Survival in early breast cancer patients is favorably influenced by a natural humoral immune response to polymorphic epithelial mucin.

PURPOSE: Polymorphic epithelial mucin (PEM or MUC1) is being studied as a vaccine substrate for the immunotherapy of patients with adenocarcinoma. The present study analyzes the incidence of naturally occurring MUC1 antibodies in early breast cancer patients and relates the presence of these antibodies in pretreatment serum to outcome of disease. MATERIALS AND METHODS: We measured immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to MUC1 with an enzyme-linked immunoassay (PEM.CIg), which uses a MUC1 triple-tandem repeat peptide conjugated to bovine serum albumin, in pretreatment serum samples obtained from 154 breast cancer patients (52 with stage I disease and 102 with stage II) and 302 controls. The median disease-specific survival time of breast cancer patients was 74 months (range, 15 to 118 months). A positive test result was defined as MUC1 IgG or IgM antibody levels equal to or greater than the corresponding rounded-up median results obtained in the total breast cancer population. RESULTS: A positive test result for both MUC1 IgG and IgM antibodies in pretreatment serum was associated with a significant benefit in disease-specific survival in stage I and II (P =.0116) breast cancer patients. Positive IgG and IgM MUC1 antibody levels had significant additional prognostic value to stage (P =.0437) in multivariate analysis. Disease-free survival probability did not differ significantly. However, stage II patients who tested positive for MUC1 IgG and IgM antibody and who relapsed had predominantly local recurrences or contralateral disease, as opposed to recurrences at distant sites in the patients with a negative humoral response (P =.026). CONCLUSION: Early breast cancer patients with a natural humoral response to MUC1 have a higher probability of freedom from distant failure and a better disease-specific survival. MUC1 antibodies may control hematogenic tumor dissemination and outgrowth by aiding the destruction of circulating or seeded MUC1-expressing tumor cells. Vaccination of breast cancer patients with MUC1-derived (glyco)peptides in an adjuvant setting may favorably influence the outcome of disease.

Vasopressin gene expression is attenuated in the fetal Brattleboro rat.

Due to a genetic defect the homozygous Brattleboro rat is unable to synthesize vasopressin gene products but still transcribes a mutant vasopressin mRNA from the gene. To study the influence of vasopressin gene products on the development of vasopressin gene expression, vasopressin mRNA levels of the supraoptic and paraventricular nucleus were measured at fetal day 20, postnatal day 1, 15 and 30 in the Wistar rat and in the heterozygous and homozygous Brattleboro rat by Northern blot analysis and in situ hybridization. In the homozygous Brattleboro rat of fetal day 20 and postnatal day 1, no or minute amounts of vasopressin mRNA were detectable but vasopressin mRNA was readily detectable at postnatal day 15 and 30. The Wistar rat and heterozygous Brattleboro rat had abundant vasopressin mRNA at fetal day 20 with increasing amounts towards postnatal day 30. The results indicate that vasopressin gene expression in the development of the homozygous Brattleboro rat is attenuated, possibly due to the absence of vasopressin gene products.

Studies on the presence of vasopressin, oxytocin and vasotocin in the pineal gland, subcommissural organ and fetal pituitary gland: failure to demonstrate vasotocin in mammals.

The demonstration of vasotocin in the mammalian pineal gland, subcommissural organ and fetal pituitary gland by bioassay has led to hypotheses regarding the function of this hormone in various reproductive processes. Preliminary examinations of the pineal gland and subcommissural organ with a specific radioimmunoassay failed to show vasotocin immunoreactivity. The presence of vasotocin, vasopressin and oxytocin in the pineal gland, subcommissural organ and fetal neurohypophysis was therefore investigated, using three specific radioimmunoassays. Frog and chicken pituitary glands were used to validate the vasotocin radioimmunoassay. Direct measurements in diluted homogenates of pituitary glands from frogs, chickens, mid-term fetal sheep and near-term fetal seals revealed the presence of vasotocin only in the frog and chicken pituitary glands, while vasopressin and oxytocin were found in the two fetal pituitary homogenates. Vasopressin and ocytocin were measured in homogenates of rat and bovine pineal glands and in preparations of the subcommissural organ of rats and rabbits after extraction with Vycor glass powder, but no specific vasotocin immunoreactivity was observed. These results indicate a discrepancy between the reported biological activity of vasotocin in the pineal gland, subcommissural organ and fetal pituitary gland and the immunoreactivity of this material, which can at present only be explained by the presence of a peptide which is structurally closely related to, but not identical with, vasotocin.

Ontogeny of vasopressin and oxytocin binding sites in the brain of Wistar and Brattleboro rats as demonstrated by lightmicroscopical autoradiography.

Vasopressin (VP) and oxytocin (OT) binding sites were localized and quantified in the developing brain of the Wistar, heterozygous (Het) and homozygous (Hom) VP-deficient Brattleboro rat using an autoradiographical technique. VP binding sites could be demonstrated from prenatal day 20 onwards in the septum and in the lateral reticular nucleus. Between this and postnatal day 15, VP binding sites appeared in all other brain areas known to contain VP binding sites in adulthood. In the caudate putamen the regional distribution of VP binding changed during development, while in some areas, for instance, the dorsal hippocampus and post cingulate cortex, the concentration of binding sites increased early but decreased with age. Comparison of VP binding between Het and Hom rats showed significant differences in the lateral reticular nucleus during development. Moreover, at postnatal day 15 there was more VP binding in the anterior commissural and suprachiasmatic nucleus and less in the central amygdala, dorsal hippocampus and post cingulate cortex of the Hom rat. This study shows, for the first time, OT binding sites in the developing rat brain. There is a considerable overlap with VP binding in the brain, sometimes with the same developmental pattern, e.g. in the anterior olfactory nucleus and caudate putamen and sometimes with a later appearance, e.g. in the central amygdala and thalamic nuclei. However most areas with VP binding sites did not show OT binding. In some areas only OT binding sites were present, for instance in the islands of Calleja and ventromedial hypothalamus. Similar to some areas with VP binding, OT binding decreased between postnatal day 5 and 15 in the dorsal hippocampus and even completely disappeared in the parietal cortex. The existence of VP binding sites in the Hom rat, together with the only occasional relationship between the previously described ontogeny of VP and OT innervation of the brain and the presently described developmental course of binding sites, indicates that the early expression of binding sites is not initiated by endogenous ligand. However, the setting of the number of VP binding sites has probably been affected by the VP deficiency of the Hom Brattleboro rat.

MUC1 (EMA) is preferentially expressed by ALK positive anaplastic large cell lymphoma, in the normally glycosylated or only partly hypoglycosylated form.

AIMS: To investigate whether MUC1 mucin, a high molecular weight transmembrane glycoprotein, also known as epithelial membrane antigen (EMA), differs in its expression and degree of glycosylation between anaplastic large cell lymphoma (ALCL) and classic Hodgkin's disease (HD), and whether MUC1 immunostaining can be used to differentiate between CD30 positive large cell lymphomas. METHODS/RESULTS: Using five different monoclonal antibodies (E29/anti-EMA, DF3, 139H2, VU-4H5, and SM3) that distinguish between various MUC1 glycoforms, high MUC1 expression (50-95% of tumour cells positive) was found in 13 of 17 anaplastic lymphoma kinase (ALK) positive systemic nodal ALCLs, and in one of 20 cases of classic HD. Scattered or focal staining (< 25% of tumour cells) was seen in two additional ALK positive systemic ALCLs, two additional classic HD cases, and in three of 20 cases of ALK negative systemic nodal ALCL. Primary cutaneous ALCL showed no staining with the anti-MUC1 antibodies. Antibodies detecting hypoglycosylated MUC1 were found to be absent in all lymphomas (SM3) or present in only six of 15 ALK positive ALCLs (VU-4H5). CONCLUSIONS: MUC1 is preferentially expressed by a subtype of systemic nodal ALCL, characterised by ALK expression, but is found in only a few cases of classic HD and ALK negative ALCL. Therefore, although MUC1 could be used in a panel of markers for CD30 positive lymphomas, it is probably not a valuable tool to differentiate between ALK negative CD30 positive large cell lymphomas. Finally, the degree of MUC1 glycosylation in lymphomas is relatively high, compared with the aberrant hypoglycosylation found in adenocarcinomas.

Body and brain growth following continuous perinatal administration of arginine- and lysine-vasopressin to the homozygous Brattleboro rat.

Using a recently developed controlled-drug-delivery implantation technique, arginine-vasopressin (AVP) or lysine-vasopressin (LVP) was administered to homozygous (HOM) Brattleboro rats throughout pregnancy in order to study the influence of compensation for the deficiency of AVP on body and brain development in their HOM offspring. This mutant is retarded in both body and brain growth from the neonatal period onwards. In one subgroup the LVP-treatment was continued postnatally by means of subcutaneous implantation in the pups. AVP treatment had no growth-stimulating effect either on pup body weight at day one or on postnatal body growth, nor did it affect noticeably the day of eye opening, or a number of brain parameters measured at one month of age. LVP treatment, in contrast, resulted in higher body weights at birth, which could be maintained postnatally if the pups were reared with a Wistar foster-mother. At one month of age body as well as brain weights were still larger in the treated pups. Although cerebellar weight was larger than in untreated Brattleboro pups in this group, cerebellar DNA content or gross morphology, known to be impaired in HOM rats, were not changed. LVP treatment of the pups, as well as maternal AVP-treatment beginning on day 15 of pregnancy, had inhibiting rather than growth-stimulating effects, high-lighting the different effects created by these two peptides at different stages of development.

The distribution of vasopressin and oxytocin in the rat brain.

Since vasopressin and oxytocin might be involved in processes of storage and retrieval of information in rats, the distribution of these hormones within the central nervous system was measured employing sensitive and specific radioimmunoassays in combination with an extraction method using Vycor glass powder. Vasopressin was detected in hypothalamus, amygdala, septum, hippocampus, nucleus parafascicularis and medulla oblongata of Wistar and Brattleboro rats, heterozygous for diabetes insipidus. It was not detectable in samples of Brattleboro rats, homozygous for diabetes insipidus. Oxytocin was found in hypothalamus, septum and nucleus parafascicularis of Wistar rats in lower amounts than vasopressin while it was present in higher amounts in the medulla oblongata. The extensive distribution of these hormones in the rat brain suggests a possible role in a variety of central nervous system processes rather than solely in the expression of acquired behavior.

Increased loss of brain DNA in the neonatal vasopressin-deficient Brattleboro rat, but not in normal rat treated with vasopressin antagonist.

In order to establish whether vasopressin (VP) influences brain cell survival, [3H]thymidine was injected in 10-day-old vasopressin-deficient Brattleboro rat pups, as well as in Wistar pups treated, neonatally, with the VP antagonist dP[Tyr(Me)2]VP followed by subsequent measurement of [3H]DNA in olfactory bulbs and cerebellum days and weeks thereafter. Results show, first of all, that the incorporation of [3H]thymidine into DNA was enhanced in the homozygous (HOM) Brattleboro, when compared with the heterozygous (HET; non-vasopressin-deficient) controls. The difference is due to the greater and prolonged tissue availability of [3H]thymidine, possibly pointing to an altered thymidine uptake and/or metabolism. Between postnatal days 25 and 39 no differences were seen in [3H]DNA content of the brain parts of the HET and Wistar control rats. For the HOM rats, however, a loss of [3H]DNA was seen (up to 8%), indicating that increased postnatal brain cell death might occur in the mutant. The antagonist treatment in Wistar rat up to 21 days of age failed to show a similar effect. It is proposed that general growth impairments, rather than VP receptor-mediated effects, lead to the brain cell loss.

Postnatal development of vasopressin mRNA content in supraoptic and paraventricular nucleus of the Wistar rat.

Levels of mRNA coding the vasopressin precursor (VP mRNA) were determined in the supraoptic and paraventricular nucleus during the first month of postnatal development of the Wistar rat by solution hybridization assay and Northern blot analysis. The supraoptic and paraventricular nuclei showed a marked increase in VP mRNA to adult levels during that period. These results indicate an upregulation of vasopressin gene expression in supraoptic and paraventricular nuclei after birth, a period in which vasopressin starts to become involved in kidney function.

Human MUC1 mucin: a multifaceted glycoprotein.

Human MUC1 mucin, a membrane-bound glycoprotein, is a major component of the ductal cell surface of normal glandular cells. MUC1 is overexpressed and aberrantly glycosylated in carcinoma cells. The role MUC1 plays in cancer progression represents two sides of one coin: on the one hand, loss of polarity and overexpression of MUC1 in cancer cells interferes with cell adhesion and shields the tumor cell from immune recognition by the cellular arm of the immune system, thus favoring metastases; on the other hand, MUC1, in essence a self-antigen, is displaced and altered in malignancy and induces immune responses. Tumor-associated MUC1 has short carbohydrate sidechains and exposed epitopes on its peptide core; it gains access to the circulation and comes into contact with the immune system provoking humoral and cellular immune responses. Natural antibodies to MUC1 present in the circulation of cancer patients may be beneficial to the patient by restricting tumor growth and dissemination: early stage breast cancer patients with a humoral response to MUC1 have a better disease-specific survival. Several MUC1 peptide vaccines, differing in vectors, carrier proteins and adjuvants, have been tested in phase I clinical trials. They are capable of inducing predominantly humoral responses to the antigen, but evidence that these immune responses may be effective against the tumor in humans is still scarce.

Neonatal treatment with vasopressin antagonist dP[Tyr(Me)2]AVP, but not with vasopressin antagonist d(CH2)5[Tyr(Me)2]AVP, inhibits body and brain development and induces polyuria in the rat.

Two vasopressin antagonists, d(CH2)5[Tyr(Me)2]AVP and dP[Tyr(Me)2]AVP, were given to Wistar rats from postnatal day 1 to 21 in order to investigate the influence on development and later diuresis. The latter antagonist significantly reduced body growth from day 3 postnatally onwards. At postnatal day 35 body, total brain, cerebellar and kidney weights were significantly reduced compared with controls. Diuresis, measured at one month of age, was four- to five-fold higher than the control group. Combined treatment with vasopressin failed to abolish the weight disturbances or polyuria. However, animals treated with the vasopressin antagonist d(CH2)5[Tyr(Me)2]AVP did not show developmental or diuretic deficits. Allometric analysis of brain/body relationship of the young animals indicated a disturbance of brain development by dP[Tyr(Me)2]AVP. Although the body and brain growth retardation induced by dP[Tyr(Me)2]AVP supports the hypothesis of a role for vasopressin in brain ontogeny, it can also be the result of a nonAVP-related toxic effect, since it could not be prevented by concomitant treatment with vasopressin.

Dendritic cells, obtained from peripheral blood precursors in the presence of PGE2, promote Th2 responses.

In order to investigate the impact of an inflammatory mediator PGE2 on the functions of maturing DC we used an in vitro model of DC generation from peripheral blood monocytes. Addition of PGE2 (10(-9) M-10(-6) M) to the cultures performed in the presence of GM-CSF and IL-4 did not alter the morphology nor high levels of expression of class II MHC and co-stimulatory molecules on arising DC, although at concentrations above 10(-8) M, the acquisition of CD1a was selectively prevented. Control DC and the DC maturing in the presence of PGE2 (PGE2-DC) induced a similar proliferation of naive Th cells. Control DC produced high amounts of IL-12, and only trace amounts of IL-10, whereas PGE2-DC produced no IL-12 and high levels of IL-10, when stimulated after the removal of PGE2. The deficient IL-12 production by PGE2-DC was observed after stimulation both in the absence and in the presence of IFN gamma, and was not compensated during further 48 h culture in the absence of PGE2. Compared to control DC, PGE2-DC induced development of Th cells secreting elevated amounts of IL-4 and IL-5, from naive precursors. These data indicate that elevated tissue levels of PGE2 may promote type 2 Th responses by impairing the ability of locally maturing DC to produce IL-12. Since Th2 responses mediate protection in Th1-related autoimmune disorders, the use of PGE2-DC in immunotherapy of such disorders may be considered.

Characterization of lesional psoriatic skin T lymphocyte clones.

T cells are considered to play a role in the pathomechanism of psoriasis. Therefore we investigated the cytokine production patterns of T cell clones that were randomly prepared from chronic plaque psoriasis lesions of 2 patients. 67% of the 49 T lymphocyte clones (TLC) expressed CD4 and 33% expressed CD8 (ratio 2:1), while gamma delta-TCR expression was absent. The production of IL-4, IFN-gamma, IL-2 and IL-6 was measured in supernatants of TLC following PHA plus PMA stimulation. Different groups of clones could be distinguished according to their IL-4/IFN-gamma production ratio. In addition to Th0 cells (low IL-4/low IFN-gamma), low IL-4/high IFN-gamma producers as well as high IL-4/low IFN-gamma producing clones were found, suggesting the presence of Th1- and Th2-like subsets. Upon stimulation, all TLC secreted low levels of IL-2 whereas a minority of the TLC secreted low levels of IL-6. These results may imply that T cells in psoriasis lesions do not show shifts towards either a Th1 or a Th2 cytokine production profile.

Vasopressin and vasopressin-antagonists applied to neonatal Wistar rats: effects on body and brain development and water metabolism.

Neonatal Wistar rats were continuously treated with the vasopressin-antagonists d(CH2)5[Tyr(Me)2]AVP, d(CH2)5DAVP or d(CH2)5[D-Ile2,Ala4]AVP, which have high anti-vasopressor or anti-anti-diuretic activity. The treatments were performed to test the hypothesis that arginine-vasopressin (AVP) might have a stimulatory effect on brain development, which is based upon the disturbed brain development of the AVP-deficient Brattleboro rat. None of the treatments with antagonists, either being given via a small drug-delivery device or by twice daily injections, inhibited body or brain development, though d(CH2)5[Tyr(Me)2]AVP treatment even had some stimulatory effect on cerebellar weight as measured at one month of age. Similar drug-delivery treatment with AVP or oxytocin (OX), or injections with 250 ng AVP, lysine-vasopressin (LVP), arginine-vasotocin (AVT) or OX had no stimulatory developmental effect. However injections with 2.5 micrograms of these peptides reduced body development, albeit transiently, of the neonatal Wistar rats. Moreover in this latter group not only the known lasting effects of AVP and LVP on body water metabolism were seen but also such effects were present after AVT and OX treatment.

Prenatal development of the Brattleboro rat is influenced by genotype and lysine vasopressin treatment of the mother.

Homozygous (Hom) Brattleboro rats suffer from severe diabetes insipidus (DI) as a consequence of the lack of arginine-vasopressin (AVP) in the brain. Compared with heterozygous (Het) AVP-synthesizing Brattleboro rats, Hom rats show disturbed body and brain development. In this study breeding experiments with Het and Hom rats were performed to determine whether prenatal conditions might contribute to the developmental disturbances in Hom pups. For this purpose Het and Hom females were mated with Hom and Het males, respectively. In addition lysine-vasopressin (LVP) was administered to half of the pregnant females, since this has previously been shown to stimulate birth weight of Hom pups. On day 1 postnatally the body and brain weight of Hom pups of nontreated Hom mothers was significantly smaller than that of the Het litter mates, whereas no difference was found between the weight of Het or Hom pups of nontreated or LVP-treated Het mothers. These results indicate an important role of the genotype of the mother in prenatal development of Hom pups. LVP administration failed to diminish the growth deficits, but increased protein and DNA content of the cerebellum of both Het and Hom pups. Notwithstanding the improved prenatal growth of Hom pups from Het mothers, postnatally retarded development was still observed: at 1 month of age there was a significant difference between the body, brain and cerebellar weight of Het and Hom pups from Het mothers. It was therefore concluded that the prenatal situation of the Hom mother, i.e. AVP-deficiency, significantly contributes to the developmental disturbance of the Hom pup, but also that growth impairment is linked to the presence of the mutation in the Hom pups themselves.

Widespread alterations in central noradrenaline, dopamine, and serotonin systems in the Brattleboro rat not related to the local absence of vasopressin.

A comprehensive study of monoamine transmitter and metabolite concentrations measured by HPLC was undertaken in female (vasopressin-deficient) Brattleboro rats as compared to Long Evans rats. Noradrenaline was significantly increased in 8 out of 13 dissected brain regions, whereas concentrations of the metabolite 3-methoxy-4-hydroxyphenylglycol were not altered. The increases were not restricted to areas which are normally innervated by vasopressin-containing neurons. Serotonin was increased in 6 and dopamine in 4 regions and this was accompanied in some areas by increases in the metabolites 5-hydroxyindolacetic acid and dihydroxyphenylacetic acid. Only in the striatum, cerebellum, and the medulla-pons no changes could be detected in any of the compounds of interest. These results show that the long term absence of vasopressin in Brattleboro rats appears to be associated with increases in monoamine transmitter contents and decreased metabolite/transmitter ratios. The regional distribution of these changes does not bear any relationship to the regional distribution of vasopressin cell bodies or nerve endings.

Modulation of T-cell cytokine secretion by accessory cell-derived products.

Several major pathological characteristics of atopic disease are causally related to CD4+ allergen-specific type 2 T-helper (Th2) cells with an aberrant cytokine secretion profile, comprising high levels of interleukin (IL)-4 and IL-5 and low levels of interferon (IFN)-gamma. Although the cytokine secretion patterns of CD4+ T-cells may be stable, they can be modulated by physiological factors which may be expected to be present during activation of these T-cells. In this review, we will focus on two secretion products of professional antigen presenting cells (APCs) and accessory cells with opposite modulatory effects on T-cell cytokine profiles, i.e. prostaglandin E2 (PGE2) and IL-12. PGE2 favours Th2-like cytokine secretion profiles by inhibiting the production of the Th1-associated cytokines, IL-2 and IFN-gamma, and in the presence of sufficient levels of IL-2, upregulating the production of the Th2-associated cytokines, IL-4 and IL-5, IL-12, on the other hand, induces and enhances IFN-gamma secretion in activated CD4+ T-cells, thereby promoting the generation of Th1 cells. PGE2 and IL-12 act via independent mechanisms and, therefore, do not mutually interfere with their modulatory effects. These data suggest that the relative contribution of PGE2 and IL-12 to the levels of secreted Th1- and Th2-associated cytokines are determined by their concentration ratio during T-cell activation.

Corticosteroids class-dependently inhibit in vitro Th1- and Th2-type cytokine production.

Corticosteroids (CS) are very potent immunosuppressive agents and are widely used to treat inflammatory diseases. On the basis of their clinical efficacy and potency CS have been divided into different classes. In the present study we investigated whether the class-associated effects of CS are correlated with a differential in vitro effect on cytokine production by T lymphocytes. Therefore, we determined the in vitro effects of CS on the production of Th1- and Th2-type cytokines. The addition of CS, in the range of 10(-9) to 10(-4) M, resulted in a class- and dose-dependent inhibition of the production of both IFN-gamma and IL-4. Notably, at the lowest doses tested, hydrocortisone and hydrocortisone 17-butyrate had a stimulatory effect on IL-4 production. CS class-dependently inhibited the IL-2 production by T cells but did not affect IL-2R expression of the T cells. Addition of rIL-2 could not completely restore the inhibitory effect of the CS on proliferation and on IFN-gamma and IL-4 production, indicating that CS act only partially via inhibition of IL-2 production. The demonstrated positive correlation between the clinical efficacy and the in vitro effects of the different classes of CS strongly suggests that the effect of CS on T-cell-mediated inflammation follows from inhibition of proliferation and cytokine production by T lymphocytes. The in vitro method used will be valuable for investigating and classifying new types of CS and other substances for applications in T-cell-mediated diseases.