From presumptive ectoderm to neural cells in an amphibian.

As an immediate consequence of neural induction during gastrulation, some neuroectodermal cells acquire the ability to develop a number of specific neuronal and astroglial features, without requiring subsequent chordamesodermal cues. Thus, cholinergic, dopaminergic, noradrenergic, gabaergic, somatostatinergic, enkephalinergic, etc. traits are expressed in cultures of neural plate and neural fold isolated from amphibian late gastrulae immediately after induction and cultured in a defined medium. These results strongly suggest that at the late gastrula stage, the neural precursor population does not yet constitute a homogeneous set of cells. It was of interest to know the origin of this heterogeneity. Is it a direct result of the process of neural induction itself, stochastic phenomena being involved or not at the cellular level, or does it reflect a pre-existing heterogeneity in the presumptive ectoderm? At the early gastrula state, presumptive ectoderm can be neuralized consecutively to its dissociation into single cells. Using this experimental model, we have demonstrated by means of immunological probes that neuralized presumptive ectodermal cells, without any intervention of the chordamesoderm (natural inducing tissue), can develop autonomously into glial and neuronal lineages. These data suggest the existence of diverse predispositions of presumptive ectodermal cells. Competent ectoderm seems to be a heterogeneous structure with cells presenting distinct neural predispositions that can emerge as a consequence of a permissive inductive signal without real specificity (such as a target tissue dissociation). Moreover, such a differentiated neuronal population includes neurons of the GABAergic and enkephalinergic phenotypes but not of the cholinergic, catecholaminergic, somatostatinergic, etc. phenotypes. These data show that the developmental program of ectodermal cells induced without interaction with the chordamesoderm appears restricted compared to the naturally induced ectoderm. Experiments are now under way to analyze such sequential neural events.

Ethylene-oxide and steam-sterilised polysulfone membrane in dialysis patients with eosinophilia.

Eosinophilia and some acute dialysis side-effects, such as itching, flushing and bronchospasm, are often associated with the presence of ethylene oxide (ETO) as dialyzer sterilizing agent. This study evaluated the effects of two different polysulfone (PS) hollow-fiber dialysers sterilized with ETO and steam in 31 chronic dialysis patients with eosinophilia. Clinical symptoms, metabolic and biochemical parameters, complement (C3a and C5a) activation and production were evaluated in each patient dialysed for two months at a time with Cuprophan dialyser, ETO-PS dialyser and steam-PS dialyser. The steam-sterilizer agent does not alter the purifying capacity of the PS membrane which maintains its superiority over Cuprophan in terms of biocompatibility. Using steam-PS, intradialytic eosinophil kinetics seems to improve. In some patients with high serum levels of ETO-specific IgE these levels tend to diminish. Generic intradialytic symptoms do not differ between the two sterilization methods, although some hypersensitivity symptoms during the first dialysis hour are considerably lower in some patients when steam-sterilized PS is used.

The pleurodele, an animal model for space biology studies.

Pleurodeles waltl, an Urodele amphibian is proposed as a model for space biology studies. Our laboratory is developing three types of experiments in space using this animal: 1) in vivo fertilization and development ("FERTILE" project); 2) influence of microgravity and space radiation on the organization and preservation of specialized structures in the neurons and muscle cells (in vitro; "CELIMENE" PROJECT); 3) influence of microgravity on tissue regeneration (muscle, bone, epidermis and spinal cord).

Structural alteration of the target plasma membrane affects reception but not expression of the neural inductive signal.

We have previously reported that incubation of presumptive neuroectoderm in a solution of lectin (soybean agglutinin or garden pea agglutinin at 50 micrograms ml-1, 30 min) prior to its association with blastoporal lip inhibits neural induction (Duprat et al., 1982). We have also shown that incubation of presumptive ectoderm immediately after its association with blastoporal lip does not prevent neural induction. The same pattern of fluorescence on the ectodermal surface was observed when incubation with lectin was carried out before or after association of ectoderm with blastoporal lip. Although a particular molecular organization of the plasma membrane of the target ectoderm appears to be essential for reception or initiation of neural induction, the subsequent transmission and expression of this neural information does not appear to be affected by structural modifications of the target membrane. Similar studies performed using the inducer Con A indicated that the experimentally induced signal differed from the natural one.

Extracellular matrix and neural determination during amphibian gastrulation.

Previous studies have shown the presence of a network of extracellular matrix on the inner surface of the ectoderm of the early gastrulae Pleurodeles waltlii. Similar dense networks have been described by other authors in another Urodele species and in Anurans. It now seems well established that this extracellular material (ECM) has important functions in the mechanisms of morphogenetic cell movements during gastrulation. The results reported here provide evidence that this ECM is not implicated in the transmission of the neuralizing signal at the gastrula stage, and therefore is not involved in the process of neural determination.

A rapid experimental method to study primary embryonic induction.

The technique described here is a combination of the sandwich-method and cell culture. It allows one to know quickly whether induction occurred or not (48 h if cell spreading is considered, 4-5 days if morphological differentiation is observed). The dissociation of the explants soon after induction does not disturb their inductive pattern. Moreover, this method allows a daily observation of the morphological events and study at the cellular and/or molecular level.

Membrane changes in neural target cells studied with fluorescent lectin probes.

The competent ectoderm of Pleurodeles waltl comprises two cell layers with characteristic differences in their morphology, their composition and the molecular arrangement of the various constituents. The use of labelled lectin probes for observations of ectoderm tissue in vitro with u.v. microscopy (epi-illumination) and the quantification of the results show the following: Differences in labelling according to the nature of the lectins (SBA, PSA, LCA and Con A). These differences provide information on the nature of the carbohydrates which are present at this stage and on the number of receptors. Differences in fluorescence intensity of the surfaces studied. The internal surface of the ectoderm is labelled more densely than the external surface. Rearrangement of the lectin receptors with a new molecular configuration, stressing the fluidity of the membrane (by the mobility of the receptors throughout the membrane) and its importance for the occurrence of neural induction. Existence of membrane glycoconjugate turnover. A difference in behavioural characteristics between the internal and the external surfaces with respect to the lectins and the formation of an extracellular matrix on the internal surface alone. The extracellular matrix seems to have a role in morphogenetic movements.

Cross-linking of membrane glycoconjugates is not a sufficient condition for neural induction by concanavalin A.

Tetravalent native concanavalin A (Con A) has a neural inducing effect on amphibian presumptive ectoderm. The divalent dimeric form of this lectin, succinylated Con A (Succ-Con A), is devoid of neuralizing action on this target tissue in Pleurodeles waltlii. These results suggested that cross-linking of Con A receptors on the cell membrane (which is not provoked by the divalent lectin) might be required for neural induction. To test this possibility, Succ-Con A binding sites were experimentally cross-linked after binding of Succ-Con A to the target cell surface, using anti-Con A antibodies. The combination of these two agents mimics the cross-linking of Con A. The results showed that cross-linking alone, either by treatment with Succ-Con A and anti-Con A antibodies, or with the lectins WGA and PHA, which also cross-link cell surface binding sites, was not able to induce neuralization. This suggested that the inductive action of Con A cannot be explained in terms of receptor cross-linking.

Neural induction and the structure of the target cell surface.

Lectins (SBA and PSA) were used to provoke crowding and structural modifications of the presumptive ectoderm cell surface in order to investigate the role of the membrane organization of the competent target cells in neural induction. Are specific characteristics of the cell surface essential for this phenomenon to occur? From amphibian gastrulae, it is possible to obtain neural induction in vitro by association of presumptive ectoderm (target cells) with chordamesoderm (inductor tissue): 4 h of contact is sufficient in Pleurodeles waltl for transmission of the inductive signal. Very quickly, the treatment of the normal ectoderm by lectins (SBA-FITC or PSA-FITC) provoked surface modifications. Lectin-treatment (50 microgram ml-1, 30 min) of presumptive ectoderm did not result in any neural induction. Lectin-treatment (50 microgram ml-1, 30 min) of presumptive ectoderm previous to its association with the natural inductor for 4 h, disturbed the phenomenon: no induction. Similar treatment followed by association with the inductor for 24 h: induction. Treatment of SBA or PSA with their respective hapten inhibitors prior to addition to ectodermal cells completely blocked the suppressive effects on induction. The structural integrity of the membrane of competent target cells is necessary for neural induction to occur. The cell membrane could thus play, directly or indirectly, an active role in the specificity of this process.

Alterations in lateral lipid mobility in the plasma membrane of urodelean ectodermal cells during gastrulation.

The mobility characteristics of lipids were studied in the plasmalemma of dissociated presumptive ectodermal cells from embryos of Pleurodeles Waltl at different stages of development, from early blastula to early neurula, using a Fluorescence Recovery After Photobleaching technique (FRAP), after incorporation of the lipophilic fluorescent probe 5N-(hexadecanoyl)-aminofluoresceine (HEDAF) into the cell plasma membrane. At all stages of development, fluorescence recovery was found to extrapolate to 100%, which suggested that the lipid phase in these plasma membranes can be regarded as dynamically homogeneous (no immobilized fraction). It appears as a continuum over a wide cell surface area, in which lipids are free to move laterally. The lateral diffusion coefficient of the probe, obtained from statistical analysis of the fluorescence recovery data, was found to decrease significantly from blastula to gastrula, slightly increasing at the neurula stage. These changes in the dynamic properties of the lipid probe HEDAF during gastrulation suggest that the lipid phase of the plasma membrane of these ectodermal cells undergo structural changes. The results lend support to the idea that the plasma membrane of these cells is actively involved in the morphogenetic movements which characterize the development of the embryo.

Neural induction and in vitro initial expression of neurofilament and tetanus toxin binding site molecules in amphibians.

Tetanus toxin (Tt) binding site and neurofilament (NIF), the intermediate-sized filaments, are neuronal markers essentially described in mammals and birds; are these molecular markers present in urodela neuronal cells and are they expressed immediately after neural induction? Our findings are based on immunofluorescent localization of NIF and Tt proteins using three previously characterized antisera against 200 kDa and 70 kDa neurofilament components and against fragment IIc derived from purified tetanus toxin. Embryonic undifferentiated neuronal cells from Pleurodeles waltlii neural plate and/or neural fold (early neurula stage) are cultured isolated in vitro without further chordamesodermal influence. At the beginning of the culture none of the undifferentiated neuronal precursors bind antibodies against NIF or Tt components. The binding is detected when phenotypical differentiation takes place (2/3-day cultures). Both the cell bodies and the cell processes are stained. After 2-3 weeks, immunostaining of the neurones is very distinctive and bright; the non-neuronal cultured cells do not exhibit any labelling. These observations indicate the early acquisition of NIF and Tt binding site expression by neuronal precursor cells (late gastrula stage).

Effect of short-term cyclosporine administration in rats on renin-angiotensin and thromboxane A2: possible relevance to the reduction in glomerular filtration rate.

A short-term treatment with Cyclosporine A (CyA) induces a decrease in glomerular filtration rate (GFR), promptly reversible after withdrawal of the drug. Several lines of evidence are now available as to indicate that this phenomenon is dependent on a hemodynamic perturbation resulting in a renal vasoconstriction. With the present work we have examined the relationship between the reduction in GFR which follows a short-term administration of CyA in rats and the biochemical changes in renin-angiotensin system and renal arachidonic acid metabolism. Our results show that CyA administration (25 mg/kg/day) for 45 days stimulates renin-angiotensin system with an increase in plasma renin activity. These changes are not accompanied by a parallel increase in the renal synthesis of vasodilatory prostaglandin E2 and prostacyclin as it occurs in other conditions of renin-angiotensin stimulation. At variance glomerular synthesis and urinary excretion of thromboxane A2 (TxA2) are increased progressively during CyA treatment. These changes in renal Tx precede the increase in serum creatinine and the decrease in GFR thus indicating that TxA2 might be an additional factor potentiating the effect of angiotensin II on glomerular hemodynamics. In conclusion the early reduction in GFR which follows daily administration of CyA in rats might be the result of a synergic action of angiotensin II and TxA2 on vascular tone and mesangial contraction which is not modulated by an increase in glomerular vasodilatory prostaglandins. If this explanation may be applied to early reduction in GFR observed in humans treated with CyA before tubular toxicity develops needs to be investigated further.

Neural induction: embryonic determination elicits full expression of specific neuronal traits.

In Pleurodeles waltl, the early neuronal differentiation of precursor cells from late gastrula stage has been studied by culture in vitro from either isolated neural plate (NP) or isolated neural fold (NF). The aim of this study was to delineate the information acquired by ectodermal target cells during neural induction. By culturing these cells in vitro either with or without the underlying chordamesoderm, we showed that in the absence of chordamesodermal influence such NP or NF cells exhibited a high degree of biochemical and morphological differentiation as revealed by the synthesis and the storage of neurotransmitters, the activity of specific enzymes, as well as by the expression of neuronal markers: specific changes in cell surface carbohydrates, tetanus toxin binding sites and neurofilament polypeptides. Remarkable changes in the cell adhesive properties were the first events observed in the different central (NP) and peripheral (NF) types. In cocultures the chordamesodermal cells exert a beneficial influence on this differentiation, specially increasing acetylcholine synthesis. There are some differences between central (NP) or peripheral (NF) neuroblast response to this further notochord or mesodermal influence.

Multicentre evaluation of the Serono SR1 assay for prostate specific antigen.

This report describes the findings of an evaluation of the validity of the new, highly automated Serono SR1 enzyme immunoassay for prostate specific antigen performed at three European sites. Within-run precision was below 5.5% at the clinically important decision point of 4 micrograms/l, less than 10% below 3 micrograms/l and less than 5% above 4 micrograms/l. Between-run precision was below 12% at each site and between-site precision was less than 14%. Dilution series were shown to be parallel to the standard curve on 12 samples with neat prostate specific antigen concentrations ranging from 20 to 88 micrograms/l (linear regression slopes and correlation coefficients of 1.00 to 1.06 and 0.972 to 1.000 respectively). Comparisons with Hybritech Tandem-E and -R and Roche Cobas Core prostate specific antigen methods used routinely at the three sites were good (linear regression slopes 1.04 to 1.13, correlation coefficients 0.96 to 0.99), but results were significantly lower than the Ciba Corning ACS180 (slope 0.66, correlation coefficient 0.99). The assay presents a precise, robust and valid diagnostic tool offering additional advantages of non-isotope automation.

Neural induction.

The molecular mechanism of neural induction of embryonic cells is an important but poorly understood problem in neuroembryology. Glycoconjugates in the target cell plasma membrane and/or its structural organization play a key role in the reception of the inductive signal. It is the competent target tissue itself which probably contains the capacity and specificity for neuralization. However, the mechanism of transmission of the signal which leads to activation of the intracellular machinery involved in the process of neural determination remains to be elucidated. With respect to the information acquired by the target cells during neural induction, and the early events in differentiation, neuronal precursor cells have been shown to have acquired the potential to display a high degree of biochemical and phenotypic differentiation, even in the absence of further embryonic influences.

Urinary albumin excretion in normal pregnancy and pregnancy-induced hypertension.

We measured the urinary excretion of albumin in 67 healthy primigravidae, at monthly intervals, from 16 to 36 weeks of gestation and 12 weeks postpartum. Of the 67 primigravidae, 55 completed a normal pregnancy and 12 developed pregnancy-induced hypertension. In the latter group, an additional measurement of urinary albumin excretion was performed at 24 weeks postpartum. The aims of the study were: to look for changes of urinary albumin excretion during the progression of normal pregnancy; to assess if microalbuminuria could be an early feature of pregnancy-induced hypertension; to evaluate the effects of physical activity on the excretion of albumin in normal pregnancy and pregnancy-induced hypertension. In contrast with glomerular hyperfiltration and increased urinary total protein, two recognized characteristics of the pregnant state, we found that normal primigravidae, during the day, excrete significantly less albumin (p between less than 0.01 and less than 0.001) in comparison with the postpartum period and nonpregnant women. Normal primigravidae, as a group, showed parallel changes of urinary albumin excretion and diastolic blood pressure throughout pregnancy and postpartum, suggesting an important physiologic role of hemodynamic factors in regulating glomerular permeability to albumin. The daytime urinary albumin excretion in patients developing pregnancy-induced hypertension was significantly higher (p between less than 0.005 and less than 0.001) than in normal pregnancy from the 28th gestational week onwards. The increased urinary albumin excretion preceded the onset of hypertension and tended to persist long after blood pressure had returned to normal levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyponatremic hypertensive syndrome and massive proteinuria in a patient with renin-producing leiomyosarcoma.

We report a case of renin-producing leiomyosarcoma associated with the hyponatremic hypertensive syndrome and nephrotic-range proteinuria. Extremely high levels of active renin and, to a greater extent, of prorenin were found in plasma and tumor tissue. Immunohistochemical and in situ hybridization studies demonstrated that the neoplastic cells were the source of renin production. The hyponatremic hypertensive syndrome and proteinuria promptly responded to treatment with angiotensin-converting enzyme inhibitors, suggesting an angiotensin II dependency of these disorders. After removal of the leiomyosarcoma, plasma concentration of active renin, but not of prorenin, normalized and the hypertension, proteinuria, and electrolyte abnormalities disappeared. However, 5 months after operation, the patient presented once again with hypertension, hypokalemia, proteinuria, and markedly increased plasma levels of both active renin and prorenin that heralded the relapse of neoplastic disease.

[Hormone evaluation in brain death]. / Alcune valutazioni ormonali nella morte cerebrale.

BACKGROUND: In this study, the level and the variation of a number of hormone and metabolic parameters during brain death treatment in potential organ donors have been monitored. METHODS: Thirty-nine consecutive brain-dead patients were enrolled in 3 Intensive Care Units of Regional Hospitals of the North of Italy. All patients were potential organ donors and free from diseases before the accident leading to death. The levels of ADH, ACTH, TSH, prolactin, cortisol, aldosterone, FT3, FT4, renin, serum lactate and plasma osmolality were measured immediately after the diagnosis of brain death (T0), certified following the Italian law of December 29, 1993, n. 578, and after 6 hours (T6). RESULTS: Hormone levels were normal in the majority of subjects, and there was no significant variation during the 6 hours of the observation period. No correlation was found between the hormone levels considered and the metabolic parameters; ADH levels were not correlated with plasma osmolality. FT3 levels were below the normal range in the majority of subjects, but were not associated with a higher lactate level, which is used as a marker of a shift toward tissue anaerobic metabolism. CONCLUSIONS: In conclusion, triiodothyronine administration to improve metabolic order and thus the function of organs for transplantation is not justified in brain-dead patients.