Rebuck Skin Window Research Articles

An amebic anti-inflammatory peptide down-regulates ex vivo IL-1β expression in patients with rheumatoid arthritis

The monocyte locomotion inhibitory factor (MLIF) is a heat-stable pentapeptide produced by Entamoeba histolytica in culture. This factor displays several anti-inflammatory properties (i.e., inhibition of locomotion and respiratory burst in monocytes, reduction of skin hypersensitivity and delay of mononuclear cells in human Rebuck skin windows) with inhibition of adhesion molecules, chemokines, and other genes including interleukin-1β (IL-1β). In animal models, it reduces carragenin-induced inflammation and delays the inflammatory process in murine collagen-induced arthritis (CIA). ObjectivesTo test, in vitro, the anti-inflammatory capacity of MLIF on a promonocytic human cell line (U-937) cells and peripheral blood mononuclear cells (PBMC) from healthy subjects and from patients with rheumatoid arthritis (RA). Material and methodsIL-1β gene expression was evaluated in cell cultures either in the presence of MLIF, lipopolysaccharide (LPS), or both. Relative gene expression and immunoreactivity of IL-1β were assayed in cells and supernatants, respectively. ResultsAmebic peptide was able to down-regulate LPS-induced expression of IL-1β, in U-937 cells without a detectable effect upon the bioavailability of the cytokine. In similar culture conditions, MLIF was capable to down-regulate baseline and LPS-induced expression of IL-β only in PBMC from patients with RA. Peptide effect on immunoreactivity of IL-1β was not statistically significant. ConclusionsMLIF exerts, in primed cells, exquisite anti-inflammatory properties that deserve to be explored mechanistically.

An amebic anti-inflammatory peptide down-regulates ex vivo IL-1β expression in patients with rheumatoid arthritis

The monocyte locomotion inhibitory factor (MLIF) is a heat-stable pentapeptide produced by Entamoeba histolytica in culture. This factor displays several anti-inflammatory properties (i.e., inhibition of locomotion and respiratory burst in monocytes, reduction of skin hypersensitivity and delay of mononuclear cells in human Rebuck skin windows) with inhibition of adhesion molecules, chemokines, and other genes including interleukin-1β (IL-1β). In animal models, it reduces carragenin-induced inflammation and delays the inflammatory process in murine collagen-induced arthritis (CIA). ObjectivesTo test, in vitro, the anti-inflammatory capacity of MLIF on a promonocytic human cell line (U-937) cells and peripheral blood mononuclear cells (PBMC) from healthy subjects and from patients with rheumatoid arthritis (RA). Material and methodsIL-1β gene expression was evaluated in cell cultures either in the presence of MLIF, lipopolysaccharide (LPS), or both. Relative gene expression and immunoreactivity of IL-1β were assayed in cells and supernatants, respectively. ResultsAmebic peptide was able to down-regulate LPS-induced expression of IL-1β, in U-937 cells without a detectable effect upon the bioavailability of the cytokine. In similar culture conditions, MLIF was capable to down-regulate baseline and LPS-induced expression of IL-β only in PBMC from patients with RA. Peptide effect on immunoreactivity of IL-1β was not statistically significant. ConclusionsMLIF exerts, in primed cells, exquisite anti-inflammatory properties that deserve to be explored mechanistically.

The Monocyte Locomotion Inhibitory Factor an Anti-Inflammatory Peptide; Therapeutics Originating from Amebic Abscess of the Liver

Entamoeba histolytica in culture produces a pentapeptide (MQCNS). This oligopeptide inhibits the in vitro and in vivo locomotion of human monocytes, hence its denomination Monocyte Locomotion Inhibitory Factor (MLIF). The original isolated peptide and its synthetic construct display similar effects, among others, being inhibition of the respiratory burst in monocytes and neutrophils, decrease of Dinitrochlorobenzene (DNCB) skin hypersensitivity in guinea pigs and gerbils, and delay of mononuclear leukocytes in human Rebuck skin windows with inhibition of vascular cell Very late antigen (VLA)-4 and Vascular adhesion molecules (VCAM) in endothelia and monocytes. The MLIF molecular mechanism of action is unknown, but data reveal its implication in Nuclear factor-kappa B (NF-κB) and Mitogenactivated protein kinase (MAPK) pathways. This could explain MLIF multiplicity of biological effects. On the other hand, the amebic peptide has been useful in treating experimental amebiasis of the liver. The amebic peptide is effective in reducing inflammation induced by carragenin and arthritis in a Collagen-induced arthritis (CIA) model. Microarray data from experimental arthritis revealed an MLIF gene expression profile that includes genes that are involved in apoptosis, cell adhesion, extracellular matrix, and inflammation / chemotaxis. MLIF could be involved in unsuspected biological factions because there is increasing data on the peptide effect on several cell activities. This review also presents uses of MLIF as described in patents.

The skin as a model to study the pathogenesis of IgE-mediated acute and late-phase responses

101. Laroche D, Chrysanthou S, Lefran~ois C, et al. Evidence of a progressive degranulation of basophil in subjects operated on without adverse reaction. Agents Actions 1992;36(suppl C):C199-C200. 102. De Paulis A, Valentini G, Spadaro G, Lupoli S, Tirri G, Marone G. Human basophil releasability. VIII. Increased basophil releasability in patients with scleroderma. Arthritis Rheum 1991;34:1289-96. 103. Yunginger JW, Nelson DR, Squillace DL, et al. Laboratory investigation of deaths due to anaphylaxis. J Forensic Sci 1991;36:857-65. 104. Takeishi T, Martin TR, Katona IM, Finkelman FD, Galli SJ. Differences in the expression of the cardiopulmonary alterations associated with anti-immunoglobulin E-induced or active anaphylaxis in mast cell-deficient and normal mice: mast cells are not required for the cardiopulmonary changes associated with certain fatal anaphylactic responses. J Clin Invest 1991;88:598-608. 105. Sampson HA, Mendelson L, Rosen JP. Fatal and nearfatal anaphylactic reactions to food in children and adolescents [Comments]. N Engl J Med 1992;327:380-4. 106. Shalit M, Schwartz LB, yon Allmen C, Atkins PC, Lavker RM, Zweiman B. Release of histamine and tryptase during continuous and interrupted cutaneous challenge with allergen in humans. J ALLERCY CLIN IMVnJNOL 1990; 86:117-25. 107. Lavker RM, Schechter NM, Guzzo C, Lazarus GS. Aggressive topical corticosteroid therapy: a novel approach to mast-cell-dependent cutaneous disorders. Dermatologica 1987;175:213-6. 108. Lavker RM, Schechter NM, Lazarus GS. Effects of topical corticosteroids on human dermis. Br J Dermatol 1986;115:101-7. 109. Jarjour NN, Calhoun WJ, Schwartz LB, Busse WW. Elevated bronchoalveolar lavage fluid histamine levels in allergic asthmatics are associated with increased airway obstruction. Am Rev Resp Dis 1991;144:83-7. 110. Broide DH, Gleich GJ, Cuomo AJ, et al. Evidence of ongoing mast cell and eosinophil degranulation in symptomatic asthma airway. J A~LEROY CLlr~ IMMUNOL, 1991; 88:637-48. 111. Liu MC, Bleecker ER, Lichtenstein LM, et al. Evidence for elevated levels of histamine, prostaglandin D2, and other bronchoconstricting prostaglandins in the airways of subjects with mild asthma. Am Rev Respir Dis 1990; 142:126-32. 112. Sedgwick JB, Calhoun WJ, Gleich GJ, et al. Immediate and late airway response of allergic rhinitis patients to segmental antigen challenge: characterization of eosinophil and mast cell mediators. Am Rev Respir Dis 1991; 144:1274-81. 113. Liu MC, Hubbard WC, Proud D, et al. Immediate and late inflammatory responses to ragweed antigen challenge of the peripheral airways in allergic asthmatics: cellular, mediator, and permeability changes. Am Rev Respir Dis 1991;144:51-8. 114. Koshino T, Teshima S, Fukushima N, et al. Identification of basophils by immunohistochemistry in the airways of post-mortem cases of fatal asthma. Clin Exp Allergy 1993;23:919-25.

Skin eosinophilia in patients with allergic asthma, patients with nonallergic asthma, and healthy controls: II: 20-hydroxy-leukotriene B4 is a potent in vivo and in vitro eosinophil chemotactic factor in nonallergic asthma

Background: In allergic and nonallergic asthma, eosinophils play an important effector role. However, because the pathogenesis of these types of asthma seems different, the mechanisms responsible for the tissue mobilization of those cells may be different. The in vivo and in vitro migratory response of eosinophils from patients with allergic and nonallergic asthma toward 20-hydroxy-leukotriene B, (20-OH-LTB 4), which is reported here, illustrates this. Methods: By means of the Rebuck skin window technique the in vivo skin mobilizing capacity of intracutaneously applied buffer, LTB 4, and 20-hydroxy (OH)-LTB 4 was evaluated in healthy subjects ( n = 6), subjects with allergic asthma ( n = 14), and subjects with nonallergic asthma ( n = 17). Also the in vitro chemotactic responsiveness of eosinophils from the circulation of both patient groups (both n = 8) toward buffer, LTB 4, and 20-OH-LTB 4 were tested by use of a microchemotaxis chamber technique. Results: Although none of the substances were capable of inducing macroscopic observable skin reactions, intracutaneously applied 20-OH-LTB 4 had an almost similar capacity to mobilize eosinophils in the skin of the subjects with nonallergic asthma as allergens had in subjects with allergic asthma. In 92% of the tested subjects with nonallergic asthma significant skin eosinophilia was observed. By contrast, LTB 4 did not induce significant skin eosinophilia in both patient groups compared with buffer solution. This in vivo eosinophil mobilizing capacity of 20-OH-LTB 4 in subjects with nonallergic asthma was confirmed by in vitro chemotaxis studies. Dose ranges of both LTB 4 and 20-OH-LTB 4 proved to be potent chemoattractants for eosinophils from patients with nonallergic asthma, but not for those of healthy subjects and those with allergic asthma. Conclusions: Our results indicate that 20-OH-LTB 4 may be involved in the tissue mobilization of eosinophils in nonallergic asthma and that in vitro 20-OH-LTB 4 (and LTB 4) may act as potent chemotactic factors on eosinophils from those patients.

Skin eosinophilia in patients with allergic and nonallergic asthma and atopic dermatitis

Skin eosinophilia induced by grass pollen or platelet-activating factor (PAF) was investigated in patients with allergic and nonallergic asthma and atopic dermatitis (AD) and in normal control subjects. Intracutaneous injection of PAF resulted in positive immediate skin reactions in all tested individuals. Since most of the patients with allergic and nonallergic asthma and eczema under investigation were characterized by a peripheral blood eosinophilia, we investigated whether PAF should be capable of mobilizing blood eosinophils to the skin of patients with nonallergic asthma and AD using the Rebuck skin window technique. Our results demonstrated that, in allergic individuals, allergen induced a much stronger skin eosinophilia than PAF and in a larger percentage of the patients (77% versus 45%). There was no correlation between skin eosinophilia induced by PAF and the occurrence of late skin reactions to allergen. Furthermore, the PAF-induced and the allergen-induced skin eosinophilia differed histologically. In nonallergic individuals, neither allergen nor PAF was capable of mobilizing eosinophils in the skin window, in spite of the presence of peripheral blood eosinophilia. Our results indicate that the allergen- and PAF-induced skin eosinophilia are different and that in patients with nonallergic asthma and AD, another chemoattractant than PAF must be responsible for eosinophil mobilization in the target organ.

Skin eosinophilia in atopic patients

The responses of six atopic and six control patients to the intracutaneous administration of platelet-activating factor (PAF) acether, of the leukocyte secretagogue and chemoattractant f-methionyl-leucyl-phenylalanine (FMLP), and of allergens were studied. Immediate wheal-and-flare skin reactions to allergens and to PAF acether in atopic patients were the same, and when reactions were compared to reactions of nonatopic patients, these reactions to PAF acether and to FMLP were of equivalent intensity. Late-phase reactions were not observed macroscopically with allergen, PAF acether, or FMLP. The cellular responses, assessed by the Rebuck skin window technique, demonstrated a late (24 hours) eosinophilic response to the allergen and to both inflammatory agents, with 33% to 44% of intensively degranulated eosinophils. In comparison, very few, if there were any, eosinophils were observed in the nonatopic patients. Most infiltrated cells were neutrophils. The accumulation of eosinophils in atopic human skin indicates a potent eosinophilotactic activity restricted to the allergic patients. A similar cellular reaction has been observed in lungs from guinea pigs injected with PAF acether or with allergen, and in patients with severe asthma. The chemoattractant activity of FMLP toward eosinophils cannot be accounted for by its histamine-releasing activity and remains to be explained.

Tissue Debris at the Injury Site Is Coated by Plasma Fibronectin and Subsequently Removed by Tissue Macrophages

Fibronectin (Fn) is a normal plasma and extracellular matrix glycoprotein that is thought to be important in reticuloendothelial system function as well as in promoting adhesion of various cell types to basement membranes and to each other. Plasma Fn levels are depressed following almost any type of trauma. It opsonizes circulating tissue debris for removal by the fixed cells of the reticuloendothelial system. It has been assumed but not proven that Fn also opsonizes tissue debris at the site of the injury for subsequent phagocytosis by tissue macrophages. In this study, rats were given intracardiac injections of Fn coupled with fluorescence isothiocyanate (Fn-FITC) and human serum albumin-rhodamine isothiocyanate (HSA-RITC). Abdominal Rebuck skin windows were then prepared. Within 24 hours, debris at the sites of injury were observed to be coated with Fn-FITC but not HSA-RITC. This Fn-labeled debris was subsequently ingested by macrophages at the site. No macrophages were found that had taken up HSA-RITC. Thus, Fn is seen to coat tissue debris and effete cells within the wound, and the coated material is subsequently removed by tissue macrophages.

Neutrophil function studies in clinical medicine.

A complete evaluation of neutrophil function including: chemotaxis; adhesion; aggregation; phagocytosis; granule content and degranulation; respiratory burst activity; and bacterial killing; is expensive and requires the services of a specialized laboratory. However, preliminary screening of a patient with a predisposition toward infection, can be carried out using simple and inexpensive methods. These include examination of blood films, which may prove helpful in the diagnosis of Chediak-Higashi syndrome and specific granule deficiency; the Rebuck skin window test, which estimates chemotactic defects; the NBT test, which screens for chronic granulomatous disease patients; and peroxidase staining of the blood film in order to estimate the content of myeloperoxidase, when myeloperoxidase deficiency is suspected. For final diagnosis and determination of genetic transmission and radical treatment, ie, bone marrow transplantation, specific tests are indicated. Neutrophil function studies have also proved useful in detecting diseases in which defects in neutrophil function are secondary to the primary disorder. Indeed, increased neutrophil chemotaxis has been reported in the active phase of diseases such as: familial Mediterranean fever; psoriasis vulgaris, Behcet's syndrome and Sweet's syndrome. In these disorders the neutrophil chemotaxis assay has aided in the diagnosis and follow-up, particularly in evaluating the response to antiinflammatory agents, such as colchicine.

Monocytes accumulate on Rebuck skin window coverslips but not in skin chamber fluid: A comparative evaluation of two in vivo migration models

Migration of leukocytes into an inflammatory site is an important step of host defense. The Rebuck skin window and the skin blister chamber technique allow study of the dynamics of the inflammation in vivo. The type of inflammation is basically different in each of these methods. Whereas the former technique provokes a late monocytic response, the latter technique provokes virtually no monocyte accumulation. The difference between these two types of inflammation has never been systematically studied. We describe a skin blister chamber technique with a novel multiwell device which allows the observation of cell accumulation under different conditions, i.e., in presence and in absence of a foreign body (coverslip). This method proved simple and reproducible with variability among volunteers exceeding variability of replicate chambers within a given subject. Furthermore, it confirms that no monocytes accumulate in blister chambers during the first 20 h of inflammation, whereas monocytes accumulate on skin window coverslips within the first 6 h of inflammation, Control experiments show that the continuous presence of the coverslip in the skin window is the critical element provoking accumulation of monocytes. A different degree of neutrophil degranulation in presence and absence, respectively, of a foreign body may be responsible for the different types of inflammatory response.

Inhibition of human monocyte locomotion by products of axenically grown E. histolytica

The supernatant fluid of axenically grown E. histolytica inhibits chemotaxis, chemokinesis and random mobility of human mononuclear phagocytes (MP) as measured in Boyden chambers. Human polymorphonuclear phagocytes (PMN) locomotion is apparently unaffected. The factor was found in comparable amounts in the supernatant fluid of axenic cultures of four E. histolytica strains that differed in their human pathogenicity and virulence, as well as in two entamoebas non-pathogenic for man. This dialysable and thermolabile MP-locomotion inhibiting entamoeba product (EP) can be absorbed out by incubation with MP, but not with lymphocytes, while partial absorption was observed using PMN. The MP-locomotion inhibitory effect of this EP was cancelled by inhibiting protein synthesis in the MP by means of cycloheximide. In vivo, this EP caused a delay in MP migration in Rebuck skin windows. The molecular weight of this EP lies between 478 and 765 by gel-sieve chromatography. Our results suggest a direct effect upon the cytoskeletal and locomotive apparatus of the MP. This MP-locomotion inhibiting EP could contribute to the paucity of the inflammatory reaction observed in the advanced stages of invasive amoebiasis and consequently also to the lack of scar tissue formation upon healing of amoebic lesions.

Human Neutrophil-Specific Granule Deficiency: A Model to Assess the Role of Neutrophil-Specific Granules in the Evolution of the Inflammatory Response

It has been suggested that neutrophil (PMN) specific granules are important in cell aggregation, locomotion, hydroxyl radical formation, and in extracellular functions such as the generation of complement-related inflammatory mediators (C5a) and the feedback regulation of myelopoiesis. In the current studies, a 9-yr-old boy with a history of recurrent infections and specific granule deficiency (absent lactoferrin, B-12 binding proteins, and characteristic specific granules on sucrose gradient centrifugation of cell homogenates) was studied to assess some of these concepts. In vivo, the patient had decreased PMN and monocyte accumulation into Rebuck skin windows but an expected febrile episode with an associated neutropenia (PMN margination) and neutrophilia (mobilization of marrow reserves) in response to intravenous endotoxin. In vitro, the patient's resting PMN showed increased ruffling, increased surface-to-volume ratio, and increased numbers of centriole-associated microtubules. His PMN showed a significant decrease in cell negative surface charge (which may relate to aggregation) in response to several stimuli and adhered better than normally to plastic. In addition, his PMN aggregated normally in response to the chemoattractant f-met-leu-phe, although the subsequent disaggregation normally seen with PMN did not occur with the patient's cells. Chemotaxis of the patient's PMN to several stimuli was abnormal, and specific saturable and displaceable binding of the chemoattractant f-met-leu-[3H]phe was decreased. Similarly, following incubation with secretagogues, there was a less than normal increase in f-met-leu-[3H]phe binding and an absence of the normal increases in PMN surface area. The patient's PMN bactericidal activity, stimulated oxygen metabolism (cytochrome-c reduction, chemiluminescence, and NBT reduction), and elicited changes in membrane potential were also abnormal. Studies assessing the mechanism for the abnormal monocyte accumulation into skin windows indicated the patient's monocyte chemotaxis was better than normal in vitro. However, the patient's PMN homogenates lacked a stimulus of monocyte locomotion and did not generate chemotactic activity normally from serum. Thus, the data indicate that specific granule constituents are not required for neutrophil margination in vivo or aggregation in vitro. However, the data support the concept that PMN-specific granules are important for PMN locomotion and oxidative metabolism. In addition, extracellular release of specific granule constituents appears to be important for amplification of the initial and subsequent phases of the inflammatory response.

Human neutrophil-specific granule deficiency: a model to assess the role of neutrophil-specific granules in the evolution of the inflammatory response

It has been suggested that neutrophil (PMN) specific granules are important in cell aggregation, locomotion, hydroxyl radical formation, and in extracellular functions such as the generation of complement- related inflammatory mediators (C5a) and the feedback regulation of myelopoiesis. In the current studies, a 9-yr-old boy with a history of recurrent infections and specific granule deficiency (absent lactoferrin, B-12 binding proteins, and characteristic specific granules on sucrose gradient centrifugation of cell homogenates) was studied to assess some of these concepts. In vivo, the patient had decreased PMN and monocyte accumulation into Rebuck skin windows but an expected febrile episode with an associated neutropenia (PMN margination) and neutrophilia (mobilization of marrow reserves) in response to intravenous endotoxin. In vitro, the patient's resting PMN showed increased ruffling, increased surface-to-volume ratio, and increased numbers of centriole-associated microtubules. His PMN showed a significant decrease in cell negative surface charge (which may relate to aggregation) in response to several stimuli and adhered better than normally to plastic. In addition, his PMN aggregated normally in response to the chemoattractant f-met-leu-phe, although the subsequent disaggregation normally seen with PMN did not occur with the patients cells. Chemotaxis of the patient's PMN to several stimuli was abnormal, and specific saturable and displaceable binding of the chemoattractant f-met-leu-[3H]phe was decreased. Similarly, following incubation with secretagogues, there was a less than normal increase in f-met-leu-[3H]phe binding and an absence of the normal increases in PMN surface area. The patient's PMN bactericidal activity, stimulated oxygen metabolism (cytochrome-c reduction, chemiluminescence, and NBT reduction), and elicited changes in membrane potential were also abnormal. Studies assessing the mechanism for the abnormal monocyte accumulation into skin windows indicated the patient's monocyte chemotaxis was better than normal in vitro. However, the patient's PMN homogenates lacked a stimulus of monocyte locomotion and did not generate chemotactic activity normally from serum. Thus, the data indicate that specific granule constituents are not required for neutrophil margination in vivo or aggregation in vitro. However, the data support the concept that PMN-specific granules are important for PMN locomotion and oxidative metabolism. In addition, extracellular release of specific granule constituents appears to be important for amplification of the initial and subsequent phases of the inflammatory response.

Severe fletcher factor (plasma prekallikrein) deficiency with partial deficiency of hageman factor (factor xii): Report of a case with observation on in vivo and in vitro leukocyte chemotaxis

A case of cross-reacting material-negative Fletcher trait with additional partial deficiency of Hageman factor (HF, Factor XII) is described. Although the patient presented with a recent history of frequent epistaxis, he had no other personal or family history of a tendency toward bleeding or infection. Similar to other cases of Fletcher trait, his plasma showed a markedly prolonged partial thromboplastin time which could be corrected by prolonged incubation with the surface-activator kaolin. Surface-induced fibrinolysis, amidolysis of alpha-N-benzoyl-proline-L-phenylalanine-L-arginine-p-nitroanilide, and cold-promoted enhancement of factor VII activity, reactions requiring the presence in the plasma of fletcher factor (prekallikrein), in addition to Hageman factor and Fitzgerald factor (high-molecular weight kininogen), were also defective. In vivo chemotaxis of polymorphonuclear leukocytes and monocytes (Rebuck's skin window technique) in response to skin abrasions was defective, but was normal when diphtheria-tetanus toxoid was also applied. In vitro leukocyte chemotaxis (Boyden chamber technique) in response to normal or patient's own serum activated with zymosan was normal. Together with previous observations that kallikrein generated chemotactic activity, possibly via activation of C5, the present observations suggest that prekallikrein activation may be important for in vivo leukocyte chemotactic response to skin abrasion. The inheritance of Fletcher trait in this patient is unclear.l Although the father was an apparent heterozygote, the mother was completely normal for Fletcher factor procoagulant activity and antigen. The mild Hageman factor deficiency in the patient did not contribute significantly to the plasma defects described and was likely inherited from the father who had a low HF procoagulant activity.

Evaluation of Congenital Neutropenic Disorders by in Vitro Bone Marrow Culture

The congenital neutropenias are a heterogenous group of diseases whose etiology and pathogenesis are largely unknown. We studied nine neutropenic patients from seven families. Evaluation included peripheral blood cell and differential cell counts, epinephrine and typhoid vaccine stimulation studies. Rebuck skin windows, and bone marrow aspirations for morphological assessment and for in vitro culture in liquid suspension and in agar plates. Parallel cultures were set up with and without colony-stimulating activity (CSA), and peripheral leukocytes were assayed for cellular production of CSA. Patients were initially classified on the basis of their clinical course: benign, mild, moderately severe, or severe disease. One patient in the moderately severe group had an immunoglobulin disorder. Morphologically normal mature granulocytes were seen in bone marrow aspirates of two patients, and maturational defects of varying degree were seen in the remaining seven. Colony formation in agar was markedly reduced below normal in three of seven, moderately reduced in two of seven, and greater than normal in two patients. Colonies in six of seven patients consisted exclusively of macrophages. Marrow from all but one of the nine patients demonstrated poor neutrophil development in suspension culture, and addition of CSA did not result in augmented granulocytic proliferation or maturation. A scheme of normal neutrophil maturation is proposed, and the nine patients were categorized according to this scheme. Four patterns of congenital neutropenia emerged: type 1 was the most benign form of disease with essentially normal clinical and in vitro parameters, and a defect considered to be due to a small committed stem cell pool, abnormal release, or excessive utilization peripherally; type 2 had mild disease with presumed defective committed stem cell differentiation along the granulocyte line; type 3 included benign to severe clinical expression with an apparent defect at the level of the committed granulocyte precursor more severe than in type 2; type 4 disease had varied clinical expression but evidence for a defect at the level of the pluripotent stem cell.

CONGENITAL LEUKOCYTE MOVEMENT DISORDER AND RECURRENT INFECTIONS

A 4½ month old black female with omphalitis at 4 days of age, recurrent infections of the skin, an acquired rectovaginal fistula, marked lymphadenopathy proximal to sites of infection and delayed wound healing had a peripheral WBC count of 70-100,000/mm3 with 70% PMN's and 10% monocytes. PMN's were absent in exudates of infected areas and a Rebuck skin window at 4, 8 and 12 hours. A chemotactic assay under agarose revealed absent directed movement and decreased non-directed movement of PMN's and monocytes. Zymosan activated serum from both patient and normal controls attracted normal cells to the same degree. No serum, plasma or cellular inhibitor to normal PMN movement was found. The patient's mean PMN velocity by direct observation was 2.4 μ/min. (control 11.9 μ/mtn.) and her mean monocyte velocity was 2.1 μ/min. (control 4.5 μ/min.). By light microscopy the patient's cells formed multiple, small lame 11ipodia without developing a single dominant lamel1ipodium; electron microscopy of the cells was normal. Associated findings included marked panhyperimmunoglobulinemia with an IgE of 2600-4300 ng/ml. Assays of the phagocytes' metabolic, phagocytic and bactericidal functions, T and B cell functions and complement system were normal. This black child with severe infections from birth, a markedly elevated IgE without allergic symptoms and defective movement of both PMN's and monocytes represents a congenital and possibly genetically determined abnormality of cell mobility.

Basophils in tuberculin and "Jones-Mote" delayed reactions of humans.

Jones-Mote reactions are delayed, erythematous, and mildly indurated cutaneous reactions originally described in humans sensitized by skin injection of heterologous proteins. Similar reactions in guinea pigs contain many basophils and are called cutaneous basophil hypersensitivity. In contrast, guinea pigs immunized with mycobacterial adjuvants have classical tuberculin-type delayed hypersensitivity reactions, which contain few basophils. This has led to a new classification of delayed responses, based largely on the presence or absence of basophils. We induced sensitization for Jones-Mote reactions in 20 normal humans by intradermal injections of keyhole limpet hemocyanin. Skin tests with KLH 1 wk later showed erythematous and indurated delyaed reactions in all subjects. Rebuck skin windows showed specific accumulations of basophils with a delayed time-course in 18 of 20 subjects. In 12 normals sensitized with oxazolone-keyhole limpet hemocyanin conjugates, skin reactions and in vitro lymphocyte stimulation showed carrier and not hapten specificity, suggesting that cutaneous responses were probably mediated by T cells. A comparative study of strongly positive PPD skin tests in patients with tuberculosis showed significant basophil accumulations in five of nine subjects. Thus, basophils occurred in human tuberculin and Jones-Mote reactions and were not a distinguishing feature of Jones-Mote reactions. We suggest that the occurrence of basophils at delayed reactions is under complex regulation and that basophil accumulations are an aspect of delayed hypersensitivity, rather than an indication of a distinctive and separate response.

Defective Polymorphonuclear Leukocyte Chemotaxis and Bactericidal Capacity in a Boy With Recurrent Pyogenic Infections

A 13-year-old boy with a history of recurrent pyogenic infections had abnormalities of polymorphonuclear leukocyte (PMN) function which probably accounted for his susceptibility to infection. PMN phagocytosis and nitroblue tetrazolium dye reduction were normal but glucose 14C oxidation was abnormally increased in resting cells. The patient's PMNs possessed decreased bactericidal activity against Staphylococcus aureus and Escherichia coli. Also documented were decreased PMN chemotactic activity and leukocyte accumulation in Rebuck skin windows. PMN random motility, PMN bone marrow reserve, PMN kinetics, lymphocyte blastogenesis, and delayed cutaneous hypersensitivity were normal. This patients represents another of the growing number of distinct granulocytopathies currently being recognized which may result in increased susceptibility to infection.

Defective neutrophil motility in children with measles

The random migration and chemotactic ability of neutrophils from ten patients with uncomplicated measles was found to be grossly impaired when compared to a group of normal children. Chemotaxis to endotoxin activated serum and to hydrolysed casein was markedly depressed, but serum from measles patients, when activated by endotoxin, generated and chemotactic activity and did not contain leukotactic inhibitors. The defect in neutrophil motility was confirmed in vivo by abnormal Rebuck skin windows. The defect was temporary, and recovery of normal chemotaxis was observed by the eleventh day after the onset of the rash.

Metabolic and functional studies of monocytes in congenital neutropenia.

Summary. Children with the severe form of congenital neutropenia usually die from infection by 2 yr of age. Metabolic and functional studies of monocytes from three patients with this disease were undertaken to understand further the role of monocytes in the host's defences against bacterial infection. The bactericidal capacity of monocytes compared favourably to the bactericidal capacity of control neutrophils (PMN). Metabolic studies of monocytes and control leucocytes showed similar stimulation of oxygen consumption, hexose monophosphate pathway activity arid 125iodide fixation during phagocytosis. Further, myeloperoxidase (MPO) activity of patients’monocytes was comparable to the activity of control leucocytes. Using the Rebuck skin window, very few of the patients’mononuclcar cells were seen to migrate during the initial 2–4 hr of observation. During this time, the PMN migration of controls was maximal. Following the relatively acellular period observed in patients, a brisk mononuclear cell infiltration occurred apparently independent of the presence of neutrophils. Epinephrine injection into patients stimulated a marked increase of blood monocytes, suggesting the existence of a marginal monocyte pool. An injection of hydrocortisone caused an unexplained 3–8‐fold decrease in circulating monocytes. The delay of phagocytic cell migration to an inflammatory site, and the apparent sensitivity of monocytes to hydrocortisone may account in part for the increased susceptibility to infection in patients with severe congenital neutropenia.