Ultrastructural features of agouti (Dasyprocta aguti) preantral follicles cryopreserved using dimethyl sulfoxide, ethylene glycol and propanediol

The objective was to develop an efficient protocol for cryopreservation of agouti (Dasyprocta aguti) ovarian tissue. Agouti ovarian fragments were placed, for 10 min, in a solution containing MEM and fetal bovine serum plus 1.5 M dimethyl sulfoxide (DMSO), ethylene glycol (EG) or propanediol (PROH); some of those fragments were subsequently cryopreserved in a programmable freezer. After exposure and/or thawing, all samples were fixed in Carnoy prior to histological analysis. To evaluate ultrastructure, follicles from the control and all cryopreserved treatments were fixed in Karnovsky and processed for transmission electron microscopy. After exposure and freezing, there was a significant decrease in the percentage of morphologically normal preantral follicles in all treatments when compared to the control (92.67 ± 2.79, mean ± SD). However, there were no significant difference when the exposure and freezing procedures were compared using the same cryoprotectant. Moreover, there was no significant difference among cryoprotectants at the time of exposure (DMSO: 64.7 ± 3.8; EG: 70.7 ± 11.2, PROH: 63.3 ± 8.5) or after freezing (DMSO: 60.6 ± 3.6, EG: 64.0 ± 11.9; PROH: 62.0 ± 6.9). However, only follicles frozen with PROH had normal ultrastructure. In conclusion, preantral follicles enclosed in agouti ovarian tissue were successfully cryopreserved using 1.5 M PROH, with satisfactory maintenance of follicle morphology and ultrastructure.     

The comparative effects of IL-1 and TNF on AMN-anti-Ly 2.1 immunoconjugate therapy

The administration of mTNF and hIL-1 was investigated for their potential to increase the anti-tumor activity of AMN-anti-Ly-2.1 against the Ly-2.1⁺ murine thymoma ITT(1) 75 NS E3. Dose response studies using mTNF alone demonstrated a single 10g iv injection produced 30% inhibition in tumor size while 3 doses of 1g administered on alternative days produced 70% tumor inhibition. By contrast, hIL-1 was unable to significantly reduce E3 tumor size using single doses up to 10g or a total of 30g administered in 3 doses (iv or ip). However, intratumor injection of hIL-1 (20g injected in 2 doses) produced 20% inhibition in tumor size. Combination therapy using AMN immunoconjugates with mTNF showed enhanced antitumor activity compared to each agent alone. Biodistribution studies revealed that anti-tumor activity, was due to increased localization (2–3 fold) of AMN immunoconjugates in the presence of mTNF- whereas huIL-1 was without effect unless accompanying toxicity was seen. Clearly for this tumor, mTNF potentiated the effects of AMN immunoconjugates. Despite the shared biological properties of these cytokines, mTNF is superior to hIL- for augmenting drug immunoconjugate.Abbreviations AMN Aminopterin - CBF₁ (C57BL6xBALB/c)F₁ mice - DMSO Dimethyl sulfoxide - E3 ITT(1)75NS E3 - HAMA human-anti-mouse antibody - i.p. intraperitoneal(ly) - I.T. intratumor - i.v. intravenous(ly) - hIL-1 recombinant human Interleukin-1-alpha - MoAb monoclonal antibody - PBS phosphate buffered saline - SE standard error - s.c. subcutaneous(ly) - mTNF recombinant murine tumor necrosis factor-alpha     

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 N₂₉₇ position in the IgG₁ 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.     

A transformation procedure for recalcitrant tomato by addressing transgenic plant-recovery limiting factors

Agrobacterium tumefaciens technology is the battle horse for tomato genetic transformation. However, tomato varieties with low regeneration capacity are very difficult to transform. In the past, tomato transformation through Agrobacterium infection was focused on varieties capable of high regeneration yield, while successful transformation of low regenerable cultivars has not been reported. The genotype response to tissue culture conditions is believed to drive the frequency of regeneration of transgenic plant, whereas the capacity for cell proliferation could determine the transformation efficiency through this technology. The Campbell-28 cultivar is an example of constraints arising from a high morphogenetic potential with low conversion compared to normal plants. In the present work the roles that contribute to improved transgenic plant recovery from this recalcitrant variety were explored for factors like Agrobacterium concentration and antibiotics for bacterial removal and transformant selection. Analysis of the efficiency from independent transformation experiments revealed a more than twofold increase of transformant regeneration after selection on ammonium glufosinate compared to kanamycin selection, showing a transformation efficiency of 21.5%.     

High accumulation in tobacco seeds of hemagglutinin antigen from avian (H5N1) influenza

Tobacco seeds can be used as a cost effective system for production of recombinant vaccines. Avian influenza is an important respiratory pathogen that causes a high degree of mortality and becomes a serious threat for the poultry industry. A safe vaccine against avian flu produced at low cost could help to prevent future outbreaks. We have genetically engineered tobacco plants to express extracellular domain of hemagglutinin protein from H5N1 avian influenza virus as an inexpensive alternative for production purposes. Two regulatory sequences of seed storage protein genes from Phaseolus vulgaris L. were used to direct the expression, yielding 3.0 mg of the viral antigen per g of seeds. The production and stability of seed-produced recombinant HA protein was characterized by different molecular techniques. The aqueous extract of tobacco seed proteins was used for subcutaneous immunization of chickens, which developed antibodies that inhibited the agglutination of erythrocytes after the second application of the antigen. The feasibility of using tobacco seeds as a vaccine carrier is discussed.     

Factors associated with diarrheal disease among children aged 1–5 years in a cholera epidemic in rural Haiti

Diarrheal illness is a major cause of morbidity and mortality among children in Haiti, and the impact of diarrheal illness was compounded by a cholera outbreak between 2010 and 2019. Our understanding of risk factors for diarrhea among children during this outbreak is limited. We conducted a secondary analysis of data collected as part of a cholera vaccine effectiveness study to identify factors associated with medically attended diarrhea among children in central Haiti from October of 2012 through November of 2016. We identified 47 children aged one to five years old who presented to medical clinics with acute, watery diarrhea, and 166 matched controls who did not have diarrhea, and we performed conditional logistic regression to identify factors associated with diarrhea. Discontinuing exclusive breastfeeding within one month of birth was associated with increased risk of diarrhea (RR 6.9, 95% CI 1.46–32.64), and diarrhea was inversely associated with reported history of supplementation with vitamin A (RR 0.05, 95% CI 0.004–0.56) and zinc (reported among 0% of cases vs. 17% of controls). Because of the concordance in supplementation patterns, it was not possible to attribute the association to vitamin A or zinc independently. While having a respondent who correctly identified ≥3 means of avoiding cholera was associated with reduced risk of diarrhea (RR 0.43, 95% CI 0.19–1.01), reported household sanitation practices and knowledge of cholera were not consistently associated with risk of diarrhea. These findings support ongoing efforts to reduce barriers to breastfeeding and promote pediatric supplementation with vitamin A and zinc in Haiti. Given the reduced efficacy of current oral cholera vaccines (OCV) among children, the results reinforce the importance of breastfeeding and micronutrient supplementation in preventing all-cause pediatric diarrheal illness generally and during cholera outbreaks.     

Interaction of Moloney murine leukemia virus matrix protein with IQGAP

The matrix protein (MA) of the Moloney murine leukemia virus (M-MuLV) was found to interact with IQGAP1, a prominent regulator of the cytoskeleton. Mutational studies defined residues of MA critical for the interaction, and tests of viruses carrying MA mutations revealed a near-perfect correlation between binding and virus replication. The replication-defective mutants showed defects in both early and late stages of the life cycle. Four viable second-site revertant viruses were isolated from three different replication-defective parental mutants, and in all cases the interaction with IQGAP1 was restored by the suppressor mutations. The interaction of MA and IQGAP1 was readily detected in vitro and in vivo. Virus replication was potently inhibited by a C-terminal fragment of IQGAP1, and impaired by RNAi knockdown of IQGAP1 and 2. We suggest that the IQGAPs link the virus to the cytoskeleton for trafficking both into and out of the cell.