M. De Ley

Human urinary protein 1: evidence for identity with the Clara cell protein and occurrence in respiratory tract and urogenital secretions.

Protein 1 (P1), a low mol mass urinary protein of unknown function, has been purified, sequenced and quantified in human biological fluids. The molecular size, subunit composition and partial amino acid sequence of P1 are similar to those of the 10 kDa Clara cell protein (CC10), a lung secretory protein. P1 is found in high concentrations in sputum, bronchoalveolar lavages, urine and semen of healthy individuals and in urine of some pregnant women. Contrary to what is claimed, P1 or CC10 is not a specific and unique product of the lung, but like its homologue in rabbits (uteroglobulin) it is also present in urogenital secretions. P1 or CC10 may act as a natural immunosuppressor protecting the respiratory and urogenital tracts from unwanted inflammatory reactions.

Distribution of metallothionein in normal and pathological human skin

The expression and distribution of metallothionein (MT) in frozen sections of normal and pathological human skin was studied using the monoclonal antibody L2E3 directed against MT derived from human fetal liver. Immunohistochemical staining of normal fetal and adult skin revealed strong reactivity in basal keratinocytes of epidermis and outer hair root sheath, hair matrix cells and the secretory coil, but not the exocrine portion of eccrine glands; myoepithelial cells around apocrine sweat glands were similarly stained. In epidermal hyperplasia, variable numbers of suprabasal keratinocytes were stained, whereas in interface dermatitis, interrupted staining was found in the basal layer. Weak or scattered staining was observed in squamous tumours, whereas basal cell carcinomas did not show consistent staining. The distribution of MT in normal skin was in line with the germinative role of basal keratinocytes and hair matrix cells, whereas its distribution in hyperplastic epidermis was in line with experimental animal data, and reflected the increase in the germinative pool in these conditions. It is concluded that monoclonal antibody L2E3 may serve as a valuable immunohistochemical marker in diagnostic cutaneous pathology since it labels basal keratinocytes selectively, and since it discriminates between eccrine and apocrine sweat glands.

Purification of human fibroblast interferon by zinc chelate chromatography.

Human interferon was prepared by superinduction of cultures of either diploid embryonic skin and muscle cells or of the osteosarcoma cell line MG-63. The interferon so obtained was concentrated and partially purified by adsorption to controlled pore glass (CPG) beads at neutral pH and desorption by glycine-HCl buffer at pH 2. After neutralization, this interferon was applied to a column of zinc chelate which was eluted with buffers of decreasing pH. Most of the proteins eluted ahead of the interferon activity, which itself eluted in two distinct peaks. The first peak occurred in the effluent fractions around pH 5.9, and the second one in fractions around pH 5.2. The interferon found in fractions of pH 5.9 contained 5% of the original contaminating proteins. In contrast, the amount of total protein in the pH 5.2 peak was so small that it could not accurately be assayed by the fluorescamine method. Consequently, the interferon in the peak fraction was estimated to have a specific activity of about 2 x 10(9) units/mg. This material was radiolabelled and analysed by electrophoresis. A major peak of about 22000 mol. wt. with only minor contaminating proteins appeared on the autoradiographs. The total recovery of the zinc chelate chromatographical procedure was nearly 100%, and the interferon recovered from each peak behaved consistently on rechromatography. Fibroblast interferon produced by most diploid cells contained less than 10% of the variant eluting at pH 5.9. MG-63 cells and high-passage cultures of some diploid cell strains produced up to 50% of this variant.

The preservation of haemocyanin under carbon monoxide.

The carbon monoxide combining capacity of haemocyanin has been in dispute for a long time. Root [I] concludes that binding occurs with Limulus haemocyanin from the solubility increase in serum of carbon monoxide by the presence of haemocyanin, and from the shift to the right of the oxygen dissociation curve. Rawlinson [2], on the contrary, does not find a difference in solubility of carbon monoxide between water and a solution’ of Palinurus vulgaris haemocyanin and consequently assumes no combination. Using r4C-labelled carbon monoxide, Vanneste and Mason [3] could demonstrate the binding of carbon monoxide by Cancer magister haemocyanin. We shall prove the formation of a compound with Helix pomatia haemwyanin by studying the competition between carbon monoxide and oxygen for the copper groups, and by measuring the solubility of carbon monoxide in a buffer with and without haemocyanin. During storage haemocyanin tiiidergoes an ageing process, consisting in a decrease of the absorbance at 346 nm [4] and a conversion of a sigmoidal to a hyperbolic oxygen dissociation curve. We shall try to prevent this transformation by storage under carbon monoxide, a method currently used for haemoglobin [5].

Interferon induction and action in human lymphoblastoid cells infected with measles virus

Virus replication, cytopathic effect and interferon production were measured in Namalva lymphoblastoid cells infected with measles viruses. Eight virus strains of different origin or passage history were compared. An inverse relationship seemed to exist between the abilities of strains to induce interferon and to replicate to high titres. Two representative strains were found to be highly sensitive to lymphoblastoid cell interferon, when tested in a line of monkey kidney cells (Vero). In contrast, Namalva cells were found to be highly insensitive to autologous interferon when challenged with measles or Semliki Forest virus (SFV).

An interferon-beta-like or interferon-inducing protein released by mitogen-stimulated human leukocytes.

Human peripheral blood leukocytes were treated with concanavalin A (Con A) to produce interferon gamma (HuIFN-gamma). On gel filtration this interferon eluted as a protein with a molecular weight of 45000. In addition to this, the culture supernatant contained an interferon-like protein of apparent molecular weight 22000 (22K factor). The antiviral activity of this protein was neutralizable by a highly specific antibody to HuIFN-beta. Yet, the 22K factor differed from classical HuIFN-beta in several characteristics: lack of activity on certain homologous and heterologous cells which are sensitive to HuIFN-beta; lack of affinity for zinc-chelate and Con A-Sepharose columns; failure to bind to an anti-HuIFN-beta antibody column. Moreover, a specific antiserum raised against the 22K factor did not neutralize HuIFN-beta. Two alternative explanations of these findings are proposed: (i) the 22K factor is an interferon whose molecular structure resembles that of the known HuIFN-beta but it is not identical to it, or (ii) the 22K factor is not an interferon but a protein that can induce the production of HuIFN-beta in certain lines of fibroblastoid cells.

Interleukin 1: amino acid sequencing reveals microheterogeneity and relationship with an interferon-inducing monokine

1 Doucet-Jaboeuf, M., Pelegrin, A., Sizes, M., Guillemain, J. and Bastide, M. (1985) Int..]. ImmunopharmacoL 7,312 2 Daurat, V., Sizes, M., Pelegrin, A., DoucetJaboeuf, M., Guillemain,J. and Bastide, M. (.June 1985) Symposium Marqueurs Inflam., Lyon, France 3 Doucet-Jaboeuf, M., Guillemain, J., Piechaczyk, M., Karouby, Y. and Bastide, M. (1982) C.R. Acad. Sci. 295,283-286 4 Doucet-Jaboeuf, M., Pelegrin, A., Cot, M. C., Guillemain, J. and Bastide, M. Abstract~ Soc. Fr. ImmunoL, May 1984 5 Mac Murray, J., Barker, J., Armstrong, J., Bozzetti, L. and Kuhn, I. (1983)Life Sci. 32, 2363-2370 6 Reinberg, A., Schuller, E., Delasnerie, N., Clench, J. and Helary, M. (1977) La Nouv. PresseMed. 6, 3819-3823 7 Doueet-Jaboeuf, M., Pelegrin, A., Cot, M. C., Guillemain, J. and Bastide, M. (1984) Ann. Rev. Chronopharm. 1,231-234

Analysis of the interferon system in African patients with acquired immunodeficiency syndrome.

Serum interferon and in vitro production of alpha and gamma interferon by peripheral blood leucocytes were examined in 21 African patients with acquired immunodeficiency syndrome (AIDS) and in 15 African patients with AIDS-related complex. Interferon was detected in the serum of 44 % of the patients with AIDS-related complex and in 70 % of the patients with full-blown AIDS, and was characterized as an acid-labile alpha interferon. When compared to healthy blood donors, the interferon response of peripheral blood leucocytes to Newcastle Disease virus was impaired in 7 of 12 patients with AIDS-related complex and in 16 of 20 AIDS patients (p<0.005). Also, production of gamma interferon following stimulation with phytohaemagglutinin was diminished in 5 of 11 patients with AIDS-related complex and in 13 of 17 patients with AIDS (p<0.005). A high correlation was observed between the presence of circulating interferon and decreased in vitro production of gamma interferon, but not of alpha interferon. These results suggest that the impairment of in vitro production of gamma interferon can be used as a preclinical marker of AIDS.

Interferon therapy in multiple myeloma: Failure of human fibroblast interferon administration to affect the course of light chain disease

Abstract Fibroblast interferon at a dosage of 28 × 10 6 U/wk failed to influence disease progression in a preterminal case of therapy-resistant light chain myeloma. In a second case, that had not previously been treated, a first course of fibroblast interferon ( 30 × 10 6 U/wk ) associated with corticosteroids remained without effect. In this patient subsequent leukocyte interferon treatment was associated with a decrease in urinary light chain excretion and normalization of calcaemia, all other parameters remaining unaltered. A third patient with light chain disease was resistant to chemotherapy ab origine . None of the disease parameters responded to either fibroblast or leukocyte interferon therapy ( 21 × 10 6 U/wk ). During therapy a downward trend occurred in the relative number of lymphocytes characterizable as B and T cells. Mitogenic reactivity remained unchanged except for a downward trend, during fibroblast interferon therapy, in reactivity to PHA and Con A after 6 days culturing. Spontaneous (background) mitogenesis upon culture showed an upward trend.

Distribution of interferon-gamma receptors in normal and psoriatic skin.

Summary Recent data suggest that imbalances in production and secretion of cytokines, in particular interferon-gamma (IFN-γ), may be crucial in the pathogenesis of psoriasis. In order to exert its role on target cells, IFN-γ has to interact with a specific cell membrane receptor termed the IFN-γ-receptor (IFN-γR). We studied the distribution of IFN-γRs in frozen skin biopsies from 25 psoriatics and 5 normal controls with two unrelated monoclonal antibodies, and compared its distribution with that of the IFN-γ-inducible HLADR- and ICAM-1 antigens. In normal skin, IFN-γRs were restricted to the basal cell layer; weak staining was found on scattered mononuclear cells in the papillary dermis. In 13/25 active Psoriatic lesions, additional suprabasal immunoreactive foci, and in 5/25 cases, diffuse immunoreactivity of the entire epidermis were seen. No striking topographical similarities between the site and number o f IFN-γR+, HLADR+ and ICAM-1 + keratinocyte foci were observed, suggesting that cytokines other than IFN-γ induce HLADR-antigens on Psoriatic keratinocytes in vivo. The restricted distribution o f IFN-γR on the germinative cell layer in normal skin confirms the role played by IFN-γ in the normal growth regulation o f the epidermis. The de novo suprabasal expression o f IFN-γR in psoriasis argues against the current hypothesis that IFN-γR are down-regulated due to a local excess o f IFN-γ or transforming growth factor alpha (TGF-α). Whether IFN-γRs in psoriatic skin are functionally normal and involved in signal transmission, remains to be studied.