ETP-46321, a dual p110α/δ class IA phosphoinositide 3-kinase inhibitor modulates T lymphocyte activation and collagen-induced arthritis.

Class IA phosphoinositide 3-kinases (PI3Ks) are essential to function of normal and tumor cells, and to modulate immune responses. T lymphocytes express high levels of p110α and p110δ class IA PI3K. Whereas the functioning of PI3K p110δ in immune and autoimmune reactions is well established, the role of p110α is less well understood. Here, a novel dual p110α/δ inhibitor (ETP-46321) and highly specific p110α (A66) or p110δ (IC87114) inhibitors have been compared concerning T cell activation in vitro, as well as the effect on responses to protein antigen and collagen-induced arthritis in vivo. In vitro activation of naive CD4(+) T lymphocytes by anti-CD3 and anti-CD28 was inhibited more effectively by the p110δ inhibitor than by the p110α inhibitor as measured by cytokine secretion (IL-2, IL-10, and IFN-γ), T-bet expression and NFAT activation. In activated CD4(+) T cells re-stimulated through CD3 and ICOS, IC87114 inhibited Akt and Erk activation, and the secretion of IL-2, IL-4, IL-17A, and IFN-γ better than A66. The p110α/δ inhibitor ETP-46321, or p110α plus p110δ inhibitors also inhibited IL-21 secretion by differentiated CD4(+) T follicular (Tfh) or IL-17-producing (Th17) helper cells. In vivo, therapeutic administration of ETP-46321 significantly inhibited responses to protein antigen as well as collagen-induced arthritis, as measured by antigen-specific antibody responses, secretion of IL-10, IL-17A or IFN-γ, or clinical symptoms. Hence, p110α as well as p110δ Class IA PI3Ks are important to immune regulation; inhibition of both subunits may be an effective therapeutic approach in inflammatory autoimmune diseases like rheumatoid arthritis.

Suppression of CD4+ T lymphocyte activation in vitro and experimental encephalomyelitis in vivo by the phosphatidyl inositol 3-kinase inhibitor PIK-75.

Class IA phosphatidyl inositol-3 kinases (PI3-K) are important targets in cancer therapy and are essential to immune responses, particularly through costimulation by CD28 and ICOS. Thus, small PI3-K inhibitors are likely candidates to immune intervention. PIK-75 is an efficient inhibitor of the PI3-K p110alpha catalytic subunits that suppresses tumor growth, and its effects on immune and autoimmune responses should be studied. Here, we describe the effect of PIK-75 on different immune parameters in vitro and in vivo. PIK-75 at concentrations commonly used in vitro (≥0.1 μM) inhibited T and B cell activation by Concanavalin A and LPS, respectively, and survival of non-stimulated spleen cells. In naive CD4+ T lymphocytes, PIK-75 induced apoptosis of resting or activated cells that was prevented by caspase inhibitors. At low nanomolar concentrations (≤10 nM), PIK-75 inhibited naive CD4+ T cell proliferation, and IL-2 and IFN-gamma production induced by anti-CD3 plus anti-CD28. In activated CD4+ T blasts costimulated by ICOS, PIK-75 (less than 10 nM) inhibited IFN-gamma, IL-17A, or IL-21 secretion. Furthermore, PIK-75 (20 mg/kg p.o.) suppressed clinical symptoms in ongoing experimental autoimmune encephalomyelitis (EAE) and inhibited MOG-specific responses in vitro. Thus, PIK-75 is an efficient suppressor of EAE, modulating lymphocyte function and survival.

Brachytherapy for isolated tongue metastasis of renal clear cell carcinoma.

INTRODUCTION: Tongue metastasis of renal cell carcinoma (RCC) is rare. Treatment is hindered by the proximity of anatomic structures involved in swallowing, speech and mastication. It is, moreover, radioresistant. CASE REPORT: We report a case of inaugural isolated tongue metastasis, where biopsy redirected diagnosis to RCC. To avoid potentially mutilating excision in a metastatic disease of poor prognosis, treatment was local, consisting in interstitial brachytherapy, enabling a high dose (65Gy) to be delivered. Despite initially complete response, recurrence at 10.5months required salvage surgery. DISCUSSION AND CONCLUSION: Brachytherapy allowed a higher dose to be delivered than with external beam radiation therapy, previously reported for similar cases. This dose, which can usually be expected to ensure local control in 90% of squamous cell carcinomas of the tongue, obtained 10months' response, insufficient to avoid salvage surgery.

[Recurrent painful ophthalmoplegia secondary to polyostotic fibrous dysplasia of the maxillary sinuses with involvement of the superior orbital fissure]. / Oftalmoplejia dolorosa recurrente secundaria a displasia fibrosa poliostotica del seno maxilar con afectacion de la hendidura esfenoidal.

INTRODUCTION: Fibrous dysplasia is a bone disease that is usually accompanied by asymptomatic lesions but which may sometimes display neurological manifestations due to the involvement of the craniofacial bones. CASE REPORT: A 25-year-old female, with a history of migraines, who visited at the age of 18 due to pain in the right retro-ocular and maxillary region, although with characteristics that were unlike those of her usual migraines, and which was associated with ipsilateral ophthalmoparesis. The condition had a self-limiting course and responded well to corticoids, although it was recurring. Examination revealed intense pain on palpation of the right-hand maxillary sinus and incomplete paralysis of the ipsilateral oculomotor nerve with palpebral ptosis. Results of complementary studies were normal, except for magnetic resonance imaging of the head and computerised axial tomography of the face, which revealed an expansive lesion with involvement of the right superior maxillary sinus and the greater wing of the sphenoid bone, with probable compromise of the superior orbital fissure, consistent with the diagnosis of fibrous dysplasia, which was confirmed by means of a pathology study. CONCLUSIONS: Fibrous dysplasia is a benign bone disorder, of unknown causation, in which normal bone tissue is replaced by amorphous conjunctive tissue. There is sometimes craniofacial involvement and a hypertrophic bone mass is formed which can fill the paranasal sinuses and the orbit, resulting in exophthalmus and visual disorders. To date the scientific literature does not include any reports of this disease manifesting as bouts of recurrent painful ophthalmoparesis which responds to corticoids, as happened in the case of our patient.

[Lepra: an uncommon cause of infectious neuropathy]. / Lepra: causa infrecuente de neuropatía potencialmente tratable.

INTRODUCTION: Lepra is an uncommon disease within our setting. However, it was considered the most frequent cause of polyneuropathy only 50 years ago. CASE REPORT: We present the case of a 37 year-old woman who consulted due to paresthesias in both hands and feet, livedo reticularis and complaints of frequent hand lesions. Examination of the skin detected nodular lesions and the neurophysiological study confirmed distal symmetric sensitive polyneuropathy with axonal predominance. The skin biopsy also showed histocytic infiltrate and mycobacterium lepra type intracytoplasmatic bacilli. This led to the diagnosis of Multibacillary lepromatous leprosy. Multiple treatment was begun, according to the World Health Organization recommendations, with good evolution. In spite of the attempts to eradicate lepra, its incidence continues to be elevated, especially in endemic areas, among which the south of Spain is included. It is generally associated to overcrowding and low social-economic level. CONCLUSIONS: This is the last endemic case in the Valencian Community. The possibility of lepra should be considered among the possible causes of sensitive polyneuropathy, above all in patients in endemic areas.

Dendritic cells: still a promising tool for cancer immunotherapy.

Dendritic cells are bone marrow-derived professional antigen-presenting cells that exert critical functions in innate and adaptive immune responses. Depending on their functional maturation status, dendritic cells trigger primary immune responses or promote immunological tolerance. This functional ambivalence has taken dendritic cells into the focus of attention of immunotherapy protocols for both vaccination and tolerance induction. The capacity of dendritic cells to generate anti-tumour immune responses has already been demonstrated, and numerous clinical trials are currently in progress to assess their therapeutic potential. In the present review we will briefly outline the types and effector functions of dendritic cells in the human system, and summarise the present state of anti-tumour immunotherapy protocols, emphasising the most relevant parameters currently evaluated in preclinical and clinical assays.

[Binocular diplopy as the presentation of a haematoma in the rectus inferior]. / Diplopía binocular como presentación de un hematoma del recto inferior.

INTRODUCTION: The presence of quick onset binocular diplopy makes it essential to carry out a comprehensive differential diagnosis. The most frequent causes in adults include vascular, post-traumatic, tumorous and myopathic pathologies. Yet, to perform a differential diagnosis we also have to take into account less common aetiologies such as demyelinating disease, carotid-cavernous fistulas and Tolosa Hunt syndrome, among others. We report the case of a patient who was admitted to our hospital because she presented an acute onset diplopy secondary to a spontaneous haematoma of the left rectus inferior. CASE REPORT: We describe the case of a 54-year-old female with a history of hypercholesterolemia, who was a smoker and allergic to iodine contrasts and who visited because of an acute onset diplopy accompanied by instability and pain in the left eye. The neurological examination revealed paresis of the extraocular muscles dependent on the left oculomotor nerve with left palpebral ptosis, paresis of the rectus inferior, with no involvement of the pupils. Other results of the exploration were within normal limits. A computerised axial tomography scan of the head showed a haemorrhage in the rectus inferior. Magnetic resonance images of the head revealed an increase in the volume of the left-side rectus inferior with signs of bleeding, and the rest of the complementary explorations were normal. The patient progressed favourably and the diplopy gradually disappeared. CONCLUSIONS: Spontaneous bleeding of the extraocular muscles, although infrequent, must be taken into account in the differential diagnosis of painful ophthalmoplegia with diplopy in patients with no underlying pathology, especially in the absence of other neurological disorders.

Tyrosinase action on monophenols: evidence for direct enzymatic release of o-diphenol.

Using gas chromatography-mass spectrometry, the direct enzymatic release of o-diphenol (4-tert-butylcatechol) during the action of tyrosinase on a monophenol (4-tert-butylphenol) has been demonstrated for the first time in the literature. The findings confirm the previously proposed mechanism to explain the action of tyrosinase on monophenols (J.N. Rodríguez-López, J. Tudela, R. Varón, F. García-Carmona, F. García-Cánovas, J. Biol. Chem. 267 (1992)). Oxytyrosinase, the oxidized form of the enzyme with a peroxide group, is the only form capable of catalysing the transformation of monophenols into diphenols, giving rise to an enzyme-substrate complex in the process. The o-diphenol formed is then released from the enzyme-substrate complex or oxidized to the corresponding o-quinone. In order to detect the enzymatic release of o-diphenol, the non-enzymatic evolution of the o-quinone to generate o-diphenol by weak nucleophilic attack reactions and subsequent oxidation-reduction was blocked by the nucleophilic attack of an excess of cysteine. Furthermore, the addition of catalytic quantities of an auxiliary o-diphenol (e.g. catechol) considerably increases the accumulation of 4-tert-butylcatechol. The enzyme acting on 4-tert-butylphenol generates the enzyme-4-tert-butylcatechol complex and 4-tert-butylcatechol is then released (with k(-2)) generating mettyrosinase. The auxiliary o-diphenol added (catechol) and the 4-tert-butylcatechol generated by the enzyme then enter into competition. When [catechol] >> [4-tert-butylcatechol], the enzyme preferentially binds with the catechol to close the catalytic cycle, while 4-tert-butylcatechol is accumulated in the medium. In conclusion, we demonstrate that the enzyme produces 4-tert-butylcatechol from 4-tert-butylphenol, the concentration of which increases considerably in the presence of an auxiliary o-diphenol such as catechol.

Analysis and interpretation of the action mechanism of mushroom tyrosinase on monophenols and diphenols generating highly unstable o-quinones.

Tyrosinase can act on monophenols because of the mixture of met- (E(m)) and oxy-tyrosinase (E(ox)) which exists in the native form of the enzyme. The latter form is active on monophenols, while the former is not. However, the kinetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex since the products of the enzymatic reaction, the o-quinones, are unstable and continue evolving to generate o-diphenols in the medium. In the case of substrates such as L-tyrosine, tyrosinase generates very unstable o-quinones, in which a process of cyclation and subsequent oxidation-reduction generates o-diphenol through non-enzymatic reactions. However, the release of o-diphenol through the action of the enzyme on the monophenol contributes to the concentration of o-diphenol in the first pseudo-steady-state [D(0)](ss). Hence, the system reaches an initial pseudo-steady state when t-->0 and undergoes a transition phase (lag period) until a final steady state is reached when the concentration of o-diphenol in the medium reaches the concentration of the final steady state [D(f)](ss). These results can be explained by taking into account the kinetic and structural mechanism of the enzyme. In this, tyrosinase hydroxylates the monophenols to o-diphenols, generating an intermediate, E(m)D, which may oxidise the o-diphenol or release it directly to the medium. We surmise that the intermediate generated during the action of E(ox) on monophenols, E(m)D, has axial and equatorial bonds between the o-diphenol and copper atoms of the active site. Since the orbitals are not coplanar, the concerted oxidation-reduction reaction cannot occur. Instead, a bond, probably that of C-4, is broken to achieve coplanarity, producing a more labile intermediate that will then release the o-diphenol to the medium or reunite it diaxially, involving oxidation to o-quinone. The non-enzymatic evolution of the o-quinone would generate the o-diphenol ([D(f)](ss)) necessary for the final steady state to be reached after the lag period.

Enzyme inactivation analysis for industrial blanching applications: comparison of microwave, conventional, and combination heat treatments on mushroom polyphenoloxidase activity.

Browning reactions in fruits and vegetables are a serious problem for the food industry. In mushrooms, the principal enzyme responsible for the browning reaction is polyphenoloxidase (PPO). Microwaves have recently been introduced as an alternative for the industrial blanching of mushrooms. However, the direct application of microwave energy to entire mushrooms is limited by the important temperature gradients generated within the samples during heating, which can produce internal water vaporization and associated damage to the mushrooms texture. A microwave applicator has been developed, whereby irradiation conditions can be regulated and the heating process monitored. Whole edible mushrooms (Agaricus bisporus) were blanched by conventional, microwave, and combined heating methods to optimize the rate of PPO inactivation. A combined microwave and hot-water bath treatment has achieved complete PPO inactivation in a short time. Both the loss of antioxidant content and the increase of browning were minor in the samples treated with this combined method when compared to the control. This reduction in processing time also decreased mushroom weight loss and shrinkage.

Thermal inactivation of mushroom polyphenoloxidase employing 2450 MHz microwave radiation.

Browning reactions in fruits and vegetables are a serious problem for the food industry. In mushrooms, the principal enzyme responsible for the browning reaction is polyphenoloxidase (PPO). A microwave applicator has been designed and used for studying mushroom PPO inactivation. The effects of microwaves and conventional heating on the kinetics of the monophenolase and diphenolase activities of PPO were studied. Conventional and microwave treatments produce different enzyme intermediates with different stability and kinetic properties. We describe how considerable time can be saved during microwave inactivation of the enzyme compared with the time needed when conventional hot-water treatment is used, resulting in greater profitability and enhanced quality. The short exposure time required for samples irradiated with microwaves is very important for maintaining the quality of mushrooms. The fast microwave treatment used resulted in an increase in antioxidant content and a considerable decrease in browning.

[Coronary involvement in Takayasu's disease. Apropos of 3 cases, of which 2 were surgically treated, and review of the literature]. / AL'atteinte coronarienne dans la maladie de Takayasu. A propos de 3 cas dont 2 opérés et revue de la littérature.

Coronary arteries like other branches of the aorta may be involved in Takayasu's disease. This complication is not rare (7% of cases) but appears to be relatively unappreciated. Three new cases are reported of main coronary artery disease, two of which were treated by coronary bypass surgery. A review of the literature of 1 130 cases of Takayasu's disease revealed 86 cases with coronary involvement, 33 of which were confirmed anatomically and 15 by coronary arteriography. The clinical manifestations, angina and/or myocardial infarction, were present in 5% and 3% respectively, of patients with Takayasu's disease. They may be the first sign of the disease and, in some cases, the only symptomatic arterial localisation. The coronary lesions are either ostial, a direct complication of the aortic disease, or on a main vessel, usually proximal. Histological studies show typical changes of stenosing inflammatory panarteritis involving mainly the media and adventitia. Thrombosis and secondary atheromatous plaques may be observed. Aneurysms are rare. Apart from cases with typical ostial lesions, the coronary angiographic appearances are not specific, but some features are suggestive of the diagnosis; the occurrence in young women; the presence of associated peripheral arterial lesions, their localisation and grouping; their radiological and/or histological characteristics. The spontaneous prognosis of these proximal lesions is usually poor and justifies surgical revascularisation by coronary bypass. Six patients, including two in this series, have been treated surgically. The associated aortic lesions may pose special technical problems which we discussed. The relatively high incidence of coronary involvement in Takayasu's disease and its often unexpected revelation by myocardial infarction or sudden death, suggest that coronary arteriography should be undertaken more often during investigation of the arterial lesions of these patients. Takayasu's disease should figure prominently amongst the causes of coronary artery disease in young women.