Deficient thromboxane synthesis and response in platelets from premature infants.

In vitro function of cord blood platelets from 35 premature infants (gestational age 32 +/- 3.2 wk) was compared with that of 12 full-term infants and 14 adult control subjects. In comparison with adult platelets, preterm platelets showed impaired aggregation, in response to thrombin, collagen, ADP, and U46619 [a stable analog of thromboxane A2 (TxA2)], and impaired [14C]serotonin secretion in response to collagen and U46619. The production of TxB2 (the stable TxA2 metabolite) in response to collagen was reduced in preterm platelets (30.2 +/- 5.5 ng/mL) compared with full-term (52.7 +/- 12.6 ng/mL) or adult control platelets (132.3 +/- 38.7 ng/mL). The deficient TxB2 production and U46619 response prompted further investigation of TxA2 receptor number and binding characteristics. Immunoblotting using an anti-TxA2 receptor antibody (anti-P2) identified a single, identical 55-kD band in solubilized membranes of control, full-term, and preterm platelets. Flow cytometry using anti-P2 produced histograms that did not differ between adults and neonates. Ligand binding studies using [3H]U46619 were carried out on 10 samples from each group. Scatchard analysis yielded a single class of binding sites with no significant difference among the Kd values (85 +/- 16 versus 99 +/- 12 versus 100 +/- 12 nM) or number of binding sites per platelet (1876 +/- 460 versus 2450 +/- 478 versus 2777 +/- 536) for preterm and full-term infants and adults. Therefore platelets of preterm infants show impaired TxA2 production and response. The poor response is not related to altered binding characteristics of the TxA2 receptor but may lie in the postreceptor signal transduction pathway.

The lysosomal granule membrane protein, LAMP-2, is also present in platelet dense granule membranes.

Lysosomal Associated Membrane Protein-2 (LAMP-2) is an inherent component of lysosomal granule membranes in diverse cell types, including platelets. We examined platelets for evidence of LAMP-2 in dense granule membranes as CD63 has previously been shown to be present in both lysosomal and dense granule membranes. Immunological techniques were used to examine the localization of LAMP-2 in control platelets and those from an individual with Hermansky-Pudlak syndrome (HPS), a condition characterised by platelet dense granule deficiency. Immunoblotting studies demonstrated that LAMP-2 was enriched in a dense granule preparation. Flow cytometry of thrombin-stimulated control platelets was consistent with biphasic surface expression of LAMP-2. The early expression was accompanied by dense granule, but minimal lysosomal granule, release. The late expression was accompanied by additional lysosomal granule release only. Thrombin stimulation of HPS platelets showed only late, lysosome-associated LAMP-2 expression. Immunoelectron microscopy indicated the presence of LAMP-2 in the membranes of serotonin-containing granules as identified by an anti-serotonin polyclonal antibody. These data indicate that LAMP-2 is present in the membranes of platelet dense granules in addition to lysosomal granules, and has a similar distribution to CD63.

Quantification of a novel dense granule protein (granulophysin) in platelets of patients with dense granule storage pool deficiency.

An antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) was developed for a novel protein granulophysin, a constituent of the platelet dense granule (DG) membrane and used to characterize patients with dense granule storage pool deficiency (delta-SPD). The assay uses two monoclonal antibodies against the protein, one of which is conjugated to peroxidase. Purified DGs, an enriched source of the protein, were used for the standard curve. Granulophysin levels were only low in forms of delta-SPD associated with albinism. Granulophysin levels in platelet homogenates of 30 patients with the Hermansky-Pudlak syndrome form of delta-SPD were 1/4 to 1/5 of levels in controls or obligate heterozygotes. Two patients with the Chediak-Higashi form of delta-SPD syndrome also had markedly reduced levels of granulophysin. Patients with other forms of delta-SPD had normal levels of granulophysin. Two sisters with delta-SPD in one family had normal granulophysin present in empty dense granule membrane vesicles. Three members of another family with delta-SPD had low DG counts but normal granulophysin levels, indicating that in this group the level of granulophysin was maintained despite the reduction in granule formation. Thus, granulophysin quantitation facilitates characterization of delta-SPD patients and may provide clues to the nature of defective granules in delta-SPD subtypes.

Deficient collagen-induced activation in the newborn platelet.

We have investigated the impaired secretion response of neonatal platelets. We compared the response of washed neonatal and adult platelets to thrombin and collagen, and to specific activators of calcium flux (inositol trisphosphate) and protein kinase C activation (oleoyl-acetyl glycerol). Neonatal platelets show no impairment of aggregation, secretion of [14C]serotonin or phosphorylation of specific intracellular proteins in response to thrombin, inositol trisphosphate, or oleoyl-acetyl glycerol. However, neonatal platelets have a markedly decreased response to collagen. To further evaluate this deficient response, we examined specific aspects of the collagen activation pathway. Collagen-platelet interaction as measured by adhesion of platelets to collagen-coated dishes showed no difference in adhesion of neonatal platelets compared to adult controls (20.1 +/- 11.6 versus 18.6 +/- 9.3%). The presence of GPIa/IIa, a Mg2(+)-dependent collagen receptor, was evaluated by flow cytometric analysis of binding of fluorescein-tagged monoclonal antibody, 6F1 (directed against GPIa/IIa). There was no difference either in the percent of platelets that bound antibody (80 versus 81%) or in the mean fluorescence intensity of the adult and neonatal samples. Phosphoinositide hydrolysis was decreased in neonatal platelets in response to collagen but normal in response to thrombin. Neonatal platelets released more arachidonic acid than adult platelets in response to thrombin (29.5 +/- 3.2 versus 19.6 +/- 1.8%) but less than adult platelets in response to 10 micrograms/mL collagen (3.2 +/- 1.1 versus 9.3 +/- 3.0%).(ABSTRACT TRUNCATED AT 250 WORDS)

Sex hormones influence growth and surfactant production in fetal lung explants.

Sex-related differences may be present during fetal lung growth and at the onset of surfactant synthesis. In this study we investigated the role of dihydrotestosterone (DHT) and estrogen (EST) on cell division and on labeled palmitate incorporation into disaturated phosphatidylcholine (DSPC) at various times of gestation. Using organ cultures of fetal rat lung from sexed littermates, it was shown that both DHT and EST reduce DNA synthesis only in tissue taken during the rapid growth phase from day 16 to 19 of gestation. From autoradiographic counts, epithelial cell division was most affected. Both hormones reduced DSPC synthesis in explants prepared at day 18, when levels are normally low. At day 19, DHT reduced palmitate incorporation into DSPC of female explants to the male level; subsequently DHT had no effect on any tissue. In contrast, the addition of EST stimulated DSPC synthesis 40% above controls in both male and female explants taken at day 20 only. The results suggest that sex differences seen in late fetal lung development may arise from the combined effects of slowed epithelial growth induced by these hormones followed by inhibition of DSPC synthesis by DHT and acceleration by EST at the crucial period when surfactant synthesis begins.

Tonsillar mapping of determinants found on normal lymphoreticular (T,B,K, immature and macrophage) and myeloblastic leukemia cells.

A monoclonal immunocytochemical method with 25 monoclonal antibodies was used to study the distribution in human tonsil of determinants expressed on T cells (mature and immature), Langerhans cells, B cells, killer/natural killer cells, macrophages, immature myeloid and lymphoblastic leukemia cells. Many of the respective determinants were found to have a discrete topographic distribution in normal reactive tonsil. The common acute lymphoblastic leukemia antigen and a determinant found on myeloblastic leukemia cells (My10) were not found in the specimens of the tonsil examined.

Diurnal stage of circadian rhythm of plasma zinc in healthy and psoriatic volunteers.

The characteristics of circadian variation of human plasma zinc concentrations in health and psoriasis are compared by the cosinor method. Sequential measurements of plasma zinc were performed at 0800 (fasting), 1000 (1 hr after breakfast), 1330 (1 hr after lunch), and 1900 (1 hr after supper) on groups of 20 patients with psoriasis, a disorder in which zinc metabolism may be disturbed, and 16 clinically healthy individuals. The limited sampling notwithstanding, groups showed a statistically significant circadian rhythm when analyzed by the cosinor method with the least-squares fit of a 24-hr cosine curve. The rhythm's timing in the clinically healthy group was similar to that derived by the same cosinor method from much denser series (covering the entire 24-hr span) published earlier by others. The diurnal portion of the circadian rhythm of human plasma zinc differed in the two groups investigated; this difference was not seen in the mean fasting plasma zinc concentrations. Circadian studies may reveal differences between groups that are not apparent by conventionally used single fasting estimates. More generally, in future studies of zinc, e.g., in skin disease, sampling time and the subjects' routine should be strictly standardized as a minimum, and rhythm characteristics should be quantified as an optimum.

Analysis of histiocytosis X infiltrates with monoclonal antibodies directed against cells of histiocytic, lymphoid, and myeloid lineage.

Surface markers expressed on histiocytosis X (HX) cells were studied using 18 monoclonal antibodies (McAbs) and an in situ indirect immunoperoxidase technique. Specimens of skin (five biopsies from three cases) and lymph node (one case) were studied. Our study confirmed previous findings of an OKT6+ HLA DR+ Leu 3a+ phenotype of HX cells in skin and indicated that lymph node HX cells can also have this phenotype. A mixture of cells expressing T markers/T subset, monocyte/macrophage, and killer/natural killer (K/NK) markers was also present in two cases. However, one case with an aggressive course showed no detectable HNK 1+ (K/NK) or Leu 2A (suppressor) cells in the cutaneous infiltrates and no detectable HNK 1+ lymphocytes in an affected lymph node. The significance of these findings in relation to prognosis and therapy is discussed.

Monoclonal antibody patterns in cutaneous lymphoid infiltrates. Their possible relevance to the diagnosis and categorization of cutaneous lymphomas.

The development of monoclonal antibodies to T cells at various stages of differentiation, B cells and histiocytes, suggests that these reagents may be of use in the diagnosis and categorization of cutaneous lymphomas. The findings to date in skin and blood are discussed.

Immunophenotype of lymphoid cells in positive patch tests of allergic contact dermatitis.

The immune phenotype of the infiltrating cells in 13 positive patch tests from 8 cases of contact dermatitis and 1 case of poison ivy was studied. An indirect immunoperoxidase technique was used in conjunction with monoclonal antibodies directed against mature T cells (Leu-1, T11), helper T cells (Leu-3A), suppressor/cytotoxic T cells (Leu-2A), killer and natural killer cells (HNK 1), B cells (B1), Langerhans cells (HLA-DR), and the common acute lymphoblastic leukemia antigen (CALLA), (J5). The majority of infiltrating mononuclear cells were Leu-1+, T11+, Leu-3A+, Leu-2A-, HLA-DR+, T9-, T10-, HNK-, B1-, J5-. Occasional T6+ cells were observed in the epidermis (including spongiotic microvesicles) and also isolated in the dermis and within the dermal mononuclear infiltrates. The phenotype was compared with cutaneous T-cell lymphoma, a disease in which contact allergy and antigenic persistence may play a role.

Monoclonal antibodies and cutaneous T cell lymphoma. Theoretical and practical considerations.

The T cell nature of mycosis fungoides and Sézary syndrome was established a decade ago. The recent advent of monoclonal antibodies to T cells and other lymphoid subpopulations has resulted in an explosion of knowledge on the biology of this group of disorders. These reagents have increased our knowledge on thymic differentiation status, pathogenesis, the phenotype of premalignant vs malignant lymphocytic infiltrates, the identification of other previously unrecognized cells within lymphomas, and the phenotype of circulating vs skin lymphocytes. Therapeutic applications may result. These new developments are discussed.

Immunoperoxidase examination of cutaneous infiltrates of mycosis fungoides and large-plaque atrophic parapsoriasis with OKT10.

A monoclonal antibody (OKT10), which was developed recently, reacts with pro-thymocytes, T cell acute lymphoblastic leukemia (ALL) cells, cells in normal bone marrow (including plasma cells), and activated T cells. Tissues from patients with cutaneous T cell lymphoma were studied for the presence of OKT10-reactive cells with the use of an indirect immunoperoxidase technic. OKT10-reactive cells were identified in three of eight cases of mycosis fungoides, one of two cases of Sézary syndrome, with an equivocal reaction in one of ten cases of large-plaque parapsoriasis and in one of seven positive patch tests (allergic contact dermatitis). The biologic and possible clinical implications are discussed.

OKT 9 reactivity in mycosis fungoides and large plaque (atrophic) parapsoriasis.

A monoclonal antibody OKT 9 which detects a determinant expressed by a variety of proliferating cell types has been recently developed. This antibody was used in conjunction with the immunoperoxidase technique to study the cutaneous lymphoid infiltrates of nine patients with mycosis fungoides, one patient with lymphomatoid papulosis, two patients with Sézary syndrome, and ten patients with large plaque atrophic parapsoriasis (a condition which may terminate in overt mycosis fungoides.) OKT 9 reactive cells were identified in all cases of mycosis fungoides examined, in one case of lymphomatoid papulosis, one of two cases of Sézary syndrome, and one of ten cases of large plaque atrophic parapsoriasis. These results suggest that further studies using OKT 9 should be performed to assess whether OKT 9 reactivity may be used as a prognostic marker in cutaneous lymphomas and prelymphomas.

Plasma zinc in psoriasis: relation to surface area involvement.

Plasma zinc, serum albumin and alkaline phosphatase (a zinc-dependent enzyme) were measured in thirty-five psoriatics and their age- and sex-matched controls. No significant difference was seen between these two groups as a whole, but psoriatics with less than 10% surface involvement had significantly higher mean plasma zinc levels than their respective control group. Psoriatic patients also showed a relationship between the extent of surface involvement and the plasma zinc level, those with more extensive involvement having lower levels than those with minimal involvement. The differences were not due to disturbances in serum albumin, and no abnormalities in serum alkaline phosphatase were observed.

Demonstration of cells bearing the OKT6 determinant in human tonsil and lymph node.

An immunoperoxidase study was carried out on human tonsil (15 specimens) and human lymph node (5 specimens) using OKT6, a monoclonal antibody which was raised against a determinant on immature thymocytes. OKT6-positive cells were identified in the crypt epithelium of all tonsils examined and in occasional clusters in the interfollicular areas of two lymph nodes. OKT6 has recently been shown to react with epidermal dendritic cells (Langerhans' cells). This study confirms that OKT6-reactive cells may be found outside the thymus. The pattern of staining obtained suggests that OKT6 reactivity belongs to a dendritic subpopulation. The significance of the finding in relation to physiology and pathology is discussed. These physiological findings may also be relevant to the immunotherapy of T-cell lymphomas.

Demonstration of OKT 6-reactive cells in mycosis fungoides.

Infiltrates of five cases of mycosis fungoides (MF) were studied for the presence of cells reactive with a monoclonal antibody, OKT 6, which detects an antigen present on relatively immature thymocytes and Langerhans cells. In situ immunohistochemical staining was used for their demonstration. OKT 6-reactive cells formed a definite component of the dermal infiltrates of all patients examined. In three patients who showed numerous Pautrier microabscesses on routine histologic examination, OKT 6-positive cells were found to form a component of these abscesses. OKT 6-reactive cells have also recently been shown to be present in varying numbers in the dermal infiltrates of large plaque (atrophic) parapsoriasis (LPAP), a condition which may terminate in MF. The significance of these findings is discussed.

In situ immunologic phenotyping of mycosis fungoides.

In situ immunologic phenotyping of the cutaneous lymphoid infiltrates of five cases of mycosis fungoides (MF) was performed. This was done by the immunoperoxidase technic using monoclonal antibodies directed against normally expressed by mature peripheral T cells and T cells subsets. A significant proportion of the infiltrates reacted with peripheral T cell antibodies. In addition, "helper" predominance was demonstrated. However, cells reacting positively with "suppressor" antibodies were also demonstrated in three cases, indicating immunologic heterogeneity of the infiltrates. The presence of a significant component of the infiltrates yielding an equivocal or negative reaction with the mature T cell antibodies, especially in the infiltrates of one case which had a fatal outcome, suggests further heterogeneity with the presence of immature cells. Future investigation with monoclonal antibodies directed against immature T cell differentiation antigens will be of interest.