Performance comparison of protein A affinity resins for the purification of monoclonal antibodies.

During the selection of protein A affinity resin for the purification of monoclonal antibodies, dynamic binding capacity (Q(dyn10%)), volumetric production rate (Pr(vol)) and 'process robustness' are essential parameters to be evaluated. In this article, empirical mathematical models describe these parameters as a function of antibody concentration in load (C0), load flow rate (u(load)) and bed height (L). These models allow us to select optimal process conditions for each of the evaluated protein A affinity resins. C0, u(load) and L largely affect dynamic binding capacity (Q(dyn10%)) and volumetric production rate (Pr(vol)). Maximum Q(dyn10%) is generally obtained at high C0 and at low u(load). Maximum Pr(vol) is obtained at high C0 and at lowest L, run at high u(load). All evaluated resins have a relatively high robustness against variations in C0. |DeltaQ(dyn10%)/deltaC0| ranges from 0.0 to 7.8. It is clear that Q(dyn10%), Pr(vol) and 'process robustness' cannot be maximized all at the same time. Furthermore, some other aspects like IgG recovery, protein A leaching, easiness to pack, easiness to clean, number of re-uses and cost of production might be important to be taken into the equation. Certain evaluation parameters may be more important than others, depending on the specific situation. Therefore, a case-by-case evaluation is recommended.

Prostatic stromal cells and testicular peritubular cells produce similar paracrine mediators of androgen action.

The role of mesenchymal-epithelial interactions in androgen action was explored using Sertoli cells as the epithelial cells and testicular peritubular cells or prostatic stromal cells as mesenchymal cells. Footsole fibroblasts served as a control. The secretion of transferrin was used as an androgen-regulated parameter of Sertoli cell function. It is demonstrated that coculture of peritubular or stromal cells with Sertoli cells markedly increases the production of transferrin. This effect requires a 4-day latent period and is maximal with low concentrations (10%) of mesenchymal cells. Stimulatory effects of androgens can only be demonstrated at suboptimal concentrations of the latter cells. Fibroblasts are inactive. At least two mechanisms contribute to these stimulatory effects. Peritubular cells and stromal cells share the ability to promote the deposition of an extracellular matrix when cocultured with Sertoli cells. When Sertoli cells are seeded on this matrix, the production of transferrin is increased. This effect requires no latent period and is independent of the presence of androgens during the period of matrix deposition. In addition, peritubular cells and stromal cells produce diffusible mediators which increase transferrin production by Sertoli cells. In both cell types, the production of these mediators is controlled by androgens, and their action is preceded by a 4-day latency period. The mediators have a comparable mol wt (45,000) and resemble P Mod-S, known to be secreted by peritubular cells. These data suggest that mesenchymal-epithelial interactions play a role in androgen-supported maintenance of adult function and that mesenchymal tissue from different androgen target tissues produces similar or identical mediators of androgen action.

Stromal cells from the rat prostate secrete androgen-regulated factors which modulate Sertoli cell function.

Testicular peritubular cells produce paracrine mediators which modulate Sertoli cell function. The production of these mediators (P Mod-S) is controlled by androgens suggesting that mesenchymal-epithelial interactions play an important role in androgen action in the testis. We investigated whether mesenchymal cells from the prostate, another androgen target tissue, produce analogous mediators. To this end rat Sertoli cell cultures were exposed to dialyzed spent media derived from testicular peritubular cells, prostatic stromal cells or footsole fibroblasts. It is demonstrated that the effects of spent media from peritubular cells and stromal cells are nearly identical: they stimulate the production of androgen binding protein and transferrin and they inhibit FSH-inducible aromatase activity. The active principle (or principles) involved is non-dialyzable, heat sensitive and trypsin sensitive. Its production is markedly stimulated by androgens. Fibroblast spent media are inactive. It is concluded that mesenchymal tissue derived from different androgen target tissues may produce identical or similar mediators of androgen action acting on epithelial cells.

Kallikrein-related protease in the rat ventral prostate: cDNA cloning and androgen regulation.

A kallikrein-related protease was purified from rat ventral prostate cytosol by means of DEAE-Sepharose chromatography, followed by gel filtration on Sephadex G-100 and CM-cellulose chromatography. Antibodies raised in rabbits against the purified protease recognize two bands on immunoblots of prostatic cytosol: a 31,000 Da band and an 18,000 Da band, which constitutes a proteolytic breakdown product of the former. The corresponding cDNA was isolated from a prostatic cDNA library, inserted in a lambda gt11 vector, using immunodetection for screening and identified as encoding a kallikrein- and tonin-related protease. Castration resulted in a marked decrease of the level of the protease and its mRNA, whereas administration of androgens to castrated animals resulted in marked stimulation. These data support the hypothesis that this protease is a member of a cluster of proteins, that are regulated in parallel by androgens in prostatic epithelial cells.

The role of cell-cell interactions in androgen action.

Androgen-regulated mesenchymal-epithelial interactions play an important role during embryonic development of the male urogenital tractus. Studies on the effects of androgens on cultured testicular cells derived from the immature rat testis indicate that, even during postnatal life, similar interactions may be instrumental for normal androgen action. Androgen receptors are found in epithelial Sertoli cells as well as in mesenchymal peritubular cells. The effects of androgens on isolated Sertoli cells, however, are limited. Coculture with peritubular cells increases the sensitivity and/or the responsiveness of a number of Sertoli cell parameters (transferrin, ABP, aromatase activity) to androgens. This effect is at least in part mediated by the secretion of one or more diffusible factors (P-Mod-S) by the peritubular cells. We investigated whether such indirect effects of androgens, relying on mesenchymal-epithelial interactions are also observed in other androgen target tissues. To this end stromal cells were isolated and cultured from the immature rat ventral prostate and the production of factors with P-Mod-S activity was monitored using Sertoli cells as the test system. Under coculture conditions these stromal cells stimulate Sertoli cell transferrin secretion in an androgen-regulated fashion, exactly as peritubular cells. This stimulatory effect is related in part to the collaborative (and androgen-independent) deposition of an extracellular matrix and in part to the secretion of an androgen-regulated diffusible mediator. This mediator has the same physicochemical characteristics as P-Mod-S and it affects other Sertoli cell parameters (ABP, aromatase activity, inhibin, cGMP) in the same way as P-Mod-S. Cultured stromal and peritubular cells look very similar and stain positive after immunostaining for alpha-smooth muscle isoactin. Tissue sections suggest that these cells may be derived from myoid peritubular cells in the testis and similar periacinar cells in the prostate. The hypothesis is advanced that P-Mod-S may be a more universal mediator of indirect effects of androgens in diverse target tissues and that this factor is derived from myoid cells closely associated with the epithelial component.

A new alternative for simultaneous immunohistochemical screening of 96 hybridoma clones for tissue-specific antibody productions selects a monoclonal antibody to insect corpus cardiacum.

In the current search for the elucidation of the true structure of hitherto unidentified 'new' insect neuropeptides we designed a novel screening method to facilitate the primary detection of neurone-specific antibody secreting mouse-mouse hybridoma clones obtained after immunization with neuronal tissue homogenates. The present procedure is principally adapted from a conventional immunohistological test and enables one to rapidly screen 96 (and even more) clones at one time for potential secretion of specific antibodies to different tissue compounds, without the necessity of having a purified antigen. It has proved to be sensitive, rapid, practical and reproducible. As such it promises to be very useful to discriminate amongst the wide range of antibodies to various kinds of materials produced by hybridomas by detecting monoclonal antibodies directed against factors contained in well-defined tissues in which one is interested. This paper also reports the successful application of this method to a primary screening of clones producing murine monoclonal antibodies to substances of insect corpora cardiaca (CC), after immunization with crude antigen preparations.

Subzonal insemination: a prospective randomized study in patients with abnormal sperm morphology.

OBJECTIVE: To evaluate in a prospective randomized study the outcome of subzonal insemination (SUZI) in patients with male subfertility. DESIGN: In a period of 7 months, 48 patients underwent IVF treatment for male subfertility reasons. Normal insemination and SUZI were performed on sibling oocytes. Patients were divided into three groups depending on the sperm morphology (strict criteria): group 1, 10% to 14%; group 2, 5% to 10%; group 3, 0% to 5%. SETTING: Private fertility center in Leuven, Belgium. MAIN OUTCOME MEASURES: The fertilization rates, cleavage rates, implantation rates, and pregnancy rates between the normally inseminated and the SUZI-treated group were compared. RESULTS: The fertilization rate with SUZI was significantly higher (32%) than after normal insemination (7%). The difference was striking in groups 2 and 3 (35% and 33% versus 11% and 4%). CONCLUSION: This study indicates that SUZI increases the fertility outcome in patients with male subfertility and that there is a marked difference in fertilization rate when morphology, using strict criteria, is < 10%.

The impact of vertical vibrations on the welfare of calves.

The objective of this study was to evaluate the heart rate, body temperature, saliva cortisol levels and behaviour of young calves during transport simulation, and to define comfort conditions for calves related to the frequency and acceleration of vibration. Calves with an average age of 22 days were vibrated in the vertical direction for 2 hours at 2, 4, 8 and 12 Hz, in combination with root mean square (RMS) acceleration magnitudes 1 or 3 m/s2. Welfare and stress were quantified by measuring changes of heart rate, body temperature, cortisol concentration in saliva and behaviour. Treatments with acceleration 3 m/s2 had the largest impact on the animals. Stress response was larger in combination with 2 Hz, especially during the first hour of the experiment. Treatment 12 Hz in combination with acceleration 3 m/s2 had initially no influence on the animals, but towards the end of the treatment the calves started to express a stress response. From these results we can conclude that certain vibrations are stressful for calves and those can impair the welfare during transport.

Morphological and functional similarities between cultured prostatic stromal cells and testicular peritubular myoid cells.

A number of androgen effects on epithelial cells may be mediated by androgen-regulated paracrine factors produced by underlying mesenchymal cells. In previous studies we demonstrated that prostatic stromal cells and testicular peritubular cells, derived from immature rats, produce mediators of androgen action with identical effects on Sertoli cells. In the present paper we further compared the morphological and functional characteristics of both mesenchymal cell types. Cultured prostatic stromal cells and testicular peritubular cells look identical under phase-contrast microscopy, share the ability to form tubular structures and "balls" when cocultured with Sertoli cells, and contain proteins immunoreactive with an antiserum against alpha-smooth muscle isoactin. Two-dimensional gel electrophoresis shows that the pattern of proteins produced by both cell types is nearly identical. Conditioned media from stromal and peritubular cells contain a factor that stimulates transferrin and cGMP production in Sertoli cells. The behavior of the active principle in the media from both cell types is comparable. On reverse-phase HPLC the elution profile of this factor is comparable for media from both cell types. In conclusion, these data point to a striking similarity in the morphological and functional characteristics of mesenchymal cells cultured from the prostate and testis.

Mitochondrial distribution after fast embryo freezing.

Mitochondrial distribution pattern after ultrarapid freezing in dimethylsulphoxide was assessed in human multipronucleate zygotes, 2-cell and 4-cell stage embryos using rhodamine 123. The mitochondrial distribution pattern was evaluated at 37 degrees C after a 30 min incubation in rhodamine 123 solution, 4 h and 24 h after thawing. Non-frozen human unfertilized oocytes, 2-cell and 4-cell embryos used as a control showed a homogeneous distribution of mitochondria throughout the cytoplasm, while there was sequestration of mitochondria from the cortex to the region surrounding the pronuclei in multipronucleate zygotes. Morphologically intact multipronucleate zygotes, 2-cell and 4-cell stage embryos after quick freeze-thawing showed the same mitochondrial distribution pattern found in the unfrozen controls. Mitochondria exhibited a typical severe aggregation (clumping) throughout the cytoplasm when non-viable single blastomeres or embryos at thawing were exposed to rhodamine 123. Our study indicates that quick freezing does not affect subcellular structures. The well-organized and specific mitochondrial distribution appeared still to be present after frozen storage, and subcellular structures seemed to be rather resistant targets for cryo-injury.

Immunocytochemical localization of human growth hormone- and prolactin-like antigenic determinants in the insects, Locusta migratoria and Sarcophaga bullata.

1. By use of the peroxidase-antiperoxidase immunocytochemical method, substances immunoreactive to antisera directed against human growth hormone (hGH) and prolactin (hPrl) were localized in the nervous system of larval and adult Locusta migratoria and of adult Sarcophaga bullata belonging to different age groups. 2. No major differences in the distribution of cerebral immunoreactive materials were observed between males and females or between juvenile and adult insects. 3. Differential immuno-labeling of alternating tissue sections demonstrated that materials resembling hGH or hPrl are present in distinct neurons in the locust, whereas neurons immunoreactive to both antisera were detected in the fleshfly (Sarcophaga).

Skeletal and dento-alveolar stability after surgical-orthodontic treatment of anterior open bite: a retrospective study.

The aim of this investigation was to assess skeletal and dento-alveolar stability after surgical-orthodontic correction of skeletal anterior open bite treated by maxillary intrusion (group A) versus extrusion (group B). The cephalometric records of 49 adult anterior open bite patients (group A: n = 38, group B: n = 11), treated by the same maxillofacial surgeon, were examined at different timepoints, i.e. at the start of the orthodontic treatment (T1), before surgery (T2), immediately after surgery (T3), early post-operatively (+/- 20 weeks, T4) and one year post-operatively (T5). A bimaxillary operation was performed in 31 of the patients in group A and in six in group B. Rigid internal fixation was standard. If maxillary expansion was necessary, surgically assisted rapid palatal expansion (SRPE) was performed at least 9 months before the Le Fort I osteotomy. Forty-five patients received combined surgical and orthodontic treatment. The surgical open bite reduction (A, mean 3.9 mm; B, mean 7.7 mm) and the increase of overbite (A, mean 2.4 mm; B, mean 2.7 mm), remained stable one year post-operatively. SNA (T2-T3), showed a high tendency for relapse. The clockwise rotation of the palatal plane (1.7 degrees; T2-T3), relapsed completely within the first post-operative year. Anterior facial height reduction (A, mean -5.5 mm; B, mean -0.8 mm) occurred at the time of surgery. It can be concluded that open bite patients, treated by posterior Le Fort I impaction as well as with anterior extrusion, with or without an additional bilateral sagittal split osteotomy (BSSO), one year post-surgery, exhibit relatively good clinical dental and skeletal stability.