurement of immunoglobulin E to natural latex proteins.

Immediate hypersensitivity to natural latex (NL) occurs in sensitized individuals after repeated exposure to products or devices containing NL components. Since allergic reactions to NL proteins are quite frequent and may be quite serious, diagnostic assays are needed to identify individuals at risk. A number of latex proteins have been considered the major antigens, but they have been incompletely characterized. There is no standard material available for skin testing. In vitro diagnostic tests, such as the radioallergosorbent test (RAST), are time consuming and their sensitivity and specificity remain to be proven. We have developed a rapid microsphere-based, fluorescence-activated flow cytometry assay for the measurement of NL protein-specific human immunoglobulin E and have compared it with both the enzyme-linked immunosorbent assay and radioallergosorbent test methods. By using the total purified NL protein fraction isolated from raw ammoniated NL sap as the antigen, the flow cytometry assay was both sensitive and specific for the detection of NL protein-specific human immunoglobulin E in the sera of sensitized pediatric patients.

Role of endothelin-1 in regulating proliferation of cultured rabbit airway smooth muscle cells.

Increased expression of the potent vasoconstrictor and bronchoactive peptide, endothelin-1 (ET-1), has recently been demonstrated in airway epithelial and endothelial cells of asthmatic patients. To identify its potential role in contributing to airway smooth muscle (ASM) hyperplasia, a characteristic feature of asthmatic airways, the mitogenic action of ET-1 was investigated in cultured rabbit ASM cells. ET-1 elicited significant dose-dependent (10(-12)-10(-6) M) increases in ASM cell number, with a mean potency (i.e., -log mean effective dose) of action of 9.82-log M. ET-1 also acutely stimulated intracellular inositol 1,4,5-trisphosphate accumulation. The latter response was blocked by phospholipase C inhibition with neomycin; however, neomycin had no effect on the promitogenic action of ET-1. By contrast, the ASM cell proliferative response to ET-1 was independently inhibited by pertussis toxin, inhibitors of phospholipase A2, cyclooxygenase, and thromboxane A2 (TxA2) synthesis, as well as blockade of the TxA2 receptor. Moreover, in complementary studies, we found that administration of the stable TxA2 mimetics, carbocyclic TxA2 (CTA2) and U-46619, induced ASM cell proliferation and that ET-1 evoked the release of endogenous TxA2 from the ASM cells. Collectively, these observations provide new evidence that 1) ET-1 is a potent mitogen of ASM cells, 2) the promitogenic effect of ET-1 is associated with activation of a pertussis toxin-sensitive G protein coupled to stimulation of phospholipase A2, and 3) the latter mediates ASM cell proliferation via the release and autocrine mitogenic action of TxA2. The findings support a potential role for ET-1 in mediating the characteristic hyperplasia of ASM in asthma.

Latex hypersensitivity reactions despite prophylaxis.

Latex rubber hypersensitivity represents a significant problem facing the medical, surgical, radiologic, and dental professions. As a tertiary care center, the Childrens Hospital of Philadelphia has a large population of patients with spina bifida and complex genitourinary anomalies; a number of these children have latex rubber allergy, which may first present as intraoperative anaphylaxis. Although there is no substitute for complete antigen avoidance, all medical products containing latex rubber may not have suitable alternatives. Therefore, we have formulated a protocol to prevent perioperative reactions through the use of prophylactic medications and the limitation of latex exposure. This regimen includes steroids, antihistamines, and bronchodilators when indicated. In four children, prophylaxis failed perioperatively because of parenteral infusion of latex rubber proteins.

Mechanisms of action of endothelin 1 in maturing rabbit airway smooth muscle.

Maturational differences in the effects and mechanisms of action of endothelin 1 (ET-1) on airway contractility were investigated in tracheal smooth muscle (TSM) segments isolated from 2-wk-old and adult rabbits. In TSM under passive tension, ET-1 elicited dose-dependent contractions, with a potency of action that was significantly greater (P less than 0.001) in the 2-wk-old vs. adult tissues (i.e., mean +/- SE - log 50% of maximal response values: 8.59 +/- 0.17 vs. 7.79 +/- 0.15 - log M, respectively). In TSM half-maximally contracted with acetylcholine (ACh), however, ET-1 elicited dual and opposing dose-dependent effects. At lower doses (less than or equal to 10(-9) M), ET-1 induced TSM relaxation that was significantly greater in the adult vs. 2-wk-old TSM segments (i.e., approximately 100 vs. 26.5% decrease in active tension, respectively). The relaxant responses were associated with significantly enhanced (P less than 0.001) ET-1-induced release of prostaglandins E2 and I2 in the adult tissues. At higher doses (greater than 10(-9) M), ET-1 induced TSM contractions that were 1) attenuated to a relatively greater extent by the Ca2+ channel blocker, nifedipine (10(-5) M) in the 2-wk-old tissues and 2) associated with significantly (P less than 0.001) enhanced ET-1-stimulated accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the immature TSM. Moreover, the TSM contractions were inhibited by the protein kinase C (PKC) antagonist, H-7, and the latter effect was more potent in the immature TSM. Collectively, these findings demonstrate that ET-1 exerts a potent duality of action in rabbit TSM which varies significantly with maturation, wherein 1) age-dependent differences in airway relaxation are associated with changes in the evoked release of bronchodilatory prostaglandins and 2) maturational differences in airway contraction are associated with changes in Ins(1,4,5)P3 accumulation and extracellular Ca2+ mobilization, coupled to differences in PKC activation.

Role of endothelin 1 in regulating rabbit airway contractility.

Endothelin 1 (ET-1) is a potent vasoconstrictor peptide recently isolated from vascular endothelial cells. Because its role and mechanisms of action in regulating airway contractility remain to be identified, we examined the contractile effects of ET-1 in isolated rabbit tracheal smooth muscle (TSM) segments. In TSM under passive tension, ET-1 elicited dose-dependent contractions with a mean +/- SE -log 50% of maximal response value of 7.82 +/- 0.13 vs. a value of 5.61 +/- 0.07 -log M for acetylcholine (ACh). In TSM half-maximally contracted with ACh, however, ET-1 exerted dual and opposing contractile effects. Lower doses of ET-1 (less than or equal to 10(-9) M) produced a 74.2 +/- 16.6% decrease in active TSM tension. This relaxant response to ET-1 was associated with an accelerated accumulation of prostaglandin (PG) I2 and PGE2 and was attenuated by cyclooxygenase inhibition with indomethacin (10(-5) M). The combination of indomethacin and removal of the airway epithelium completely inhibited the TSM relaxant response to ET-1. In contrast, higher doses of ET-1 (greater than 10(-9) M) induced airway contractions that were attenuated by the Ca2+ channel blockers nifedipine (10(-5) M) and diltiazem (10(-5) M) and ablated in Ca2(+)-free buffer. Moreover, ET-1-induced TSM contractions were inhibited by the protein kinase C (PK-C) antagonists 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, and staurosporine.(ABSTRACT TRUNCATED AT 250 WORDS)

Heat-killed Salmonella typhi induces the release of prostaglandins and leukotrienes from mouse macrophages.

Mouse macrophages pre-labeled with [3H]arachidonic acid (20:4) were shown to release metabolites generated by the lipoxygenase and cyclo-oxygenase pathways following in vitro addition of heat-killed Salmonella typhi. These metabolites were maximally released after 60-90 min of incubation and consisted of prostaglandins (85%), leukotriene C (6%), di-HETEs, leukotrienes D and E (4%), mono-HETEs (2%) and other metabolites (3%). Of the metabolites generated by the cyclo-oxygenase pathway (prostaglandins), 6-keto PGF1 alpha and PGE2 were generated at a ratio of 1.2 to 1. The significance and importance of these results are discussed.

Prostaglandins mediate the ocular hypotensive action of the angiotensin converting enzyme inhibitor MK-422 (enalaprilat) in African green monkeys.

MK-422 (enalaprilat) (0.0005-0.5%) significantly reduced intraocular pressure (IOP) in African Green monkeys. Studies utilizing unilateral instillation of MK-422 and its inactive R-isomer indicated a local site of action within the eye which is dependent upon inhibition of angiotensin converting enzyme, also known as kininase II. Tonography showed a small increase (21%) in conventional aqueous humor outflow facility which did not entirely account for the IOP lowering effect of MK-422. Pretreatment with indomethacin or pilocarpine specifically attenuated the ability of MK-422 to lower IOP suggesting that biosynthesis of prostaglandins and uveoscleral outflow pathways are important in mediating the ocular hypotension. The data indicate that MK-422 may lower IOP in monkeys by virtue of its ability to prevent the breakdown of bradykinin and thereby promote the formation of endogenous prostaglandins in the eye.

The selective binding and transmigration of monocytes through the junctional complexes of human endothelium.

Human monocytes show a high affinity for vascular endothelium both in vitro and in vivo. To explore monocyte-endothelial interaction in greater detail, we have developed a new in vitro model for growth of human endothelial cells (EC). Human umbilical vein EC (HUVEC) cultured upon collagen gels form confluent monolayers of EC that bind silver at their intercellular border similar to cells in situ. Intercellular junctional structures, both adherens and tight junctions, were identified. In contrast, HUVEC grown on plastic surfaces did not stain with silver. The silver-staining characteristic of EC-collagen monolayers was reversible and related to their in vitro maturation and senescence. Silver staining of EC borders provided a grid by which the location of monocyte binding to the luminal surface of individual EC could be assessed. Using this technique, we found that monocytes preferentially bound to the margins of EC, in approximation to the silver-staining junctions. These results suggest that EC determinants recognized by monocytes occur in a unique topographical distribution on the apical face of EC. After binding, monocytes migrated through the EC monolayers at high basal rates. The lack of penetration of collagen gels in the absence of an EC monolayer suggested the generation of EC-specific chemotactic signal(s). Monocytes were observed to pass between EC without evidence of disruption of the monolayer. Silver stain remained present during all phases of migration, and under transmission electron microscopy, junctional complexes were found proximal to monocytes that had just completed their passage through the monolayer. After orientation to the basal surface of the EC monolayer, monocytes migrated randomly into the underlying collagen gel. Monocyte adherence, penetration, migration, and long term survival can be studied under these conditions.