Frank C. Schmalstieg

Abnormalities of polymorphonuclear leukocyte function associated with a heritable deficiency of high molecular weight surface glycoproteins (GP138): common relationship to diminished cell adherence.

Investigations of polymorphonuclear leukocyte (PMN) function were performed in a 5-yr-old white female with delayed umbilical cord separation, impaired pus formation, and a severe defect of PMN chemotaxis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated an almost total deficiency of a high molecular weight glycoprotein(s) (GP138) in the granule and membrane fractions of the patients cells, and NaB3H4-galactose oxidase labeling demonstrated the absence of a major glycoprotein complex on the surface of her PMNs. Monoclonal antibodies (MAb) were employed in flow cytometry experiments to demonstrate that two previously characterized glycoproteins (Mo1 and LFA1) were undetectable on the surface of the patients PMNs and monocytes. Immunoprecipitation of 125I-labeled patient cells with subunit specific MAbs confirmed that the alpha-subunits of Mo1 (155 kD) and LFA1 (177 kD) and their common beta-subunit (94 kD) were totally deficient. Functional analyses of patient PMNs demonstrated severe impairment of adherence- and adhesion-dependent cell functions including spreading, aggregation, orientation in chemotactic gradients, antibody-dependent cellular cytotoxicity, and phagocytosis of particles (Oil-Red-0-paraffin, zymosan) selectively opsonized with C3-derived ligands. Patient PMNs demonstrated a normal capacity to rosette with IgG or C3b-coated sheep erythrocytes, but rosette formation with C3bi-coated erythrocytes was profoundly diminished. Adhesion-independent functions including shape change, N-formyl-methionyl-leucyl-3H-phenylalanine binding, and O-2 generation or secretion elicited by soluble stimuli were normal. Membrane fluidity, surface charge, and microtubule assembly were also normal. These findings provide new evidence that critical PMN surface glycoproteins are required to facilitate multiple adhesion-dependent cellular functions of the inflammatory response.

Interleukin-10 in human milk.

ABSTRACT: The concentrations of immunoreactive IL-10 in the aqueous fraction of 20 specimens of human milk obtained during the first 80 h of lactation and stored at –60°C ranged from 66 to 9301 pg/mL (mean ± SD, 3304 ± 3127 pg/mL). IL-10 was present also in the lipid layer of milk. Gel filtration revealed that IL-10 was located in a high molecular weight fraction, where certain other cytokines in human milk have been found. In addition, immunoreactive IL-10 in milk increased after treatment with sodium taurocholate. Bioactive IL-10 was demonstrated by the finding that human milk inhibited [3H]thymidine uptake by human blood lymphocytes and that inhibition was partly overcome by concomitant incubation with antibodies to human IL-10. IL-10 mRNA but no protein product was found in cultured human mammary epithelial cells. Some IL-10 was associated with preparations of human milk leukocytes, but the data did not suggest that the cells were producing the cytokine. Bioactive IL-10 in a possible protected compartment suggests that IL-10 in human milk may have immunomodulating, antiinflammatory effects on the alimentary tract of the recipient infant.

Heparin nebulization attenuates acute lung injury in sepsis following smoke inhalation in sheep.

Pseudomonas pneumonia is a common complication of smoke inhalation injury. Airway casts formed from clotted mucous occur frequently in this condition. A recent report shows that intravenous heparin improves oxygenation and reduces lung damage in a sheep model of smoke inhalation. We hypothesized that nebulized heparin could be an effective means of reducing cast formation. Female sheep (n = 19) were surgically prepared for a study of acute lung injury (ALI). After a tracheotomy, 48 breaths of cotton smoke (<40°C) were inflated into the airway. Afterwards, live Pseudomonas aeruginosa (5 × 1011 CFU) was instilled into the lung. All sheep were mechanically ventilated with 100% O2 and were divided into four groups: a heparin-nebulized group (n = 5; animals received aerosolized heparin [10,000 I.U.] 1 h after the bacterial instillation and subsequently every 4 h thereafter), an intravenous heparin group (n = 5,300U/kg/23 h, infusion was started 1 h after the injury), a saline-nebulization group (n = 5; animals received inhaled nebulized saline), and a sham injury group (n = 4, treated in the same fashion, but no injury). The animals were sacrificed after 24 h of mechanical ventilation, and lung samples were harvested. Sheep exposed to lung injury presented with typical hyperdynamic cardiovascular changes and a corresponding drop in PaO2. These changes were significantly attenuated in the heparin groups. Histological changes consisting of cellular infiltrates, lung edema, congestion, and cast formation were reduced by heparin. These data suggest that nebulized inhaled heparin is a beneficial therapy for sepsis-induced ALI.

Tumor necrosis factor-α in human milk

ABSTRACT: We previously demonstrated that certain biologic activities in human milk were partially blocked by antibodies directed against human tumor necrosis factor-α (TNF-α). In this study, immunochemical methods were used to verify the presence of TNF-α in human milk obtained during the first few days of lactation. Gel filtration revealed the presence of TNF-α by RIA in molecular weight fractions between 80 and 195 kD. TNF-α could not be detected consistently by conventional Western blotting or cytotoxic assays. Although immunoreactive bands were detected by a Western blot-125I protein A technique in TNF-α-positive fractions from gel filtration, those bands proved to be nonspecific. TNF-α in milk was reliably quantified by the competitive RIA. Those studies revealed that the concentrations of TNF-α in milk were 620 ± 183 pg/mL. Although RNA to TNF-α was detected in milk leukocytes by Northern blotting, little TNF-α was found in those cells before or after stimulation with N-formyl-l-methionyl-l-leucyl-l-phenylalanine or 4β-phorbol-12β-myristate-13α-acetate. The origin of this cytokine in human milk remains unclear. Nevertheless, this study suggests that TNF-α is present in early human milk in sufficient quantities to exert possible biologic effects upon the mammary gland of the mother or the immune system of the infant.

Cytokines in human milk: Properties and potential effects upon the mammary gland and the neonate

Epidemiologic and immunologic studies of breastfed and nonbreastfed infants and investigations of certain biologic activities in human milk led to the identification of immunomodulating agents in human milk. Among them were the cytokines interleukin-1β (IL-1β); IL-6, IL-8, IL-10, granulocyte-colony stimulating factor, macrophage-colony stimulating factor (M-CSF), tumor necrosis factor-α, interferon-γ, epithelial growth factor (EGF), transforming growth factor-α (TGF-α), and TGF-β2. Inteferon-γ may originate from T cells in milk; EGF, TGF-α, TGF-β, M-CSF, IL-6, and IL-8 may be produced by mammary gland epithelium. Based upon their known functions, we hypothesize that cytokines influence the development and immunologic function of the mammary gland and the neonate. Thosein vivo functions remain to be defined by future investigations.

Production of interleukin-6 and interleukin-8 by human mammary gland epithelial cells

The production of transforming growth factor-beta 2 (TGF-beta 2), interleukin-1 beta (IL-1 beta), IL-6, IL-8, and tumor necrosis factor-alpha (TNF-alpha) by spontaneously immortalized human mammary gland epithelial cells of non-malignant origin and the effect of prolactin upon the production of those cytokines were investigated. Cells were cultured on plastic with epithelial growth factor, insulin, and hydrocortisone. Cytokines were quantified by enzyme-immunoassays. The cells produced IL-6 and IL-8, but no detectable TGF-beta 2, IL-1 beta, or TNF-alpha. Although prolactin enhanced the uptake of [3H]thymidine, it did not alter the production of cytokines/interleukins. Because of the marked production of IL-8 by mammary epithelium and a past report of TGF activity in human milk, those agents were quantified in human milk. The mean +/- S.D. concentrations of IL-8 and TGF-beta 2 in human milk obtained in the first 3 days of lactation were 3684 +/- 2910 and 130 +/- 108 pg/ml, respectively. Thus, IL-8 and TGF-beta 2 are normal constituents in human milk, and human mammary gland epithelium may be responsible for producing some of the IL-6 and IL-8 in human milk.

Analysis of Adenosine and Other Adenine Compounds in Patients With Immunodeficiency Diseases

Abstract Adenine compounds can be measured in picomole amounts using liquid chromatography of the fluorescent 1, N6-etheno derivatives. The limit of detection for the etheno derivatives in tissue extracts, however, is tissue-dependent due to interference by nucleotides and fluorescent components which are normally present. Prior to derivatization nucleotides were partially removed from extracts of lymphocytes and erythrocytes by treatment with Dowex AG1-X2 anion exchange resin. Samples were analyzed using either a Partisil PXS 10/25 SCX column eluted with 100 mM NH4H2PO4, pH 4.5, at a flow rate of 2 ml/min; or using two μBondapak/C18 reversed-phase columns eluted with 5 mM KH2PO,4:25% methanol (V/V) pH 7.5, at a flow rate of 1 ml/min. Adenosine was found to be 0.07 nmole/ml in normal adult human plasma. The urine of a child with severe combined immunodeficiency disease associated with absence of adenosine deaminase contained a normal amount of adenosine (5–6 nmole/ml), but contained a high level (∼60 nmol...

Missense mutation in exon 7 of the common gamma chain gene causes a moderate form of X-linked combined immunodeficiency.

Clinical and immunologic features of a recently recognized X-linked combined immunodeficiency disease (XCID) suggested that XCID and X-linked severe combined immunodeficiency (XSCID) might arise from different genetic defects. The recent discovery of mutations in the common gamma chain (gamma c) gene, a constituent of several cytokine receptors, in XSCID provided an opportunity to test directly whether a previously unrecognized mutation in this same gene was responsible for XCID. The status of X chromosome inactivation in blood leukocytes from obligate carriers of XCID was determined from the polymorphic, short tandem repeats (CAG), in the androgen receptor gene, which also contains a methylation-sensitive HpaII site. As in XSCID, X-chromosome inactivation in obligate carriers of XCID was nonrandom in T and B lymphocytes. In addition, X chromosome inactivation in PMNs was variable. Findings from this analysis prompted sequencing of the gamma c gene in this pedigree. A missense mutation in the region coding for the cytoplasmic portion of the gamma c gene was found in three affected males but not in a normal brother. Therefore, this point mutation in the gamma c gene leads to a less severe degree of deficiency in cellular and humoral immunity than that seen in XSCID.

Interleukin-6 in human milk

Interleukin-6 (IL-6) in human milk collected during the first 2 days of lactation was investigated by a competitive radioimmunoassay (RIA) and column chromatography. The concentrations of IL-6 in the aqueous phase of fresh human milk were 151 +/- 89 pg/ml. The concentrations of IL-6 in milk increased after storage at 4 degrees C and decreased after storage at -20 degrees C (P < 0.01). Column chromatography revealed two molecular weight peaks of IL-6 in human milk, the first > or = 100 kDa and the second between 25 and 30 kDa. The 25-30-kDa peak corresponded to known isoforms of human IL-6 and to the elution pattern for 125I-labeled recombinant human IL-6, whereas the higher molecular weight peak may be in keeping with a bound or compartmentalized form of that cytokine. The precise molecular forms of this protein, the compartmentalization of or binding proteins for this cytokine and the in vivo effects of IL-6 in human milk upon the mammary gland or the recipient infant remain to be explored.

Activated neutrophils and neutrophil activators in human milk: Increased expression of CD11b and decreased expression of L-selectin

Human milk neutrophils and macrophages were examined by flow cytometry to determine whether they displayed phenotypic markers of activation. The markers were CDllb and L‐selectin, which are increased or shed, respectively, from the surface of activated neutrophils. Phenotypic features of milk neutrophils and macrophages were similar to blood neutrophils stimulated with fMLP: plasma membrane expression of CDllb was increased and L‐selectin was decreased. After blood neutrophils were incubated in acellular milk, their expression of CDllb increased and L‐selectin decreased. The activation was not affected by trypsin but was significantly decreased by treating acellular milk with chloroform or ether. Sedimentation studies suggested that particulate fractions of milk were active. Further, the activation was partly blocked by treating target blood neutrophils with cytochalasin B. Thus, human milk neutrophils are activated, and the activation may be due partly to phagocytosis of membranous structures in milk.