The M4 insulator, the TM2 matrix attachment region, and the double copy of the heavy chain gene contribute to the enhanced accumulation of the PHB-01 antibody in tobacco plants.

The expression of recombinant proteins in plants is a valuable alternative to bioreactors using mammalian cell systems. Ease of scaling, and their inability to host human pathogens, enhance the use of plants to generate complex therapeutic products such as monoclonal antibodies. However, stably transformed plants expressing antibodies normally have a poor accumulation of these proteins that probably arise from the negative positional effects of their flanking chromatin. The induction of boundaries between the transgenes and the surrounding DNA using matrix attachment regions (MAR) and insulator elements may minimize these effects. With the PHB-01 antibody as a model, we demonstrated that the insertion of DNA elements, the TM2 (MAR) and M4 insulator, flanking the transcriptional cassettes that encode the light and heavy chains of the PHB-01 antibody, increased the protein accumulation that remained stable in the first plant progeny. The M4 insulator had a stronger effect than the TM2, with over a twofold increase compared to the standard construction. This effect was probably associated with an enhancer-promoter interference. Moreover, transgenic plants harboring two transcriptional units encoding for the PHB-01 heavy chain combined with both TM2 and M4 elements enhanced the accumulation of the antibody. In summary, the M4 combined with a double transcriptional unit of the heavy chain may be a suitable strategy for potentiating PHB-01 production in tobacco plants.

Comparative in vitro and experimental in vivo studies of the anti-epidermal growth factor receptor antibody nimotuzumab and its aglycosylated form produced in transgenic tobacco plants.

A broad variety of foreign genes can be expressed in transgenic plants, which offer the opportunity for large-scale production of pharmaceutical proteins, such as therapeutic antibodies. Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) recombinant IgG1 antibody approved in different countries for the treatment of head and neck squamous cell carcinoma, paediatric and adult glioma, and nasopharyngeal and oesophageal cancers. Because the antitumour mechanism of nimotuzumab is mainly attributed to its ability to interrupt the signal transduction cascade triggered by EGF/EGFR interaction, we have hypothesized that an aglycosylated form of this antibody, produced by mutating the N(297) position in the IgG(1) Fc region gene, would have similar biochemical and biological properties as the mammalian-cell-produced glycosylated counterpart. In this paper, we report the production and characterization of an aglycosylated form of nimotuzumab in transgenic tobacco plants. The comparison of the plantibody and nimotuzumab in terms of recognition of human EGFR, effect on tyrosine phosphorylation and proliferation in cells in response to EGF, competition with radiolabelled EGF for EGFR, affinity measurements of Fab fragments, pharmacokinetic and biodistribution behaviours in rats and antitumour effects in nude mice bearing human A431 tumours showed that both antibody forms have very similar in vitro and in vivo properties. Our results support the idea that the production of aglycosylated forms of some therapeutic antibodies in transgenic plants is a feasible approach when facing scaling strategies for anticancer immunoglobulins.

Systematic review of case reports concerning adults suffering from neutropenic enterocolitis.

INTRODUCTION: Neutropenic enterocolitis (NEC) is a well recognised clinical-pathological and life-threatening complication in patients suffering from several conditions, including solid and haematological malignancies or aplastic anaemia. OBJECTIVE: This review was aimed at evaluating overall NEC mortality rate, describing clinical diagnostic findings and therapeutical interventions reported in the literature and generating a hypothesis regarding factors influencing mortality and surgical intervention. MATERIALS AND METHODS: An advanced search was made in Medline, Embase, Lilacs and Google. Additional strategies included manual search of specific journals. Reports were considered if they described case definition, inclusion and exclusion criteria. RESULTS: 275 cases were selected; 109 were from individual data and 40 from grouped data. Comparing data between case reports and case series revealed no significant differences related to mortality, surgical intervention, sex or age. Higher mortality (chi2 = 7.51 p = 0.006) was found in women (50%) compared to men (28%). No significant difference was found between antibiotic combinations and mortality (chi(2) = 12.85 df 13 p = 0.45). Mortality (chi2 = 3.89 df 1, p = 0.049), surgical intervention (chi2 = 7.64 df 1, p = 0.006) and duration of diarrhoea (chi2 = 4.71 df 1, p = 0.043) were significantly different in 26.4% of individuals using antifungal agents; death occurred in 81% of patients! who did not receive such medication compared to 19% individuals reported as being treated with antifungal agents. CONCLUSION: The current evidence suggests that antifungal agents should be used early in patients suffering from NEC. However, this hypothesis must be evaluated in multi-centric, randomised controlled trials.

Systematic review of case reports concerning adults suffering from neutropenic enterocolitis

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Introduction. Neutropenic enterocolitis (NEC) is awell recognised clinical-pathological and life-threateningcomplication in patients suffering from severalconditions, including solid and haematologicalmalignancies or aplastic anaemia.Objective. This review was aimed at evaluatingoverall NEC mortality rate, describing clinical diagnosticfindings and therapeutical interventions reportedin the literature and generating a hypothesisregarding factors influencing mortality and surgicalintervention.Materials and methods. An advanced search wasmade in Medline, Embase, Lilacs and Google. Additionalstrategies included manual search of specificjournals. Reports were considered if they describedcase definition, inclusion and exclusion criteria.Results. 275 cases were selected; 109 were from individualdata and 40 from grouped data. Comparingdata between case reports and case series revealedno significant differences related to mortality, surgicalintervention, sex or age. Higher mortality (÷2 =7.51 p = 0.006) was found in women (50%) comparedto men (28%). No significant difference wasfound between antibiotic combinations and mortality(÷2 = 12.85 df 13 p = 0.45).Mortality (÷2 = 3.89 df 1, p = 0.049), surgical intervention(÷2 = 7.64 df 1, p = 0.006) and duration of diarrhoea(÷2 = 4.71 df 1, p = 0.043) were significantly differentin 26.4% of individuals using antifungal agents;death occurred in 81% of patients! who did not receivesuch medication compared to 19% individualsreported as being treated with antifungal agents.Conclusion. The current evidence suggests that antifungalagents should be used early in patients sufferingfrom NEC. However, this hypothesis must beevaluated in multi-centric, randomised controlledtrials

Subcutaneous panniculitis-like T-cell lymphoma and haemophagocytic

Se presenta el caso de un hombre de 57 años con una historia de tres meses de evolución caracterizada por la aparición de varios nódulos subcutáneos localizados en una pequeña área del abdomen y de la región inguinal, acompañados de ulceración, pancitopenia y fiebre. El examen patológico revelo un infiltrado atípico de células linfoides localizado en la región subcutánea. Estas células fueron identificadas por inmunohistoquímica como linfocitos T, descritos con importante cariorrexis,angioinvasión y necrosis grasa, compatible con un linfoma T subcutáneo. Esta rara entidad pertenece al subgrupo de los linfomas T periféricos y se acompaña ocasionalmente del síndrome hemofagocítico. El paciente presentó pobre respuesta a la quimioterapia y múltiples complicaciones infecciosas durante su hospitalización.

Mortalidad y complicaciones asociadas a nefrostomías percutáneas en pacientes con cáncer avanzado de cuello uterino / Mortality and complications associated with percutaneous nephrostomy in patients with uretral obstruction related to advanced cervical cancer

La nefrostomía percutánea es una técnica bien establecida que permite la rápida resolución de la obstrucción ureteral y la recuperación de la función renal en pacientes con uropatía obstructiva. Sin embargo, su papel en el manejo de la falla renal secundaria a la obstrucción de la vía urinaria por neoplasias abdominales avanzadas es controvertido. Describir las variables demográficas, las complicaciones asociadas a la intervención, el curso de la uropatía obstructiva y varios desenlaces relacionados con la morbilidad y la mortalidad secundarios a la derivación percutánea de la vía urinaria. Se incluyeron 106 casos con cáncer avanzado de cuello uterino y obstrucción ureteral tratada con nefrostomía percutánea. Las pacientes se dividieron en dos grupos; en el A se incluyeron 37 mujeres con cáncer localmente avanzado de cuello uterino, recientemente diagnosticado y sin tratar, y en el grupoB, 69 pacientes con tumores recurrentes en progresión. Se incluyeron en el estudio 106 mujeres con edades entre 24 y 75 años (media=46,2, DEñ13,1); 9 tenían cáncer estado IIB de cuello uterino (8,5 por ciento); 68, tumores estado IIIB (64porciento), y 29 (27,5porciento) se clasificaron en el estado IVA. Ciento dos mujeres recibieron radioterapia pélvica más braquiterapia, y 4 fueron tratadas concomitantemente con quimioterapia más radioterapia. En 90 pacientes se diagnosticó obstrucción urinaria bilateral y en 16 fue unilateral. El 90porciento de las pacientes tenía pruebas anormales de función renal en el momento de la realización del procedimiento. El 13,2porciento de las pacientes presentó sangrado después del procedimiento; 26porciento, alteraciones hidroelectrolíticas; 12porciento, abscesos intra o perirrenales, y 6porciento, infección en el sitio de inserción. La mediana de permanencia de las nefrostomías fue de 5,2 y 6,9 meses para los grupos A y B, respectivamente. Durante este tiempo, 53porciento de los sujetos requirió 1 o más cambios del catéter y se documentaron 147 episodios de infección de vías urinarias (3,12 infecciones por cada 100 días-catéter). Las infecciones recurrentes fueron más frecuentes en las pacientes del grupo B (p=0,03), en aquéllas con albúmina menor de 2,5 mg/dl (p=0,011), en las que se había diagnosticado fístula recto o vesicovaginal (p=0,02) y en las requirieron más de dos cambios de los catéteres (p=0,05).

Development of a highly efficient system for assessing recombinant gene expression in plant cell suspensions via Agrobacterium tumefaciens transformation.

A transient gene-expression system was developed and used to characterize promoter strength, to verify suitability of bacterial gene modifications for expression in plant cells, and to express active antibody molecules. The system is based on suspension tobacco cells transformed by Agrobacterium in a transient way. Conditions such as pre-culture of tobacco cells and the co-cultivation period were identified as determinants to achieve high expression levels. Under established conditions the activity strength of CaMV (cauliflower mosaic virus) 35 S and ToMoTV (tomato mottle taino virus) AL1 promoters were compared. A modified cry gene sequence from Bacillus thuringiensis was expressed and detected by Western-blot analysis. A monoclonal antibody against anti-(hepatitis B virus surface antigen) was produced in such quantities as to allow testing of biological activity and preliminary characterization.