The role of radiotherapy in the management of primary cutaneous neuroendocrine tumors (Merkel cell or trabecular carcinoma): experience at the Peter MacCallum Cancer Institute (Melbourne, Australia).

Clinical presentation, treatment, and radiation response data are presented for 20 patients with primary cutaneous neuroendocrine tumors (Merkel cell or trabecular carcinoma). Thirty-six sites were irradiated, 26 sites were local recurrences after surgery or metastases, only 3 primary tumors were irradiated de novo. In 22 out of 23 sites (96%) a complete response of measurable tumor was observed and 1 partial response (4%), an overall response rate of 100%. Thirteen sites were irradiated prophylactically with no measurable disease present and no recurrences have been seen in these areas. There was only 1 recurrence in an irradiated site (after a low radiation dose). Forty percent (8/20) either had distant metastases at presentation (5 patients) or developed them after radiotherapy (3 patients) and five of these patients have died of metastatic disease. Follow-up time ranged from 1-77 months, and actuarial 5-year survival was 63%. In view of these findings we would advocate the wider study of primary radiotherapy after biopsy or excision biopsy for the primary lesion, and prophylactic nodal irradiation with the object of avoiding extensive surgery in these often elderly patients.

Simultaneous quantitation of cytokine mRNAs in interleukin-1 beta stimulated U373 human astrocytoma cells by a polymerisation chain reaction method involving co-amplification with an internal multi-specific control.

The measurement of cytokine mRNA levels is of fundamental importance in the understanding of diverse pathological states. We present a simplification of a polymerase chain reaction-based technique which permits the simultaneous measurement of up to 20 cytokine mRNAs, together with those of several other cellular products, including beta 2-microglobulin and beta-actin. The technique makes use of internal standards bearing multiple PCR primer sites which are identical to those on the mRNAs to be assayed. Known quantities of the standards are added to the cellular RNA and the mixture is co-reverse transcribed and co-amplified. The simplifications described here are based on the fact that each pair of amplicons accumulates in a constant ratio even in the plateau phase of amplification. As a result, no preliminary experiments to determine the limits of the exponential phase of amplification are necessary; the same number of cycles may be chosen for all the mRNAs to be measured, whatever their level in the mixture might be; pipetting errors are avoided since all calculations are based upon the relative quantities of co-amplified material. Here we illustrate the method through a quantitative study of the expression of cytokine mRNAs in U373 human astrocytoma cells before and after stimulation with IL-1 beta. Quantitation was carried out either by incorporating radioactivity in the amplicons or by fluorescence measurements after propidium iodide staining. Only very low numbers of transcripts for IL-6, IL-8, CSF-1, MCP-1 and either Gro alpha or Gro beta were detectable in unstimulated cells. The levels of these cytokine mRNAs increased dramatically following IL-1 beta stimulation and, in addition, transcription of IL-1 beta, TNF alpha, GM-CSF, G-CSF, Gro gamma and MCP-1, some of which have not previously been detected in U373, was initiated in the stimulated cells. At the same time we found that transcripts for IL-2, IL-3, IL-4, IL-5, IFN gamma, huMlP1 alpha and huMlP1 beta were totally absent in this cell line. These results suggest a potentially important role for astrocytes in the local amplification of inflammatory responses in the brain.

Molecular cloning of the MCP-3 chemokine gene and regulation of its expression.

We have isolated a cDNA (NC28) transcribed from a mRNA which is transiently induced in U937 promonocytic cells by PMA and super-induced by cycloheximide. NC28 cDNA encodes a new member of the chemokine family, MCP-3, recently purified from MG-63 osteosarcoma cells by Van Damme et al. [1]. The MCP-3 protein sequence shows 74% identity with human monocyte chemoattractant protein 1 (MCP-1) and, like MCP-1, recombinant MCP-3 protein shows chemotactic activity for monocytes but not for neutrophils. However the secreted MCP-3 protein differs from MCP-1 in being N-glycosylated. The 3' noncoding regions of MCP-3 and MCP-1 mRNAs are more diverged (44%), allowing specific cDNA probes to be made, and indicating that the two genes are evolutionarily distant. Sequence comparisons of the 3' noncoding regions suggest that MCP-3 may be the human homologue of the mouse MARC gene [2], and that MCP-1 and MCP-3 genes arose by a gene duplication event before the mammalian radiation. Both MCP-1 and MCP-3 mRNAs are expressed by PBMC, principally by monocytes, with MCP-1 mRNA being expressed at levels 2-4 times that of MCP-3 mRNA. However, while MCP-1 mRNA is also expressed at high levels in fibroblast or astrocytoma cell lines after IL-1 and TNF stimulation, MCP-3 mRNA is expressed only at very low levels in these cells. The cellular origin of MCP-3 is thus more restricted than that of MCP-1. In our experiments on PBMC, LPS is not a consistent inducer of MCP-1 and MCP-3 mRNAs. In some experiments, it actually decreases levels of these two mRNAs, while concomitantly increasing IL-6 and TNF-alpha mRNA levels. Levels of MCP-1 and MCP-3 mRNAs in PBMC are both increased by IFN-gamma, although IL-6 mRNA is not induced. They are also increased by PHA-P and are decreased, in most cases, by IL-13 [3]. MCP-1 and MCP-3 mRNAs are thus co-ordinately regulated in monocytes in response to a number of inducing or inhibitory agents, in a manner differing in several respects from that of other monokines such as IL-6.

Levamisole in patients with recurrent herpes infection.

The effect of the immunomodulating drug levamisole was tested in 33 patients with frequently recurring attacks of herpes labialis or herpes genitalis. All patients had suffered monthly recurrent attacks for at least six months, but were otherwise healthy. Patients were randomly allocated to receive levamisole tablets, 2.5 mg/kg orally, on two consecutive days each week for 26 weeks, or placebo tablets taken for a similar time. The tablets were reversed for a second consecutive six-month period. Seven of 21 patients (33%) with recurrent herpes genitalis infection showed complete response and 10 (47%) showed a partial response while receiving levamisole. Three of 21 patients (14%) showed a partial response on placebo. Six of 12 patients (50%) with herpes labialis showed complete or partial responses, with three partial responses on placebo. Frequent minor drug side effects were seen, and therapy was ceased in one patient. No episodes of leucopenia or agranulocytosis were encountered. Levamisole produces a significantly better reduction in frequency, duration and severity of herpes attacks than placebo, particularly after the initial eight weeks of administration.

Depressed exocytosis by rheumatoid neutrophils in vitro.

Polymorphonuclear leucocytes and monocytes have been isolated from the peripheral blood of patients with rheumatoid arthritis and healthy controls using Percoll gradient centrifugation. These cells have been tested for their ability to release superoxide anion and to undergo exocytosis following stimulation with heat-aggregated IgG. Monocytes from patients and controls react similarly in these assays but neutrophils from the patients release less beta-glucuronidase than do normal cells. This could not be related either to the expression of cell surface Fc receptors or to oxidative metabolism and does not appear to be an effect of the administration of non-steroidal anti-inflammatory drug therapy. It is suggested that rheumatoid neutrophils are not inherently defective but are less responsive to activation in vitro because of prior exposure to immune complexes in vivo.

Age-related changes in localization of injected radiolabelled lymphocytes in the lymph nodes of antigen-stimulated mice.

The localization of i.v. injected syngeneic lymph node cells, radiolabelled with 51Cr or 75Se-L-selenomethionine, was studied in male CBA/H mice aged between 3 and 30 months. The following results were obtained. (1) Localization of cells from young adult donors was greater in the s.c. lymph nodes of old than of young recipients, the main increase being between 15 and 17 months of age. Increases in lymph node weight and DNA-synthesis were also seen at this time; but the rise in cell localization was significant even when calculated per unit of tissue weight. Splenic localization either declined slightly with age or, like the liver, showed no significant change. (2) Local antigenic stimulation by a single injection of sheep erythrocytes into one front footpad, 24 hr before lymph node cell injection, resulted in increased localization in the regional lymph nodes of 3-17 month old, but rarely of 24-30 month old mice. (3) No consistent differences in localization were observed between lymph node cells from 4-month and 25-month old donors. Both age-related and antigen-related increases in cell localization were at least partly attributable to an enhanced rate of entry of lymphocytes from the blood to the lymph nodes. Although the changes underlying the decline in antigen-related localization of cells in old recipients have still to be clarified, it is probable that the defective immune responses of old mice result partly from this decline.

Specific immune response in human skin carcinoma.

Eight out of nine patients with squamous cell carcinoma of skin have shown immunological reactivity against their own tumour cells by one or more tests with their sera or peripheral blood lymphocytes. The tests included membrane and cytoplasmic immunofluorescence, and, with cultured tumour, complement-dependent serum cytotoxicity and lymphocyte attack. One case examined in depth had an unusually conspicuous lymphocyte and plasma cell reaction on histological examination, and was positive by all four tests; a time-lapse cinephoto-micrographic record over seven days was obtained of the attack on the carcinoma cells in culture by the patient's lymphocytes.

A human lymphocyte homing receptor, the hermes antigen, is related to cartilage proteoglycan core and link proteins.

Lymphocyte interactions with high endothelial venules (HEV) during extravasation into lymphoid tissues involve an 85-95 kd class of lymphocyte surface glycoprotein(s), gp90Hermes (CD44). We report here the cloning of cDNA for gp90Hermes expressed in a mucosal HEV-binding B lymphoblastoid cell line, KCA. Northern hybridization revealed the presence of three invariant RNA bands at 1.5, 2.2, and 4.5 kb in mucosal HEV-, lymph node HEV-, or dual-binding cells. The deduced amino acid sequence predicts a mature protein with a C-terminal cytoplasmic tail, a hydrophobic transmembrane domain of 23 amino acids, and an N-terminal extracellular region of 248 amino acids. A proximal extracellular domain is the probable region of O-glycosylation and chondroitin sulfate linkage and displays at least two of the three immunodominant epitope clusters of native gp90Hermes. A distal region contains the majority of potential N-glycosylation sites and cysteines, and exhibits a striking homology to tandemly repeated domains of the cartilage link and proteoglycan core proteins. No significant similarities were found to the immunoglobulin, integrin, or cadherin gene families. Thus gp90Hermes represents a novel class of integral membrane protein involved in lymphocyte-endothelial cell interactions and lymphocyte homing.