Melanocyte transformation requires complete loss of all pocket protein function via a mechanism that mitigates the need for MAPK pathway activation.

Deregulation of p16INK4A is a critical event in melanoma susceptibility and progression. It is generally assumed that the major effect of loss of p16 function is mediated through the CDK-cyclin pathway via its influence on the pocket protein (PP) pRb. However, there are also two other PPs, p107 and p130, which, when phosphorylated by CDK-cyclin complexes, play a role in permitting cell progression. Cohorts of mice carrying melanocyte-specific knockouts (KOs) of various combinations of the three PPs were generated. Mice null for pRb, p107, p130 or any combination of double mutants did not develop melanoma. Surprisingly, melanocyte-specific loss of all three PPs facilitated melanoma development (median age of onset 308 days, penetrance 40% at 1 year). Tumorigenesis was exacerbated by Trp53 co-deletion (median age of onset 275 days, penetrance 82% at 1 year), with cell culture studies indicating that this difference may result from the apoptotic role of Trp53. Melanomas in PP;Trp53-deficient mice lacked either Ras or Braf mutations, and hence developed in the absence of constitutive MAPK pathway activation. The lag period between induction of total PP or PP/Trp53 KO and melanoma development indicates that additional genetic or epigenetic alterations may account for neoplastic progression. However, exome sequencing of PP;Trp53 KO melanomas failed to reveal any additional recurrent driver mutations. Analysis of the putative mutation signature of the PP;Trp53 KO melanomas suggests that melanocytes are primed for transformation via a mutagenic mechanism involving an excess of T>G substitutions, but not involving a preponderance of C>T substitutions at CpG sites, which is the case for most spontaneous cancers not driven by a specific carcinogen. In sum, deregulation of all three PPs appears central to neoplastic progression for melanoma, and the customary reference to the p16INKA/CDK4/pRB pathway may no longer be accurate; all PPs are potentially critical targets of CDK-cyclins in melanoma.

Prophylactic but not therapeutic activity of a monoclonal antibody that neutralizes the binding of VEGF-B to VEGFR-1 in a murine collagen-induced arthritis model.

OBJECTIVE: To assess the therapeutic potential of a mAb that neutralizes the binding of VEGF-B to VEGFR-1, to inhibit the pathogenesis of CIA in mice. METHODS: CIA was induced in C57BL6/J and DBA-1 mice by intradermal injection of chick collagen type II (CII) in adjuvant. A neutralizing VEGF-B mAb or an isotype control mAb was then administered by intraperitoneal injection twice weekly beginning either post CII booster injection but prior to or immediately following clinical disease diagnosis. RESULTS: Neutralizing VEGF-B mAb inhibited the development of CIA in C57BL6/J mice in a dose-dependent manner when administered following the CII booster injection, but prior to clinical disease diagnosis. This result was also confirmed in DBA-1 strain mice. In contrast, the neutralizing VEGF-B mAb had no measurable effect on disease severity or progression when treatment commenced from the day of clinical disease diagnosis. CONCLUSIONS: Treatment with an mAb that neutralizes the binding of VEGF-B to VEGFR-1 exhibits prophylactic but not therapeutic actions in a mouse model of RA. These data indicate that while VEGF-B/VEGFR-1 signalling is involved in the early development of arthritis it may not be required for maintenance or progression of established disease.

Lack of augmentation of tumor spectrum or severity in dual heterozygous Men1 and Rb1 knockout mice.

To identify possible genetic interactions between the mechanisms of tumor suppression of menin and pRb, we intercrossed mice with targeted deletions of Men1 and Rb1, and compared tumor development in cohorts of animals carrying single or dual mutations of these tumor-suppressor genes. In mice lacking one copy of Men1, pancreatic islet and anterior pituitary adenomas are common. In animals lacking one copy of Rb1, intermediate pituitary and thyroid tumors occur at high frequency, with less frequent development of pancreatic islet hyperplasia and parathyroid lesions. In mice heterozygous for both Men1 and Rb1, pancreatic hyperplasia and tumors of the intermediate pituitary and thyroid occurred at high frequency. Serum measurements of calcium and glucose did not vary significantly between genotypic groups. Loss of heterozygosity at the Rb1 locus was common in pituitary and thyroid tumors, whereas loss of menin was observed in pancreatic and parathyroid lesions. The tumor spectrum in the double heterozygotes was a combination of pathologies seen in each of the individual heterozygotes, without decrease in age of onset, indicating independent, non-additive effects of the two mutations. Together with the lack of increased tumor spectrum, this suggests that menin and pRb function in a common pathway of tumor suppression.

Elemental signals regulating eosinophil accumulation in the lung.

In this review we identify the elemental signals that regulate eosinophil accumulation in the allergic lung. We show that there are two interwoven mechanisms for the accumulation of eosinophils in pulmonary tissues and that these mechanisms are linked to the development of airways hyperreactivity (AHR). Interleukin-(IL)-5 plays a critical role in the expansion of eosinophil pools in both the bone marrow and blood in response to allergen provocation of the airways. Secondly, IL-4 and IL-13 operate within the allergic lung to control the transmigration of eosinophils across the vascular bed into pulmonary tissues. This process exclusively promotes tissue accumulation of eosinophils. IL-13 and IL-4 probably act by activating eosinophil-specific adhesion pathways and by regulating the production of IL-5 and eotaxin in the lung compartment. IL-5 and eotaxin co-operate locally in pulmonary tissues to selectively and synergistically promote eosinophilia. Thus, IL-5 acts systemically to induce eosinophilia and within tissues to promote local chemotactic signals. Regulation of IL-5 and eotaxin levels within the lung by IL-4 and IL-13 allows Th2 cells to elegantly co-ordinate tissue and peripheral eosinophilia. Whilst the inhibition of either the IL-4/IL-13 or IL-5/eotaxin pathways resulted in the abolition of tissue eosinophils and AHR, only depletion of IL-5 and eotaxin concurrently results in marked attenuation of pulmonary inflammation. These data highlight the importance of targeting both IL-5 and CCR3 signalling systems for the resolution of inflammation and AHR associated with asthma.

Early pregnancy factor suppresses experimental autoimmune encephalomyelitis induced in Lewis rats with myelin basic protein and in SJL/J mice with myelin proteolipid protein peptide 139-151.

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties. During pregnancy, it appears in maternal serum within 6-24 h of fertilization, is present for at least the first two-thirds of pregnancy in all species studied and is essential for embryonic survival. It is a homologue of chaperonin 10, a heat shock protein, but, unlike other members of this family, EPF has an extracellular role. As it has the ability to modulate CD4+ T cell-dependent immune responses, its role in treatment of experimental autoimmune encephalomyelitis (EAE) was investigated. EAE is a CD4+ T cell-mediated disease, the best available animal model of multiple sclerosis (MS). Two models of EAE were investigated, acute EAE induced in Lewis rats by inoculation with myelin basic protein (MBP-EAE) and chronic relapsing EAE induced in SJL/J mice by inoculation with myelin proteolipid protein peptide (residues 139-151) (PLP-EAE). EPF, delivered intraperitoneally or orally to rats or intraperitoneally to mice, suppressed clinical signs of disease. Mice with PLP-EAE were also treated with interferon-beta, with and without EPF. Both EPF and IFN-beta suppressed clinical signs of EAE and, when administered together, gave greater suppression than when given separately. These findings suggest that EPF may be a potential candidate for use in treatment of MS and may be of use in combined therapy with IFN-beta.

The effect of IL-5 and eotaxin expression in the lung on eosinophil trafficking and degranulation and the induction of bronchial hyperreactivity.

The mechanisms regulating the selective migration and degranulation of eosinophils in the asthmatic lung and the subsequent development of airways hyperreactivity (AHR) have not been fully delineated. In this investigation, we have employed a novel transgene model to facilitate the dissection of the contributions of IL-5 and/or eotaxin to eosinophil function in the absence of complex tissue signals derived from the allergic lung. Gene transfer of IL-5 and/or eotaxin to the lungs of naive mice induced a pronounced and selective airways eosinophilia, but did not result in eosinophil degranulation or AHR. Airways eosinophilia occurred independently of the induction of a blood eosinophilia, but was markedly augmented by the coexpression of both cytokines and/or by the transient mobilization of eosinophils from the bone marrow by the administration of i.v. IL-5. However, for eosinophil degranulation and AHR to occur, the inhalation of Ag was required in association with IL-5 and eotaxin expression. Investigations in IL-5-deficient mice linked eosinophilia, and not solely IL-5 and eotaxin, with the induction of AHR. Furthermore, eosinophil degranulation and AHR were dependent on CD4+ T cells. Importantly, this investigation shows that IL-5 regulates eosinophilia within the lung as well as in the circulation and also amplifies eotaxin-induced chemotaxis in the airway compartment. Moreover, the interplay between these cytokines, CD4+ T cells, and factors generated by Ag inhalation provides fundamental signals for eosinophil degranulation and the induction of AHR.

Cellular and molecular regulation of eosinophil trafficking to the lung.

Airway inflammation is central to the pathogenesis of allergic asthma, and molecules that mediate this process obviously represent targets for therapy. In the present article, we discuss our experiments, which point to CD4+ T cells and IL-5-driven eosinophilia as potential targets for the relief of bronchial hyperreactivity in late-phase asthma.

Relationship between interleukin-5 and eotaxin in regulating blood and tissue eosinophilia in mice.

The mechanism of cooperation between IL-5 and eotaxin for the selective accumulation of eosinophils at sites of allergic inflammation is unknown. In this investigation we have used IL-5 deficient mice to define the relationship between this cytokine and eotaxin in the regulation of blood eosinophilia and eosinophil homing and tissue accumulation. Both IL-5 and eotaxin could independently induce a rapid and pronounced blood eosinophilia in wild type mice when administered systemically. In contrast, only eotaxin induced a pronounced blood eosinophilia in IL-5 deficient mice. The eosinophilic response induced by intravenous eotaxin in wild type mice did not correlate with a significant reduction in the level of bone marrow eosinophils, whereas intravenous IL-5 resulted in depletion of this store. These results suggest the existence of two mechanisms by which eosinophils can be rapidly mobilized in response to intravenous eosinophil chemoattractants; first, mobilization of an IL-5 dependent bone marrow pool, and second, an eotaxin-induced sequestration of eosinophils from tissues into the blood. Subcutaneous injection of eotaxin induced a local tissue eosinophilia in wild type mice but not in IL-5 deficient mice. Furthermore, tissue eosinophilia in wild type mice, but not in IL-5 deficient mice, was enhanced by adoptive transfer of eosinophils or the administration of intravenous IL-5. However, pretreatment of IL-5 deficient mice with intraperitoneal IL-5 for 72 h restored eosinophil homing and tissue accumulation in response to subcutaneous eotaxin. We propose that eotaxin secreted from inflamed tissue may play an important role in initiating both blood and tissue eosinophilia in the early phases of allergic inflammation. Furthermore, IL-5 is not only essential for mobilizing eosinophils from the bone marrow during allergic inflammation, but also plays a critical role in regulating eosinophil homing and migration into tissues in response to eotaxin and possibly other specific chemotactic stimuli.