Pharmacokinetic and tumor-seeking properties of recombinant and nonrecombinant anti-carcinoembryonic antigen antibody fragments

Production of recombinant antibody fragments in bacterial expression systems results in intentional or fortuitous differences compared to the original products prepared by hybridoma technology. These differences may have significant effects not only on antigen-binding properties but also on pharmacokinetic and tumor-seeking properties. Our major goal was to investigate some of these possible differences. We produced in Escherichia coli an rFab' fragment containing only 1 cysteine residue in the hinge region; the fragment was derived from a mouse MAb (F6) specific for CEA. The rFab' had a slightly lower m.w. and a higher isoelectric point relative to the corresponding nonrecombinant fragment (pFab'). This was explained by the absence of N-glycosylation on the V kappa domain of rFab'. V kappa glycosylation had no significant effect on antibody-binding affinity and kinetics. However, rFab' was eliminated from the circulation much faster than pFab', and the maximal dose accumulated in the tumor was reduced relative to pFab'. Thus, glycosylation appears to modify the targeting efficiency of antibody fragments. rF(ab')(2) fragments were obtained either spontaneously from the culture supernatant of E. coli or by chemical cross-linking [rcF(ab')(2)]. We observed improved tumor targeting with rcF(ab')(2) compared to rF(ab')(2), which could be explained by the greater stability of the thioether compared to the disulfide linkage. These results demonstrate that a single cysteine residue in the hinge region of rFab' is particularly well suited to prepare stable, chemically coupled, bivalent or bispecific antibodies, avoiding intrahinge disulfide bonding and thus achieving higher production yields.     

Oxygène, stress oxydant et supplémentations antioxydantes ou un aspect différent de la nutrition dans les maladies respiratoires

Dioxygen is an element essential to our survival, our life, our development, our capacity of adaptation. Nevertheless, dioxygen is also at the origin of toxicity, acidity, deterioration, degeneration. Indeed, when the metabolism of dioxygen is altered, as in the respiratory diseases, an “oxidative stress” can appeared and induced metabolic anomalies associated with important consequences. Oxidative stress is defined as an imbalance between pro-oxidant (reactive oxygen species) and antioxidant factors, in favour of the former. The genomic, metabolic and functional modifications induced by oxidative stress were implied in the development of various degenerative diseases. Antioxidant treatments, in a nutritional or pharmacological way, appeared consequently as new potent therapies. In this review, the role of dioxygen in the cellular metabolism, and in the production of the reactive oxygen species is discussed. The negative effects of the oxidative stress on the organism are reported. Finally, the results of the studies on the nutritional antioxidant treatment firstly in the degenerative diseases, secondly in a chronic respiratory disease, the chronic obstructive pulmonary disease, are discussed in a last part.     

Pure-tone threshold description of an elderly French screened population.

OBJECTIVES: To describe the distribution of pure-tone hearing thresholds of a Caucasian population living in the south of France aged 70 years and older. To establish age- and sex-adjusted normative hearing thresholds based on results of subjects free of noise and ototoxic drug exposure and to compare them with hearing thresholds of exposed (E) subjects. DESIGN: Cross-sectional analysis of a longitudinal epidemiologic cohort study. SETTING: Montpellier suburb, south of France. PARTICIPANTS: A total of 778 subjects 70 years old and older were examined. Noise exposure, ototoxic medication use, and medical history were collected. Hearing thresholds were obtained via pure-tone audiometry. After excluding patients with ear-related disease, 659 subjects were further analyzed (270 men and 389 women). Noise or ototoxic medication exposure was found in 364 subjects (E subjects), whereas 295 had no exposure (nonexposed [NE] subjects). METHODS: Median pure-tone thresholds, lower deviation, and upper deviation were calculated for the NE subjects with a statistical method similar to the ISO 7029 norm and were compared with thresholds of E subjects. RESULTS: Hearing thresholds, especially in high frequencies, increased with age more for women than for men. Median thresholds of E subjects were significantly higher than those for the NE sample in men. CONCLUSION: Age- and sex-adjusted hearing thresholds could well be useful in the study of the impact of environmental and genetic factors on hearing loss in the elderly. The next step would be to quantify the impact of noise, ototoxic drug exposure, and genetics using these age- and sex-adjusted thresholds.     

Increasing digesta viscosity using carboxymethylcellulose in weaned piglets stimulates ileal goblet cell numbers and maturation

Intestinal mucin, a family of glycoproteins secreted by goblet cells, is the main constituent of the mucus protecting the gastrointestinal tract. For optimal mucosal protection, both the quantitative and qualitative characteristics of mucin are essential. To evaluate how viscosity influences ileal apparent digestibility and mucin biology, a highly viscous nonfermentable soluble polysaccharide, carboxymethylcellulose (CMC), was fed to weaned piglets for 15 d. The ileal crude mucin concentration was determined by ethanol precipitation, and changes in goblet cell subtypes were analyzed by the histochemistry of ileal and colonic tissues. As expected, CMC increased the viscosity of ileal digesta and the moisture of feces (P < 0.001). The crude mucin concentration and output at the ileum were higher (P < 0.05) in pigs fed CMC than those fed the control diet. Increasing intestinal content viscosity in pigs fed CMC had no significant effects on the ileal apparent digestibility of dry matter, organic matter, nitrogen, and minerals. The number of total ileal goblet cells per villus also was higher (+30%, P < 0.05) in pigs fed the CMC diet compared with controls. This increase was essentially accounted for by increased numbers of acidic and acidic sulfated mucin-containing cells (+30%, P < 0.05). Trends (P = 0.06) toward decreased numbers of neutral and acidic mucin-containing cells in ileal crypts were also noted. In conclusion, increasing intestinal content viscosity in weaned piglets fed CMC increased the ileal mucin output and numbers and maturation of goblet cells in ileal villi without effects on the apparent digestibility of the diet.     

Captopril-induced eruptions: occurrence over a 3-year period

With an informatized sample of 123 patients taking Captopril and treated for a total duration of 1,321 month/patients, the frequency of the dermatological symptoms induced by the drug is related. Pruritus is found in 10,5 p. 100 and rashes in 2,4 p. 100 of cases. The results are compared with those of preceeding reports, although some authors have found a higher percentage because of highin doses of the drug. The clinical features of the rashes and the proposed mechanisms are also reported.     

Short-term survival of newborn neurons in the adult olfactory bulb after exposure to a complex odor environment

In the olfactory bulb of adult mice, new neurons are continually integrated into existing neuronal networks. Previous studies have demonstrated that exposure to a complex odor environment increases the incorporation of newborn bulbar neurons without modifying the proliferation rate. Whether this incorporation is transient or leads to the long-lasting presence of new neurons has not yet been answered. Because a transient increase of new neurons impacts olfactory information processing differently than a long-lasting increase, we conducted experiments to investigate the time course of survival and cell death of newly generated bulbar neurons following exposure to an enriched olfactory environment. Dividing cells were labeled with bromodeoxyuridine (BrdU) and were counted at several survival time points thereafter. Interestingly, whereas the number of surviving BrdU-labeled cells was elevated at the time when animals were withdrawn from their enriched housing, this number returned to control level 1 month later. Similarly, when olfactory memory was investigated, we found that the improvement of short-term memory, induced by enriched odor exposure, lasted less than 1 month. These findings indicate not only that the recruitment of newborn neurons closely followed the degree of environment complexity, but also that olfactory memory is tightly associated with the level of ongoing neurogenesis in the adult olfactory bulb.     

Lentivector-mediated delivery of GDNF protects complex motor functions relevant to human Parkinsonism in a rat lesion model

Although viral vector-mediated delivery of glial cell-line derived neurotrophic factor (GDNF) to the brain has considerable potential as a neuroprotective strategy in Parkinson's disease (PD), its ability to protect complex motor functions relevant to the human condition has yet to be established. In this study, we used an operant task that assesses the selection, initiation and execution of lateralized nose-pokes in Lister Hooded rats to assess the efficacy with which complex behaviours are protected against neurotoxic lesions by prior injection of a lentiviral vector expressing GDNF. Unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) caused rats to attempt fewer trials and to make more procedural errors. Lesioned rats also developed a pronounced ipsilateral bias, with a corresponding drop in contralateral accuracy. They were also slower to react to contralateral stimuli and to execute movements bilaterally. Rats that were pre-treated 4 weeks prior to lesion surgery with an equine infectious anaemia virus (EIAV) vector carrying GDNF [EIAV-GDNF, injected into the striatum and above the substantia nigra (SN)] performed significantly better on all of these parameters than control rats. In addition to the operant task, EIAV-GDNF successfully rescued contralateral impairments in the corridor, staircase, stepping and cylinder tasks, and prevented drug-induced rotational asymmetry. This study confirms that GDNF can protect against 6-OHDA-induced impairments in complex as well as simple behaviours, and reinforces the use of EIAV-based vectors for the treatment of PD.     

Fibrillar type I collagens enhance platelet-dependent thrombin generation via glycoprotein VI with direct support of alpha2beta1 but not alphaIIbbeta3 integrin

The role of collagens and collagen receptors was investigated in stimulating platelet-dependent thrombin generation. Fibrillar type-I collagens, including collagen from human heart, were most potent in enhancing thrombin generation, in a way dependent on exposure of phosphatidylserine (PS) at the platelet surface. Soluble, non-fibrillar type-I collagen required pre-activation of integrin alpha2beta1 with Mn2+ for enhancement of thrombin generation. With all preparations, blocking of glycoprotein VI (GPVI) with 9O12 antibody abrogated the collagen-enhanced thrombin generation, regardless of the alpha2beta 1 activation state. Blockade of alpha2beta1 alone or antagonism of autocrine thromboxane A2 and ADP were less effective. Blockade of alphaIIbbeta3 with abciximab suppressed thrombin generation in platelet-rich plasma, but this did not abolish the enhancing effect of collagens. The high activity of type-I fibrillar collagens in stimulating GPVI-dependent procoagulant activity was confirmed in whole-blood flow studies, showing that these collagens induced relatively high expression of PS. Together, these results indicate that: i) fibrillar type-I collagen greatly enhances thrombin generation, ii) GPVI-induced platelet activation is principally responsible for the procoagulant activity of fibrillar and non-fibrillar collagens, iii) alpha2beta1 and signaling via autocrine mediators facilitate and amplify this GPVI activity, and iv) alphaIIbbeta3 is not directly involved in the collagen effect.