Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity.

Cytokines and their receptors regulate haemopoiesis by controlling cellular growth, survival and differentiation. Thus it is not surprising that mutations of cytokine receptors contribute to the formation of haemopoietic disorders, including cancer. We recently identified transforming properties of IL27R, the ligand-binding component of the receptor for interleukin-27. Although wild-type IL27R exhibits transforming properties in haemopoietic cells, in the present study we set out to determine if the transforming activity of IL27R could be enhanced by mutation. We identified three mutations of IL27R that enhance its transforming activity. One of these mutations is a phenylalanine to cysteine mutation at residue 523 (F523C) in the transmembrane domain of the receptor. The two other mutations identified involve deletions of amino acids in the cytoplasmic juxtamembrane region of the receptor. Expression of each of these mutant IL27R proteins led to rapid cytokine-independent transformation in haemopoietic cells. Moreover, the rate of transformation induced by these mutants was significantly greater than that induced by wild-type IL27R. Expression of these IL27R mutants also induced enhanced activation of JAK (Janus kinase)/STAT (signal transducer and activator of transcription) signalling compared with wild-type. An activating deletion mutation of IL27R enhanced homodimerization of the receptor by a mechanism that may involve disulfide bonding. These transforming IL27R mutants displayed equal or greater transforming activity than bona fide haemopoietic oncogenes such as BCR-ABL (breakpoint cluster region-Abelson murine leukaemia viral oncogene homologue) and JAK2-V617F. Since IL27R is expressed on haemopoietic stem cells, lymphoid cells and myeloid cells, including acute myeloid leukaemia blast cells, mutation of this receptor has the potential to contribute to a variety of haemopoietic neoplasms.

Effectiveness of a modified therapeutic protocol for the management of vernal keratoconjunctivitis based on Bonini's graded clinical severity.

Purpose: To evaluate the effectiveness of a modified therapeutic protocol used for vernal keratoconjunctivitis (VKC) based on severity as per Bonini grading system. Methods: This was a prospective observational clinical study conducted with 123 eyes of 63 patients. A meticulous clinical examination was performed, and data was documented in all the cases. Patients on known systemic atopy and antiallergic therapy were excluded from the study. Eyes with a clinical diagnosis of VKC were segregated based on Bonnini's grading system. A treatment protocol was created depending on the grade of VKC. Therapeutic responses were documented at 3 weeks, 3 months, 6 months, 12 months, and 24 months. Grading of the eyes was performed in each visit. Results: The mean age of the patients was 8.85 years with a standard deviation of 4.48 years. Males were predominant, and 95.24% had bilateral manifestation. The palpebral component was the most common form of manifestation. Itching was the most common manifestation, followed by congestion, discharge, and papillae in a decreasing order. Also, 68% of patients were in grade 2, 14% in grade 3, 12% in grade 1, and the rest were in grade 4. Following the treatment protocol, 70% showed signs of significant improvement in grade by the end of 6 weeks, reaching 90% at the end of 6 months (P = 0.074) and 92% at the end of 12 months (P = 0.002). Also, 52.4% versus 77.8% of patients had no recurrence in the pre- versus posttreatment protocol and it was statistically significant (P = 0.001). Conclusion: Grading of VKC gives a clear evaluation of the severity and progression of the condition. Besides, significant improvement in the grades was observed with fewer incidences of recurrences following execution of the therapeutic protocol. Hence, it is essential to maintain a treatment protocol in our clinical practice to provide grade-based therapy and monitor accurate changes in the clinical condition.

Activation of JAK2-V617F by components of heterodimeric cytokine receptors.

The JAK2-V617F mutation is an important etiologic factor for the development of myeloproliferative neoplasms. The mechanism by which this mutated tyrosine kinase initiates deregulated signals in cells is not completely understood. It is believed that JAK2-V617F requires interactions with homodimeric cytokine receptors to elicit its transforming signal. In this study, we demonstrate that components of heterodimeric cytokine receptors can also activate JAK2-V617F. Expression of IL27Ra, a heterodimeric receptor component, enhanced the activation of JAK2-V617F and subsequent downstream signaling to activation of STAT5 and ERK. In addition, expression of components of the interleukin-3 receptor, IL3Ra and the common beta chain, activated JAK2-V617F as well as STAT5 and ERK. Importantly, expression of IL27Ra functionally replaced the requirement of a homodimeric cytokine receptor to promote the activation and transforming activity of JAK2-V617F in BaF3 cells. Tyrosine phosphorylation of IL27Ra was not required to induce activation of JAK2-V617F or STAT5, or to enhance the transforming activity of JAK2-V617F. Expression of IL3Ra or the common beta chain in BaF3 cells also enhanced the ability of JAK2-V617F to transform these hematopoietic cells. However, the heterodimeric receptor component IL12RB1 did not enhance the activation or transforming signals of JAK2-V617F in BaF3 cells. IL27Ra also activated the K539L and R683G JAK2 mutants. Together our data demonstrate that in addition to homodimeric receptors, some heterodimeric receptor components can support the activation and transforming signals of JAK2-V617F and other JAK2 mutants. Therefore, heterodimeric receptors may play unappreciated roles in JAK2 activation in the development of hematopoietic diseases including myeloproliferative neoplasms.

Transformation of hematopoietic cells and activation of JAK2-V617F by IL-27R, a component of a heterodimeric type I cytokine receptor.

From a patient with acute myeloid leukemia (AML), we have identified IL-27Ra (also known as TCCR and WSX1) as a gene whose expression can induce the transformation of hematopoietic cells. IL-27Ra (IL-27R) is a type I cytokine receptor that functions as the ligand binding component of the receptor for IL-27 and functions with the glycoprotein 130 (gp130) coreceptor to induce signal transduction in response to IL-27. We show that IL-27R is expressed on the cell surface of the leukemic cells of AML patients. 32D myeloid cells transformed by IL-27R contain elevated levels of activated forms of various signaling proteins, including JAK1, JAK2, STAT1, STAT3, STAT5, and ERK1/2. Inhibition of JAK family proteins induces cell cycle arrest and apoptosis in these cells, suggesting the transforming properties of IL-27R depend on the activity of JAK family members. IL-27R also transforms BaF3 cells to cytokine independence. Because BaF3 cells lack expression of gp130, this finding suggests that IL-27R-mediated transformation of hematopoietic cells is gp130-independent. Finally, we show that IL-27R can functionally replace a homodimeric type I cytokine receptor in the activation of JAK2-V617F, a critical JAK2 mutation in various myeloproliferative disorders (MPDs). Our data demonstrate that IL-27R possesses hematopoietic cell-transforming properties and suggest that, analogous to homodimeric type I cytokine receptors, single-chain components of heterodimeric receptors can also enhance the activation of JAK2-V617F. Therefore, such receptors may play unappreciated roles in MPDs.

Infantile simple dacryops: clinicopathological correlation and immunocharacterization.

Simple dacryops is a cystic lesion of the ductal structures of the palpebral lobe of the lacrimal gland and is the commonest lacrimal ductal cystic lesion in adults. We report the case of an 18-month-old girl with a painless swelling of the right temporal upper eyelid that was first noted at 4 months of age. An early surgical excision was planned to avoid deprivational amblyopia. We describe clinicopathological and immunohistochemical correlations in our case, highlighting the immuno-characterization of adjacent lacrimal gland duct and acinar structures present within the cyst wall and management of this relatively rare entity.

Thyroid eye disease survey: An anonymous web-based survey in the Indian subcontinent.

Purpose: To evaluate the current practice patterns in the treatment of thyroid eye disease (TED) in Indian subcontinent through a web-based survey of members of Oculoplastics Association of India (OPAI). Methods: This was an online web-based questionnaire survey disseminated via monkeysurvey.com to all ratified active members of OPAI between May 1, 2016 and June 30, 2016. Questions encompassed the background, training, region, and experience of oculoplastic surgeons along with the management protocol of TED. Results: Of the 435 emails sent to OPAI members, 9 bounced and 180 (42.3%) responded within the study period. A large majority (96%) of respondents were oculoplastic surgeons practicing in India and the remaining practiced within South-East Asia. Two-thirds of respondents were oculoplastic surgeons with less than 10 years of clinical experience; 82% were fellowship trained in Oculoplasty. Almost all (99%) favored a multidisciplinary management of TED. A large majority routinely grade the severity (89%) and activity (87%) of disease before management. While corticosteroid remained the treatment of choice, 54% preferred immune-modulators as the second-line of therapy for recalcitrant TED. Three-quarters did not use orbital radiotherapy as a management modality in active TED owing to concerns over its efficacy and/or safety. Conclusion: The survey gives useful insights to the practice patterns of TED management in Indian subcontinent. Multidisciplinary approach and grading of disease severity and activity were the rule rather than exception among OPAI members. Immune modulation was the preferred steroid-sparing agent in recalcitrant disease. Orbital radiotherapy was an uncommon treatment choice.

Functional role of JNK in neuritogenesis of PC12-N1 cells.

JNKs, also known as SAPKs, are activated in response to a wide variety of factors including growth factors, cytokines, UV radiation, and heat shock. In the rat pheochromocytoma PC12 cells, the JNK signaling pathway mediates diverse functions such as differentiation and apoptosis. We have previously shown that activated JNK is required for later stages of neuritogeneis induced by NGF in a variant PC12 cell line (N1). Here, the functional role of JNK in N1 cells is further investigated. We show that NGF treatment, which induces extensive neurite branching and cell soma enlargement in the N1 cells, stimulates a biphasic activation of JNK. The first phase of activation is rapid and transient, beginning at 15 min after NGF exposure and lasting approximately 45 min. The second phase of activation is sustained, beginning at 9-12 h of NGF treatment and lasting for at least 24 h. Similar biphasic pattern of JNK activation is also observed in the parental PC12 cells. Using the specific JNK inhibitor SP600125, we show that the biphasic activation is necessary for neurite outgrowth and branching, and that inhibition of either phase suppresses neuritogenesis in the N1 cells. These results suggest that dynamic JNK activation may play a key role in neurite outgrowth during neuronal development.

The PIM inhibitor AZD1208 synergizes with ruxolitinib to induce apoptosis of ruxolitinib sensitive and resistant JAK2-V617F-driven cells and inhibit colony formation of primary MPN cells.

Classical myeloproliferative neoplasms (MPNs) are hematopoietic stem cell disorders that exhibit excess mature myeloid cells, bone marrow fibrosis, and risk of leukemic transformation. Aberrant JAK2 signaling plays an etiological role in MPN formation. Because neoplastic cells in patients are largely insensitive to current anti-JAK2 therapies, effective therapies remain needed. Members of the PIM family of serine/threonine kinases are induced by JAK/STAT signaling, regulate hematopoietic stem cell growth, protect hematopoietic cells from apoptosis, and exhibit hematopoietic cell transforming properties. We hypothesized that PIM kinases may offer a therapeutic target for MPNs. We treated JAK2-V617F-dependent MPN model cells as well as primary MPN patient cells with the PIM kinase inhibitors SGI-1776 and AZD1208 and the JAK2 inhibitor ruxolitinib. While MPN model cells were rather insensitive to PIM inhibitors, combination of PIM inhibitors with ruxolitinib led to a synergistic effect on MPN cell growth due to enhanced apoptosis. Importantly, PIM inhibitor mono-therapy inhibited, and AZD1208/ruxolitinib combination therapy synergistically suppressed, colony formation of primary MPN cells. Enhanced apoptosis by combination therapy was associated with activation of BAD, inhibition of downstream components of the mTOR pathway, including p70S6K and S6 protein, and activation of 4EBP1. Importantly, PIM inhibitors re-sensitized ruxolitinib-resistant MPN cells to ruxolitinib by inducing apoptosis. Finally, exogenous expression of PIM1 induced ruxolitinib resistance in MPN model cells. These data indicate that PIMs may play a role in MPNs and that combining PIM and JAK2 kinase inhibitors may offer a more efficacious therapeutic approach for MPNs over JAK2 inhibitor mono-therapy.

The calcium-responsive transactivator recruits CREB binding protein to nuclear bodies.

The calcium-responsive transactivator (CREST) is required for the normal development of neuronal dendritic trees. Here we report that CREST is localized to sub-nuclear structures in the rat neuroendocrine pheochromocytoma PC12 cells. A yellow fluorescence protein-CREST fusion protein was expressed in HEK 293 and PC12 cells and the recombinant protein was exclusively targeted to nuclear bodies. A similar result was obtained with a Flag-tagged CREST. Deleting the N-terminal 148 or the C-terminal 79 amino acid sequences had no effect on targeting, whereas removing 164 amino acid residues from the C-terminus abolished nuclear body localization. We found that CREST did not co-localize with promyelocytic leukaemia oncoprotein (PML) body and was not targeted to PML bodies. Overexpression of CREST markedly increased the number of nuclear bodies positive for the histone acetyltransferase CREB binding protein (CBP). Double immunofluorescence staining of Flag-CREST and CBP suggested that CREST and CBP had a high degree of co-localization within the nuclear bodies. Deletion of the CBP binding domain of CREST inhibited the recruitment of CBP to CREST nuclear bodies. These results suggest that CBP recruitment to nuclear bodies by CREST may play an important role in CREST-mediated calcium-responsive transactivation, and CREST nuclear body may function as an assembly site for activators/co-activators in gene transcription control.

A multifunctional domain of the calcium-responsive transactivator (CREST) that inhibits dendritic growth in cultured neurons.

The calcium-responsive transactivator (CREST) is targeted to nuclear bodies and is required for the normal development of neuronal dendritic trees. Here we report the identification of a multifunctional domain (MFD) of CREST that is involved in transcription transactivation, nuclear body targeting, and dimerization. MFD is located near the C terminus of CREST from amino acid 251 to 322 and is required and sufficient for CREST homodimerization. When fused with a GAL4 DNA-binding domain, MFD was effective in transcription transactivation of a luciferase reporter system. A C-terminal 339-401 amino acid sequence of CREST was shown to contain a nuclear localization signal (NLS), which was able to direct a yellow fluorescence protein (YFP) to nucleus. A CREST deletion mutant containing both the MFD and NLS, which spanned the C-terminal amino acid sequence 251-401, was able to target YFP to the nucleus and nuclear bodies. However, MFD alone failed to target YFP and was largely cytosolic. The addition of a SV40 NLS to MFD domain restored nuclear body targeting. When YFP-MFD was expressed in cultured rat embryonic cortical neurons, it was effective in inhibiting depolarization-induced dendritic growth, suggesting that CREST dimerization may be necessary for its function in neuronal dendritic development.

An immunohistochemical method that distinguishes free from complexed SNAP-25.

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complexes composed of target (t-) SNAREs syntaxin and SNAP-25 and vesicle SNARE synaptobrevin play an essential role in neurosecretion. It is hypothesized that a transient intermediate complex between the t-SNAREs is formed during the assembly of the ternary complex. The existence of the t-SNARE binary complexes in vivo, however, has not been demonstrated. By using an affinity absorption scheme with preformed syntaxin-SNAP-25 complexes, we isolated antibodies capable of distinguishing free SNAP-25 from those associated with syntaxin. By semiquantitative immunohistochemistry, we estimated that, in cultured cerebellar neurons, the majority of SNAP-25 existed as complexes. Compared with the cultured neurons, PC12 cells expressed significantly less syntaxin, and we found that SNAP-25 was primarily in free forms. In contrast, a PC12 line that stably expressed a recombinant syntaxin showed a marked increase in SNAP-25 complexes. By using fluorescence resonance energy transfer (FRET) techniques, we observed FRET between cyan fluorescence protein-syntaxin and yellow fluorescence protein-SNAP-25 fusion proteins expressed in COS-7 and PC12 cells, suggesting a physiological interaction between syntaxin and SNAP-25. Our results demonstrate that, unlike what was previously hypothesized, syntaxin and SNAP-25 exist preferably as stable binary complexes in neurons. These findings offer novel insight into the mechanisms underlying the initiation and regulation of SNARE complex assembly.