The hemolysin-producer coelomocytes in Holothuria polii

Using sodium metrizoate discontinuous gradients, two hemolysin-producer amebocyte populations have been separated from total circulating Holothuria polii coelomocytes. The amebocytes of population 1 are responsible for the production of the calcium-dependent and temperature-labile hemolysin, whereas those of population 2 produce the calcium-independent and temperature-stable one. The intracytoplasmic hemolysins were evidenced also by immunofluorescence. Petaloid and filipodial amebocytes were the only positive cell types.     

Effects of antisense mediated inhibition of cathepsin K on human osteoclasts obtained from peripheral blood.

Cathepsin K is a cystein protease that displays a proteolytic activity against Type I collagen and is abundantly and selectively expressed in osteoclasts where it plays a critical role in bone degradation. Its direct role in bone tissue has been defined by knock-out mice studies and inhibiting strategies in animals models. However, direct proof of cathepsin K function in human osteoclast model in vitro is lacking. The aim of this study is to analyze cathepsin K expression and localization in human osteoclasts obtained from peripheral blood and to examine cathepsin K function in these cells by antisense oligodeoxynucleotide (AS-ODN) strategy. AS-ODN was added to the culture of osteoclast precursors induced to differentiate by RANKL and M-CSF. AS-ODN treatment produced a significant down-regulation of cathepsin K mRNA (>80%) and protein expression, as verified respectively by Real-time PCR and by immunocytochemistry or Western blot. The cathepsin K inhibition caused an impairment of resorption activity as evaluated by a pit formation assay ( p = 0.045) and by electron microscopy, while the acidification process was unaffected. We demonstrated that antisense strategies against cathepsin K are selectively effective to inhibit resorption activity in human osteoclasts, like in animal models.     

Giant cavernous hemangiomas: Diagnosis and surgical strategies

From January, 1972 to June, 1989, 51 patients with liver hemangiomas (32 females and 19 males, mean age 35 years) were evaluated for surgical treatment. Diameters of the masses were 5 cm to 20 cm (median 8.5 cm). Nine of the patients had already been treated for cancer. Twenty-two (43.1%) of the 51 patients were symptomatic and 29 (56.9%) patients were asymptomatic. In 34 patients (66.7%) a definite diagnosis of hemangioma was made by scintiscan and/or ultrasound and/or computed tomography and/or angiography while in the remaining 17 (33.3%) patients the diagnosis was uncertain. The most common indications for resection were the presence of a symptomatic angioma, a symptomatic mass with an uncertain diagnosis, and/or lack of a definite pre-operative diagnosis. Surgery was performed on 25 patients. Ten anatomic and 15 atypical resections or enucleations were performed. There were no postoperative deaths. Two further patients, operated for probable hemangioma, were found to have primary hepatic malignancies. In the 26 unresected patients, no complications were observed during follow-up. In 3 patients, hemangioma enlargement was detected by ultrasound, but there were no symptoms. As cavernous liver hemangiomas are now more reliably diagnosed and their natural history is usually uneventful, surgery can be avoided in most cases. However, when a non-resection policy is adopted, an exact diagnosis is essential in order to rule out primary or metastatic cancer. Surgical exploration and treatment should be limited to symptomatic or complicated cases as well as to patients with an uncertain diagnosis.

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Resumen Cincuenta y un pacientes con hemangiomas del higado (32 mujeres y 19 hombres, edad promedio 35 años) fueron valorados en cuanto a tratamiento quirúrgico en el período enero 1972 a junio de 1989; el diámetro de las lesiones oscilo entre 5 y 20 cm (promedio 8.5 cm). Nueve de los pacientes ya habían sido tratados por cáncer; 22/51 (43.1%) estaban sintomáticos y 29/51 (56.9%) eran asintomáticos. En 34/51 (66.7%) se hizo el diagnóstico definitivo de hemangioma mediante escintigrafia y/o ultrasonido y/o tomografía computadorizada y/o angiografia, en tanto que en los otros 17 pacientes (33.3%) el diagnóstico resultó incierto. Las indicaciones más comunes para resección fueron: presencia de un angioma sintomático, una masa asintomática con diagnóstico incierto y/o ausencia de diagnóstico preoperatorio definitivo. Se practicó cirugía en 25/51 pacientes, habiéndose realizado 10 resecciones anatómicas y 15 resecciones atípicas o enucleaciones. No hubo muertes postoperatorias. Otros dos pacientes operados por probable hemangioma demostraron tener neoplasias malignas hepáticas primarias. En los 26 pacientes no resecados no se observaron complicaciones durante el seguimiento; en tres casos se detectó ensanche del hemangioma en el examen con ultrasonido, pero no se presentaron síntomas. Puesto que actualmente los hemangiomas cavernosos del hígado pueden ser diagnosticados con mayor certeza y puesto que su historia natural generalmente está libre de complicaciones es posible evitar la cirugía en la mayoría de los casos. Sin embargo, cuando se pretenda adoptar una política de no resección es esencial establecer el diagnóstico exacto para excluir la posibilidad de un cáncer primario o metastásico. La exploración y el tratamiento quirúrgicos debe permanecer limitados a los hemangiomas sintomáticos o complicados, y en ningún caso deben significar riesgo para el paciente.

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Résumé De Janvier 1972 à Juin 1989, 51 patients ayant un hémangiome du foie (32 femmes et 19 hommes, âge moyen 35 ans) ont été examinés en vue d'une exérèse chirurgicale. Le diamètre de la lésion variait entre 5 et 20 cm (médiane = 8.5 cm). Neuf des patients avaient déjà été traités pour un cancer; 22/51 (43.1%) étaient symptomatiques alors que 29/51 (56.9%) étaient asymptomatiques. Chez 34/51 patients (66.7%), le diagnostic définitif d'hémangiome a été confirmé par scintigraphie et/ou échographie et/ou tomodensitométrie, alors que chez les 17 autres (33.3%), le diagnostic était uncertain. Les indications d'une résection les plus fréquentes étaient: la présence d'un angiome symptomatique, une masse symptomatique avec un diagnostic uncertain, et/ou absence de diagnostic définitif préopératoire. Vingt-cinq des 51 patients ont été opérés. Il y a eu 10 résections anatomiques et 15 résections atypiques ou énucléations. Il n'y a pas eu de mortalité postopératoire. Deux autres patients, traités pour ce que l'on soupçonnait être une probalbe hémangiome du foie, avaient en fait un cancer hépatique. Chez les 26 patients non résequés, il n'y avait pas eu de complication. Chez trois patients, on a mis en évidence une augmentation de volume par l'échographie, mais ces modifications ne s'accompagnait d'aucune symptomatologie. Comme on peut faire le diagnostic d'hémangiome carverneux du foie avec plus de fiabilité qu'avant, et comme on sait que leur histoire naturelle est généralement bénigne, on peut le plus souvent surseoir à l'exérèse chirurgicale. Cependant, il importe de toujours faire le diagnositc avec certitude, de façon à éliminer un cancer primitif ou sécondaire du foie.

     

Genome and transcriptome scale portrait of sigma factors in Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease (JD), a chronic gastroenteritis of ruminants and other animals, including primates. Many evidences suggested association of MAP to Crohn's disease, a chronic granulomatous gastrointestinal disease of humans with strong similarities with JD. The present study attempts to evaluate global gene regulation in MAP, which has not been addressed previously, despite the availability of MAP genome sequence. For this purpose, we investigated: (i) the presence of sigma factors and their relationship to sigma factors of other mycobacteria (M. avium subsp.avium, M. tuberculosis, M. bovis, M. leprae and M. smegmatis), and (ii) their expression during different growth conditions and in vitro infection of intestinal epithelial Caco2 cells. MAP genome contains 19 putative sigma factor, but only 12 belong to gene families common to other mycobacteria. Gene expression was evaluated with Real-Time PCR during growth in 7H9 medium and mycobactin J, in 7H9 medium plus mycobactin J and lisozyme, and during infection of Caco2 cells: very different expression patterns were observed and, on the whole, only 7 sigma factors were found to be expressed. sigJ was upregulated during the infection of Caco2 cells. Even if only few sigma factors were expressed in the three conditions tested, the overall high numbers of MAP sigma factors suggests a noteworthy flexibility of this pathogen. Thus, this first report on expression of MAP sigma factors opens the way to an extensive characterization of global gene regulation, as a key to understand strategies of survival and mechanisms of infections used by this organism.     

Adoptive immunotherapy of feline immunodeficiency virus with autologous ex vivo-stimulated lymphoid cells modulates virus and T-cell subsets in blood

The potential of immunotherapy with autologous virus-specific T cells to affect the course of feline immunodeficiency virus (FIV) infection was explored in a group of specific-pathogen-free cats infected with FIV a minimum of 10 months earlier. Popliteal lymph node cells were stimulated by cocultivation with UV-inactivated autologous fibroblasts infected with recombinant vaccinia viruses expressing either FIV gag or env gene products, followed by expansion in interleukin-2. One or two infusions of both Gag- and Env-stimulated cells resulted in a slow increase in FIV-specific gamma interferon-secreting T cells in the circulation of cats. In the same animals, viral set points fluctuated widely during the first 2 to 3 weeks after adoptive transfer and then returned to pretreatment levels. The preexisting viral quasispecies was also found to be modulated, whereas no novel viral variants were detected. Circulating CD4(+) counts underwent a dramatic decline early after treatment. CD4/CD8 ratios remained instead essentially unchanged and eventually improved in some animals. In contrast, a single infusion of Gag-stimulated cells alone produced no apparent modulations of infection.     

Alteration of acylphosphatase levels in familial Alzheimer''s disease fibroblasts with presenilin gene mutations

Acylphosphatase (ATPase), an enzyme that modulates the activity of Ca²⁺-ATPase by hydrolysing its phosphorylated moiety, has been found to be significantly higher in cultured skin fibroblasts from donors affected by early onset familial Alzheimer's disease (EOFAD) with PS-1 and PS-2 gene mutations. Of the two known isoenzymes of acylphosphatase, only the erythrocyte one accounts for the total increase in activity. No relevant alteration was observed in phosphotyrosine phosphatase activity (PTPase), in Ca²⁺-ATPase and Na⁺,K⁺-ATPase activities of the same cells as compared to age-matched controls. This finding could suggest a possible explanation for the calcium-dependent biochemical alterations previously described in Alzheimer's disease fibroblasts.     

Mannoprotein from Cryptococcus neoformans promotes T-helper type 1 anticandidal responses in mice

We previously demonstrated that mannoprotein (MP) from Cryptococcus neoformans (CnMP) stimulates interleukin-12 production by human monocytes, thus fostering a T-helper type 1 (Th1) protective anticryptococcal response. In this paper we show that CnMP was also able to induce a Candida albicans-directed protective Th1 response. This was demonstrated for mice immunized with CnMP by induction of a delayed-type hypersensitivity (DTH) reaction to C. albicans MP (CaMP) as well as induction of gamma interferon production by CD4(+) and CD8(+) splenic T cells stimulated in vitro with CaMP. CnMP-immunized mice were also partially protected from lethal systemic challenge with C. albicans, as shown by prolonged median survival times and decreased fungal burden in the kidney. Much evidence supports the validity of these cross-reactive and functional Th1 responses: (i) a non-cross-reactive C. albicans antigen, such as enolase, did not produce a DTH response to CaMP; (ii) passive adoptive transfer of T cells primed with CnMP induced a DTH reaction; (iii) C. neoformans extract elicited a DTH response to CaMP; and (iv) a monoclonal antibody (7H6) directed against a major and immunodominant T-cell-stimulatory 65-kDa MP (MP65) of C. albicans also recognized discrete 100-kDa constituents of C. neoformans extracts, as well as secretory constituents of the fungus. These results suggest the presence of common Th1 antigenic determinants in the mannoproteic material of C. neoformans and C. albicans epitopes, which should be considered in devising common strategies for immunoprophylactic or immunotherapeutic control of the fungi.     

Neurological phenotype of Potocki–Lupski syndrome

Potocki–Lupski syndrome is a condition mainly characterized by infantile hypotonia, developmental delay/intellectual disability (DD/ID), and congenital anomalies, caused by duplications of the 17p11.2 region, encompassing RAI1 gene. Its clinical presentation is extremely variable, especially for what concerns the cognitive level and the behavioral phenotype. Such aspects, as well as the dysmorphic/malformative ones, have been covered by previous studies; otherwise neurological features have never been systematically described. In order to delineate the neurological phenotype of Potocki–Lupski Syndrome, we collect an 8‐patients cohort. Developmental milestones are delayed and a mild to moderate cognitive impairment is present in all patients, variably associated with features of autism spectrum disorder, behavioral disturb, and sleep disturb. Hypotonia appears a less frequent finding than what previously reported, while motor clumsiness/coordination impairment is frequent. EGG registration demonstrated a common pattern with excess of diffuse rhythmic activity in sleep phases or while the patient is falling asleep. Brain MRI did not reveal common anomalies, although unspecific white matter changes may be present. We discuss such findings and compare them to literature data, offering an overview on the neurological and cognitive‐behavioral presentation of the syndrome.     

Selected RD1 Peptides for Active Tuberculosis Diagnosis: Comparison of a Gamma Interferon Whole-Blood Enzyme-Linked Immunosorbent Assay and an Enzyme-Linked Immunospot Assay

We recently set up a gamma interferon (IFN-γ) enzyme-linked immunospot assay (ELISPOT), using selected early secreted antigenic target 6 (ESAT-6) peptides, that appears specific for active tuberculosis (A-TB). However, ELISPOT is difficult to automate. Thus, the objective of this study was to determine if the same selected peptides may be used in a technique more suitable for routine work in clinical laboratories, such as whole-blood enzyme-linked immunosorbent assay (WBE). For this purpose, 27 patients with A-TB and 41 control patients were enrolled. Our WBE, using the already described selected peptides from ESAT-6 plus three new ones from culture filtrate protein 10, was performed, and data were compared with those obtained by ELISPOT. Using our selected peptides, IFN-γ production, evaluated by both WBE and ELISPOT, was significantly higher in patients with A-TB than in controls (P < 0.0001). Statistical analysis showed a good correlation between the results obtained by WBE and ELISPOT (r = 0.80, P < 0.001). To substantiate our data, we compared our WBE results with those obtained by QuantiFERON-TB Gold, a whole-blood assay based on region of difference 1 (RD1) overlapping peptides approved for TB infection diagnosis. We observed a slightly higher sensitivity with QuantiFERON-TB Gold than with our WBE (89% versus 81%); however, our test provided a better specificity result (90% versus 68%). In conclusion, results obtained by WBE based on selected RD1 peptides significantly correlate with those generated by ELISPOT. Moreover, our assay appears more specific for A-TB diagnosis than QuantiFERON-TB Gold, and thus it may represent a complementary tool for A-TB diagnosis for routine use in clinical laboratories.     

Reactive Compatibilization of PA6/LDPE Blends with Glycidyl Methacrylate Functionalized Polyolefins

Summary: Blends of polyamide‐6 (PA6) and low density polyethylene (LDPE) were compatibilized by melt mixing with various polyolefins functionalized with glycidyl methacrylate (GMA), i.e., GMA grafted LDPE (LDPE‐g‐GMA), GMA grafted styrene‐ethylene/butylene‐styrene block copolymer (SEBS‐g‐GMA) and ethylene‐co‐glycidyl methacrylate copolymer (E‐GMA). Blends with PA6/LDPE composition ratios of 25/75 and 75/25 wt.‐%/wt.‐% were prepared in a Brabender internal mixer and their properties were evaluated by SEM, rheological measurements and DSC. Morphological investigation by SEM showed a neat improvement of phase dispersion and interfacial adhesion in all compatibilized blends when compared to PA6/LDPE binary blends. The variation of the dispersed phase size was analyzed as a function of blend composition, compatibilizer concentration and GMA content. The emulsification curves of compatibilized blends showed that the equilibrium size of dispersed particles at the saturation concentration of copolymer was lower when PA6 was the major component. The finest dispersion of the LDPE phase (<0.25 μm) was observed in the presence of SEBS‐g‐GMA copolymer. LDPE‐g‐GMA and E‐GMA displayed a similar compatibilizing efficiency. In all cases, the blends with a polyamide matrix presented a marked rise in torque and melt viscosity with increasing compatibilizer content. These effects were accounted for by a reaction between the epoxide groups of LDPE‐g‐GMA and the carboxyl/amine end‐groups of PA6, leading to the formation of an interchain graft copolymer. The phase transition processes of PA6 in the blends were influenced by the compatibilizer content and the interfacial interactions between the polymer components, suggesting a different role for the compatibilizer at the PA6/LDPE interface.

SEM micrograph of PA6/LDPE 25/75 blend compatibilized with 2.5 phr SEBS‐g‐GMA.