Serum and cutaneous transcriptional expression levels of IL31 are minimal in cutaneous T cell lymphoma variants.

AIM: Recent studies suggested a role for IL31 in the pathogenesis of pruritus and disease severity in patients with cutaneous T cell lymphomas (CTCL). However, discrepant results were reported for IL31 serum levels, transcriptional expression levels or immunohistochemistry studies and its relation to pruritus intensity and/or disease severity in CTCL. Most studies did not distinguish between different CTCL variants. We investigated IL31 serum levels in different subtypes of CTCL, including Mycosis Fungoides (MF) (typically not pruritic), Folliculotropic Mycosis Fungoides (FMF) and Sézary syndrome (SS) (both often pruritic). METHODS: From 54 CTCL patients (17 SS, 21 FMF and 16 classic MF) serum samples were analyzed with a high sensitivity V-PLEX immunoassay for IL31. The study group included 35/54 (65%) patients with complaints of pruritus. Thirty-five patients had advanced stage disease (≥stage IIB). A visual analog scale score (VAS score) for pruritus was available in 29 CTCL patients (7 SS, 9 FMF and 13 classic MF) and in other cases complaints of pruritus were retrieved from medical records. qPCR analyses for IL31 expression were performed in lesional skin biopsies from 8 CTCL patients. Serum samples from 4 healthy individuals without pruritus and from 5 atopic dermatitis (AD) patients with severe pruritus were included as controls. RESULTS: In 11/54 (20%) of CTCL patients low serum levels of IL31 were detected (mean 0.48 pg/mL, range 0.20-1.39 pg/mL) including 6/17 (35%) SS patients (mean 0.57 pg/mL) and 5/21 (24%) FMF patients (mean 0.33 pg/mL). All 11 patients with detectable levels of IL31 reported complaints of moderate to severe pruritus and 9/11 patients presented with advanced stage disease (≥IIB). qPCR analyses resulted in lowly expressed IL31 expression levels in 4 of 8 patients; these patients all suffered from pruritus and advanced stage disease. CONCLUSIONS: Translational and transcriptional expression levels of IL31 were very low or undetectable in CTCL patients. Detectable low IL31 serum levels were exclusively observed in SS and FMF patients and not in patients with classic MF. However, these marginal IL31 levels in a small proportion of CTCL patients do not support an essential role for IL31 in CTCL patients.

Clinical and pathogenic aspects of the severe cutaneous adverse reaction epidermal necrolysis (EN).

The severe cutaneous adverse reaction epidermal necrolysis (EN) which includes toxic epidermal necrolysis and the milder Stevens-Johnson syndrome is characterized by epidermal loss due to massive keratinocyte apoptosis and/or necroptosis. EN is often caused by a drug mediating a specific TCR-HLA interaction via the (pro)hapten, pharmacological interaction or altered peptide loading mechanism involving a self-peptide presented by keratinocytes. (Memory) CD8 + T cells are activated and exhibit cytotoxicity against keratinocytes via the perforin/granzyme B and granulysin pathway and Fas/FasL interaction. Alternatively drug-induced annexin release by CD14 + monocytes can induce formyl peptide receptor 1 death of keratinocytes by necroptosis. Subsequent keratinocyte death stimulates local inflammation, activating other immune cells producing pro-inflammatory molecules and downregulating regulatory T cells. Widespread epidermal necrolysis and inflammation can induce life-threatening systemic effects, leading to high mortality rates. Research into genetic susceptibility aims to identify risk factors for eventual prevention of EN. Specific HLA class I alleles show the strongest association with EN, but risk variants have also been identified in genes involved in drug metabolism, cellular drug uptake, peptide presentation and function of CD8 + T cells and other immune cells involved in cytotoxic responses. After the acute phase of EN, long-term symptoms can remain or arise mainly affecting the skin and eyes. Mucosal sequelae are characterized by occlusions and strictures due to adherence of denuded surfaces and fibrosis following mucosal inflammation. In addition, systemic pathology can cause acute and chronic hepatic and renal symptoms. EN has a large psychological impact and strongly affects health-related quality of life among EN survivors.

Array-CGH analysis of cutaneous anaplastic large cell lymphoma.

This chapter describes a study in which the pattern of numerical chromosomal alterations in cutaneous anaplastic large cell lymphoma (C-ALCL) tumor samples was defined using array-based comparative genomic hybridization (CGH). First, the array-based CGH technique applied is outlined in detail. Next, its application in the analysis of C-ALCL tumor specimens is described. This approach resulted in the identification of highly recurrent chromosomal alterations in C-ALCL that include gain of 7q31 and loss on 6q16-6q21 and 13q34, each affecting 45% of the patients. The pattern characteristic of C-ALCL differs markedly from chromosomal alterations observed in other CTCL such as mycosis fungoides and Sézary syndrome and yielded several candidate genes with potential relevance in the pathogenesis of C-ALCL.

Rapamycin impairs UV induction of mutant-p53 overexpressing cell clusters without affecting tumor onset.

Because of its antitumor effect, the immunosuppressant rapamycin holds great promise for organ transplant recipients in that it may lower their cancer risk. In a mouse model, we showed previously that rapamycin inhibits the outgrowth of primary skin carcinomas induced by UV radiation. However, the tumors that did grow out showed an altered p53 mutation spectrum. Here, we investigated whether this shift in p53 mutations already occurred in the smallest tumors, which were not affected in onset. We found that rapamycin did not alter the mutational spectrum in small tumors and in preceding microscopic clusters of cells expressing mutant-p53. However, rapamycin did reduce the number of these cell clusters. As this reduction did not affect tumor onset, we subsequently investigated whether rapamycin merely suppressed expression of mutated p53. This was not the case, as we could demonstrate that switching from a diet with rapamycin to one without, or vice versa, did not affect the number of existing mutant-p53 expressing cell clusters. Hence, rapamycin actually reduced the formation of mutant-p53 cell clusters. In wild-type and p53-mutant mice, we could not measure a significant enhancement of UV-induced apoptosis, but we did observe clear enhancement in human skin equivalents. This was associated with a clear suppression of HIF1α accumulation. Thus, we conclude that rapamycin reduces the formation of mutant-p53-expressing cell clusters without affecting tumor onset, suggesting that tumors grow out of a minor subset of cell clusters, the formation of which is not affected by rapamycin.

Quantitative expression profiling of G-protein-coupled receptors (GPCRs) in metastatic melanoma: the constitutively active orphan GPCR GPR18 as novel drug target.

G-protein-coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. Utilizing a robust quantitative PCR array, we quantified expression of 94 human GPCRs, including 75 orphan GPCRs and 19 chemokine receptors, and 36 chemokine ligands, in 40 melanoma metastases from different individuals and benign nevi. Inter-metastatic site comparison revealed that orphan GPR174 and CCL28 are statistically significantly overexpressed in subcutaneous metastases, while P2RY5 is overexpressed in brain metastases. Comparison between metastases (all three metastatic sites) and benign nevi revealed that 16 genes, including six orphan receptors (GPR18, GPR34, GPR119, GPR160, GPR183 and P2RY10) and chemokine receptors CCR5, CXCR4, and CXCR6, were statistically significantly differentially expressed. Subsequent functional experiments in yeast and melanoma cells indicate that GPR18, the most abundantly overexpressed orphan GPCR in all melanoma metastases, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival. GPR18 and five other orphan GPCRs with yet unknown biological function may be considered potential novel anticancer targets in metastatic melanoma.

Early and late effects of the immunosuppressants rapamycin and mycophenolate mofetil on UV carcinogenesis.

Increased skin cancer risk in organ transplant recipients has been experimentally emulated with enhanced UV carcinogenesis from administering conventional immunosuppressants. However, newer generation immunosuppressive drugs, rapamycin (Rapa) and mycophenolate mofetil (MMF), have been shown to impair angiogenesis and outgrowth of tumor implants. To ascertain the overall effect on UV carcinogenesis, Rapa and MMF were admixed into the food pellets of hairless SKH1 mice receiving daily sub-sunburn UV dosages. With immunosuppressive blood levels neither of the drugs affected onset of tumors (<2 mm), but in contrast to MMF, Rapa significantly increased latency of large tumors (>or=4 mm, medians of 190 vs 125 days) and reduced their multiplicity (1.6 vs 4.5 tumors per mouse at 200 days). Interestingly, tumors (>2 mm) from the Rapa-fed group showed a reduction in UV-signature p53 mutations (39% vs 90%) in favor of mutations from putative base oxidation. This shift in mutation spectrum was not essentially linked to the reduction in large tumors because it was absent in large tumors similarly reduced in number when feeding Rapa in combination with MMF, possibly owing to an antioxidant effect of MMF. Significantly fewer tumor cells were Vegf-positive in the Rapa-fed groups, but a correspondingly reduced expression of Hif1alpha target genes (Vegf, Ldha, Glut1, Pdk1) that would indicate altered glucose metabolism with increased oxidative stress was not found. Remarkably, we observed no effect of the immunosuppressants on UV-induced tumor onset, and with impaired tumor outgrowth Rapa could therefore strongly reduce skin carcinoma morbidity and mortality rates in organ transplant recipients.

Reduced IL-1Ra/IL-1 ratio in ultraviolet B-exposed skin of patients with polymorphic light eruption.

Polymorphic light eruption (PLE) is a putative delayed-type allergic reaction to (solar) ultraviolet (UV) exposure. Inadequate immune suppression after UVB-induced sunburn appears to be associated with reduced trafficking of Langerhans cells (LCs) out of and neutrophils into the epidermis of patients sensitive to UVB provocation of PLE. Therefore, we investigated whether pro-inflammatory and chemotactic cytokines are differentially expressed in UVB-irradiated skin of UVB-provocable PLE patients (n = 6) and age- and gender-matched healthy controls (n = 6). Interstitial interleukin-1alpha (IL-1alpha), IL-1beta, IL-1Ra, IL-4, IL-8, tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein 1-alpha (MIP-1alpha), MIP-1beta and monocyte chemotactic protein-1 (MCP-1) were measured in suction blister fluid raised 16 h after exposure to 0, three and six minimal erythemal UVB doses. In unirradiated skin, the IL-1Ra levels were significantly lower in the PLE patients than in controls (P < 0.05). IL-8 and TNF-alpha levels increased strongly upon UVB irradiation in both groups. No differential shifts in cytokine profiles were found that could explain a reduced trafficking of Langerhans cells and neutrophils in PLE patients. Dose-trend analyses showed that UVB irradiation caused significant increases in IL-1alpha in both groups, and that the levels of IL-1alpha and IL-1beta were on average twofold higher in the PLE group (P = 0.03 and P = 0.004, respectively.). Accordingly, the ratios of IL-1Ra over IL-1alpha and over IL-1beta were overall lower in the skin of PLE patients (P = 0.015 and P < 0.001, respectively.). This shift in cytokines in UVB-irradiated skin of PLE patients reveals an amplified early pro-inflammatory cytokine response, which may contribute to the allergic reaction to UVB radiation.

Profiling of apoptosis genes identifies distinct types of primary cutaneous large B cell lymphoma.

Two distinct primary cutaneous large B cell lymphomas are recognized: primary cutaneous follicle centre lymphoma (PCFCL), characterized by an excellent prognosis, and primary cutaneous large B cell lymphoma, leg-type (PCLBCL leg-type), with an unfavourable prognosis. To determine whether inhibition of the apoptosis pathways may underlie the difference in clinical outcome between PCFCL and PCLBCL leg-type, we investigated the expression of only apoptosis-related genes by microarray expression profiling. Unsupervised cluster analysis was carried out using 169 genes involved in apoptosis on a group of 21 previously untreated patients diagnosed with primary cutaneous large B cell lymphoma. Cluster analysis resulted in two separate groups which showed large overlap with the PCFCL and PCLBCL leg-type. One group was characterized by high expression levels of pro- and anti-apoptotic genes. The other group was characterized by high expression levels of apoptosis-inducing cytotoxic effector genes, possibly reflecting a cellular cytotoxic immune response. Our results suggest that the clinically favourable PCFCLs are characterized by a relatively intense cellular cytotoxic immune response and that PCLBCL leg-types are characterized by constitutive activation of the intrinsic mediated apoptosis pathway, with concomitant downstream inhibition of this apoptosis pathway. Thus, strategies neutralizing the function of apoptosis-inhibiting proteins might be effective in PCLBCL leg-type.

Differential expression of thymus and activation regulated chemokine and its receptor CCR4 in nodal and cutaneous anaplastic large-cell lymphomas and Hodgkin's disease.

Recent studies demonstrated that Hodgkin/Reed-Sternberg (H/RS) cells in Hodgkin's disease (HD) express thymus and activation-regulated chemokine (TARC), whereas reactive lymphocytes surrounding H/RS cells express its ligand, CC-chemokine receptor 4 (CCR4). Because in vitro studies showed that CCR4 expression is a marker for lymphocytes bearing a T-helper 2 (Th2) phenotype, it was suggested that expression of TARC is a new immune escape mechanism in HD. To find out whether this mechanism might also be operative in CD30+ malignant lymphomas other than HD, TARC and CCR4 expression was investigated by immunohistochemistry on paraffin and frozen-tissue sections of 39 nodal CD30+ anaplastic large cell lymphomas (ALCL), including 27 ALK-negative and 12 ALK-positive ALCL, 25 primary cutaneous CD30+ ALCL, including 11 patients with lymphomatoid papulosis, and 31 cases of HD. TARC was expressed by the neoplastic cells in 12/27 (44%) nodal ALK-negative ALCL and all cases of classic HD, but not in nodal ALK-positive ALCL (0/12) and only rarely in primary cutaneous CD30+ ALCL (3/25). In contrast, CCR4 was expressed by the neoplastic cells in 9/9 cutaneous CD30+ ALCL, and in 9/15 (60%) nodal ALK-negative ALCL, but only in 1/4 (25%) nodal ALK-positive ALCL and not by the H/RS cells in HD (0/8). Apart from three cases of HD showing 10 to 15% CCR4-positive lymphocytes surrounding TARC-positive H/RS cells, CCR4-positive reactive T cells were few (<5%) in all other cases studied. Our results demonstrate a differential expression of TARC and CCR4 in different types of CD30+ malignant lymphomas. The small number of CCR4-positive reactive T cells in most cases studied argues against an important role of TARC expression in the evasion of antitumor responses.

Proteomic analysis of skin irritation reveals the induction of HSP27 by sodium lauryl sulphate in human skin.

BACKGROUND: There is an increasing need for screening of mild irritants in vitro to reduce animal testing. OBJECTIVES: Proteomics were used to search for new markers of which the expression changes after mild irritation. METHODS: Sodium lauryl sulphate (SLS) was applied topically on excised human skin. Epidermal proteins were isolated from SLS-treated skin specimens that showed hardly any morphological changes. The proteins were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and proteins that significantly increased or decreased after SLS treatment in a dose-dependent way were characterized by mass spectrometry. Subsequently, immunohistochemistry was performed on skin samples treated with SLS in vivo and nonanoic acid (NAA) or benzalkonium chloride (BC) in vitro to evaluate one of the identified proteins for its predictive value. RESULTS: We identified seven proteins as potentially new epidermal markers for skin irritation. Among these seven proteins, the 27 kDa heat shock protein (HSP27) was identified as the most prominently upregulated protein. A strong nuclear HSP27 staining was seen in the SLS-treated skin, whereas in the vehicle controls only cytoplasmic staining was observed. Moreover, nuclear staining was also observed after topical application of SLS in vivo and after exposure to NAA and BC in vitro. CONCLUSIONS: Our findings suggest that HSP27 may serve as a sensitive marker of skin irritation and eventually as a novel tool in clinics for testing the sensitivity of the patient for a panel of irritants.

Processing of natural and recombinant CXCR3-targeting chemokines and implications for biological activity.

Chemokines comprise a class of peptides with chemotactic activity towards leukocytes. The potency of different chemokines for the same receptor often varies as a result of differences in primary structure. In addition, post-translational modifications have been shown to affect the effectiveness of chemokines. Although in several studies, natural CXCR3-targeting chemokines have been isolated, detailed information about the proteins and their possible modifications is lacking. Using a combination of liquid chromatography and mass spectrometry we studied the protein profile of CXCR3-targeting chemokines expressed by interferon-gamma-stimulated human keratinocytes. The biological implications of one of the identified modifications was studied in more detail using calcium mobilization and chemotaxis assays. We found that the primary structure of human CXCL10 is different from the generally accepted sequence. In addition we identified a C-terminally truncated CXCL10, lacking the last four amino acids. Native CXCL11 was primarily found in its intact mature form but we also found a mass corresponding to an N-terminally truncated human CXCL11, lacking the first two amino acids FP, indicating that this chemokine is a substrate for dipeptidylpeptidase IV. Interestingly, this same truncation was found when we expressed human CXCL11 in Drosophila S2 cells. The biological activity of this truncated form of CXCL11 was greatly reduced, both in calcium mobilization (using CXCR3 expressing CHO cells) as well as its chemotactic activity for CXCR3-expressing T-cells. It is concluded that detailed information on chemokines at the protein level is important to characterize the exact profile of these chemotactic peptides as modifications can severely alter their biological activity.

Differential expression of CXCR3 targeting chemokines CXCL10, CXCL9, and CXCL11 in different types of skin inflammation.

Recruitment of activated T-cells to the skin is a common feature in a wide variety of inflammatory skin diseases. As CXCR3 activating chemokines CXCL10 (IP-10), CXCL9 (Mig), and CXCL11 (IP-9/I-TAC) specifically attract activated T-cells, this study addressed the question of whether differences in the expression of these chemokines correlate with the site and cellular composition of the skin infiltrates in different types of inflammatory skin disease. Skin biopsies from lichen planus, chronic discoid lupus erythematosus, allergic patch test reactions, psoriasis, and Jessner's lymphocytic infiltration of the skin were investigated for chemokine expression using RNA in situ hybridization, and for the expression of CXCR3 using immunohistochemistry. The results showed differential expression of CXCL10, CXCL9, and CXCL11, which correlated with differences in the localization and cellular composition of the infiltrates. Whereas CXCL10 and CXCL11 were mainly expressed by basal keratinoctyes, CXCL9 mRNA expression was located predominantly in the dermal infiltrates. Correlation with immunohistochemical data suggested that macrophages and activated keratinocytes were the main producers of these chemokines. CXCR3 was expressed by a majority of both CD4+ and CD8+ infiltrating T-cells, suggesting a functional interaction between locally produced chemokines and CXCR3-expressing T-cells. In conclusion, these findings indicate that these CXCR3 activating chemokines play a significant role in the recruitment and maintenance of T-cell infiltrates in the inflammatory skin diseases studied.

Cross reactivity of three T cell attracting murine chemokines stimulating the CXC chemokine receptor CXCR3 and their induction in cultured cells and during allograft rejection.

Recent work identified the murine gene homologous to the human T cell attracting chemokine CXC receptor ligand 11 (CXCL11, also termed I-TAC, SCYB11, ss-R1, H174, IP-9). Here, the biological activity and expression patterns of murine CXCL11 relative to CXCL9 (MIG) and CXCL10 (IP-10/crg-2), the other two CXCR3 ligands, were assessed. Calcium mobilization and chemotaxis experiments demonstrated that murine CXCL11 stimulated murine CXCR3 at much lower doses than murine CXCL9 or murine CXCL10. Murine CXCL11 also evoked calcium mobilization in CHO cells transfected with human CXCR3 and was chemotactic for CXCR3-expressing human T lymphocytes as well as for 300--19 pre-B cells transfected with human or murine CXCR3. Moreover, murine CXCL11 blocked the chemotactic effect of human CXCL11 on human CXCR3 transfectants. Depending on cell type (macrophage-like cells RAW264.7, J774A.1, fetal F20 and adult dermal fibroblasts, immature and mature bone marrow-derived dendritic cells) and stimulus (interferons, LPS, IL-1 beta and TNF-alpha), an up to 10,000-fold increase of CXCL9, CXCL10 and CXCL11 mRNA levels, quantified by real-time PCR, was observed. In vivo, the three chemokines are constitutively expressed in various tissues from healthy BALB/c mice and were strongly up-regulated during rejection of allogeneic heart transplants. Chemokine mRNA levels exceeded those of CXCR3 and IFN-gamma which were induced with similar kinetics by several orders of magnitude.

Absence of T(H)2 cytokine messenger RNA expression in CD30-negative primary cutaneous large T-cell lymphomas.

BACKGROUND: Previous studies demonstrating that the neoplastic cells in Sézary syndrome and tumor stage mycosis fungoides express interleukin 4 (IL-4), IL-5, and IL-10 have resulted in the concept that cutaneous T-cell lymphomas are derived from CD4(+) T cells with a T(H)2 type cytokine profile. OBJECTIVE: To determine the cytokine profile in CD30(-) primary cutaneous large T-cell lymphomas, which represent a subgroup of cutaneous T-cell lymphoma with an aggressive clinical behavior (5-year survival rate of 15%). DESIGN AND METHODS: Seven biopsy specimens were taken from 4 patients with CD30(-) primary cutaneous large T-cell lymphomas and studied for the expression of T(H)1 (IL-2 and interferon gamma) and T(H)2 (IL-4, IL-5, IL-10) cytokines using a reverse transcription-polymerase chain reaction technique. Skin biopsy specimens from patients with Sézary syndrome, mycosis fungoides, atopic dermatitis, or psoriasis were included as controls. RESULTS: In the 7 CD30(-) primary cutaneous large T-cell lymphomas showing an almost pure population of large tumor cells (>90%), no expression of IL-4 was found, and IL-5 was only found in 1 of 7 cases. In control biopsy specimens, expression of IL-4 and/or IL-5 was demonstrated in atopic dermatitis (3/3), tumor stage mycosis fungoides (2/2), and Sézary syndrome (3/3), but not in plaque stage mycosis fungoides. CONCLUSION: Our results demonstrate that CD30(-) primary cutaneous large T-cell lymphomas do not produce T(H)2 cytokines, illustrating that not all cutaneous T-cell lymphomas have a T(H)2 cytokine profile.

The antipsoriatic drug dimethylfumarate strongly suppresses chemokine production in human keratinocytes and peripheral blood mononuclear cells.

BACKGROUND: The effectiveness of systemic treatment of psoriasis with fumaric acid esters has been proven, but their mode of action at the cellular and molecular level has not yet been fully elucidated. OBJECTIVES: To study the effect of dimethylfumarate (DMF) on the production of the chemokines CXCL1, CXCL8, CXCL9, CXCL10 and CXCL11, formerly known as GROalpha, interleukin-8, Mig, IP-10 and IP-9/I-TAC, respectively, in human keratinocytes and peripheral blood mononuclear cells (PBMC). METHODS: Cultured keratinocytes were stimulated with interferon (IFN) -gamma to produce CXCL9, CXCL10 and CXCL11 and with phorbol myristate acetate to produce CXCL1 and CXCL8 in the absence and presence of DMF (5, 15 and 45 micromol L(-1)). PBMC were stimulated with either IFN-gamma to produce CXCL9 and CXCL10 or lipopolysaccharide to produce CXCL8, in the absence and presence of DMF (5, 15 and 45 micromol L(-1)). RNA preparations from isolated keratinocytes were analysed by Northern blotting; protein production by keratinocytes and PBMC was monitored by an enzyme-linked immunosorbent assay. RESULTS: Northern blot analysis on isolated keratinocyte RNA preparations showed a dose-dependent inhibition of CXCL1, CXCL8, CXCL9, CXCL10 and CXCL11 transcription by DMF. At 45 micromol L(-1) the inhibition was almost complete. In addition, keratinocytes and PBMC showed in the presence of DMF a dose-dependent inhibition of CXCL8, CXCL9 and CXCL10 protein production. CONCLUSIONS: These results show the ability of DMF to inhibit the production of chemokines that may be critically involved in the development and perpetuation of psoriatic lesions. This might explain, at least in part, the beneficial effects of treatment with fumaric acid esters in psoriasis patients.

Discrepancy between molecular structure and ligand selectivity of a testicular follicle-stimulating hormone receptor of the African catfish (Clarias gariepinus).

A putative FSH receptor (FSH-R) cDNA was cloned from African catfish testis. Alignment of the deduced amino acid sequence with other (putative) glycoprotein hormone receptors and analysis of the African catfish gene indicated that the cloned receptor belonged to the FSH receptor subfamily. Catfish FSH-R (cfFSH-R) mRNA expression was observed in testis and ovary; abundant mRNA expression was also detected in seminal vesicles. The isolated cDNA encoded a functional receptor since its transient expression in human embryonic kidney (HEK-T) 293 cells resulted in ligand-dependent cAMP production. Remarkably, African catfish LH (cfLH; the catfish FSH-like gonadotropin has not been purified yet) had the highest potency in this system. From the other ligands tested, only human recombinant FSH (hrFSH) was active, showing a fourfold lower potency than cfLH, while hCG and human TSH (hTSH) were inactive. Human CG (as well as cfLH, hrFSH, eCG, but not hTSH) stimulated testicular androgen secretion in vitro but seemed to be unable to bind to the cfFSH-R. However, it was known that hCG is biologically active in African catfish (e.g., induction of ovulation). This indicated that an LH receptor is also expressed in African catfish testis. We conclude that we have cloned a cDNA encoding a functional FSH-R from African catfish testis. The cfFSH-R appears to be less discriminatory for its species-specific LH than its avian and mammalian counterparts.

NPY in invertebrates: molecular answers to altered functions during evolution.

As in Lymnaea stagnalis NPY plays a key role in regulating energy flows but has no effect on food intake, two important questions arise: 1) How is the amount of food consumed related to energy storage? 2) Can we give a molecular explanation for this alteration in function of NPY during evolution? Recent data have shown that also in Lymnaea a leptin-like factor is produced by glycogen storing cells which inhibits food intake, a Lymnaea storage feedback factor (LySFF). So, food consumption seems in balance with the amount of energy stored in this animal. We suppose that NPY neurons in Lymnaea have receptors for LySFF so that their activity in regulating energy homeostasis reflects the amount of stored energy. By comparing the molecular structure of NPYs in invertebrates it became clear that only molluscan and arthropod NPY are synthesized from a prohormone similar to vertebrate NPYs and should be considered as real invertebrate homologs of NPY. Based on pharmacological data we suppose that the identified Lymnaea NPY receptor is a Y1 subtype. This might explain that LyNPY has no effect on food intake in Lymnaea as this function of NPY in mammals is regulated through the Y5 subtype receptor.

Parasites flicking the NPY gene on the host's switchboard: why NPY?

It was investigated whether up-regulation of the NPY gene by the schistosome Trichobilharzia ocellata in its snail host Lymnaea stagnalis redirects the host's energy flows. We cloned the cDNA encoding Lymnaea NPY (LyNPY), purified and sequenced the peptide, and used synthesized peptide for physiological and morphological studies. Increasing the LyNPY titer in nonparasitized snails (mimicking parasitosis) by 1) implantation of slow-release pellets and 2) injections suppressed reproductive activity and reduced growth in a dose- and time-dependent manner without affecting food intake. When the LyNPY titer was back to normal, reproduction and growth were resumed, coinciding with a transient increase of food intake serving to replenish glycogen stores. Observations on double-immunostained whole mount preparations of brains support these data. A close association was found between LyNPY-positive axons and axons both from ovulation hormone-producing neurons and molluscan insulin-like peptide-producing neurons involved in regulation of growth. As no synaptic(-like) contacts were observed, it is supposed that LyNPY acts nonsynaptically. No morphological interaction was found between LyNPY-positive axons and motoneurons innervating the feeding apparatus. Our data explain why it is an advantageous strategy for endoparasites to up-regulate the highly conserved NPY gene in their host.