Use of pressure-hold test for sterilizing filter membrane integrity in radiopharmaceutical manufacturing

The bubble point test is the de facto standard for postproduction filter membrane integrity test in the radiopharmaceutical community. However, the bubble point test depends on a subjective visual assessment of bubbling rate that can be obscured by significant diffusive gas flows below the manufacturer's prescribed bubble point. To provide a more objective means to assess filter membrane integrity, this study evaluates the pressure-hold test as an alternative to the bubble point test. In our application of the pressure-hold test, the nonsterile side of the sterilizing filter is pressurized to 85% of the predetermined bubble point with nitrogen, the filter system is closed off from the pressurizing gas and the pressure is monitored over a prescribed time interval. The drop in pressure, which has a known relationship with diffusive gas flow, is used as a quantitative measure of membrane integrity. Characterization of the gas flow vs. pressure relationship of each filter/solution combination provides an objective and quantitative means for defining a critical value of pressure drop over which the membrane is indicated to be nonintegral. The method is applied to sterilizing filter integrity testing associated with the commonly produced radiopharmaceuticals, [¹⁸F]FDG and [¹¹C]PIB. The method is shown to be robust, practical and amenable to automation in radiopharmaceutical manufacturing environments (e.g., hot cells).     

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.

     

Hypokaliemic rhabdomyolysis associated with liquorice ingestion: report of an atypical case

We here present an unusual case of hypokaliemic rhabdomyolysis, characterised by a sthenic deficit exclusively involving the distal muscles of the upper limbs and secondary to chronic glycyrrhizic acid intoxication, and by the absence of even ictal arterial hypertension.We discuss the etiopathogenetic bases and the risks related to the development of this secondary hypokaliemic syndrome, also in relation to other concomitant risk factors such as prolonged physical exercise and exposure to low temperatures.The study was supported in part by the Scuola di Specializzazione in Neurologia, Università degli Studi di Ferrara.     

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.