Experimental protocol for clinical analysis of cerebrospinal fluid by high resolution proton magnetic resonance spectroscopy.

High resolution magnetic resonance spectroscopy (MRS) of cerebrospinal fluid (CSF) is a non-destructive analytical method which allows rapid and simultaneous detection of molecules involved in intermediary and oxidative metabolic pathways. We developed a protocol suitable for routine MRS analysis of lyophilized CSF samples. This procedure guarantees sample integrity, from CSF collection to spectrum acquisition. MRS analysis of blood serum was included in our protocol as a complementary method to CSF analysis. This protocol can contribute to establish MRS of CSF as a new analytical tool to better understand the metabolic processes involved in neurological diseases.

Further assignment of resonances in 1H NMR spectra of cerebrospinal fluid (CSF).

A number of previously unidentified 1H NMR signals detected in CSF spectra of patients with various neurological and metabolic diseases are assigned to metabolites, drugs and drug excipients. Two-dimensional 1H NMR spectroscopy (COSY and J-resolved) is employed to resolve resonances which are hidden by superimposed peaks in one-dimensional spectra. Assignments obtained by making use of 2-D techniques, and of a 1-D 1H NMR data base created for ca. 150 authentic compounds, enable us to clarify the nature of complex signal patterns found in crowded spectral regions of CSF such as the aliphatic methyl region at ca. 1.0 ppm.

Unexpected citrate toxicity and severe hypocalcemia during apheresis.

BACKGROUND: The citrate anticoagulant used during apheresis procedures is considered a safe medication because it is rapidly metabolized by the donor. However, acute, life-threatening hypocalcemia is possible if the infusion rate of citrate is increased. CASE REPORT: A 54-year-old woman with metastatic breast cancer, but otherwise in good health, had just begun a fifth collection of hematopoietic peripheral blood progenitor cells by leukapheresis. The instrument's self-loading apheresis kit was primed uneventfully. Seven minutes into the procedure, the patient developed signs and symptoms suggesting severe hypocalcemia, including muscle spasms, chest pain, and hypotension. The citrate bag was discovered to have emptied, and a section of the anticoagulant tubing was protruding outside of the rotary pump. The patient's ionized calcium level was 0.64 mmol per L (normal range, 1.18-1.38 mmol/L). In subsequent experiments where the anticoagulant tubing was either improperly loaded at the outset or partially pulled out of the rotary pump, no instrument alarms sounded. CONCLUSION: Citrate toxicity and life-threatening hypocalcemia can occur if the anticoagulant line of an apheresis instrument is not properly housed in its rotary pump or becomes disengaged during the procedure. Instrument manufacturers are encouraged to consider designs that allow the direct measurement of the volume of citrate delivered. In the interim, periodic visual and tactile confirmation of tubing placement during apheresis procedures is prudent.

Cerebrospinal fluid metabolic profiles in multiple sclerosis and degenerative dementias obtained by high resolution proton magnetic resonance spectroscopy.

We have analyzed the cerebrospinal fluid (CSF) of 19 patients with multiple sclerosis (MS), 12 patients with degenerative dementia and 17 control patients using in vitro high resolution proton magnetic resonance spectroscopy (MRS) at 400 MHz. The CSF metabolic profile is slightly modified in MS patients (increased lactate and fructose concentrations, decreased creatinine and phenylalanine concentrations) and is not correlated with the intensity of the intrathecal inflammation. Proton MRS of CSF does not differentiate relapsing-remitting MS and primary progressive MS. We have not detected any specific abnormal resonance in native or lyophilized CSF. The CSF metabolic profile of demented patients is much more altered (increased concentration of lactate, pyruvate, alanine, lysine, valine, leucine-isoleucine, tyrosine, glutamine) and is in agreement with a brain oxidative metabolism impairment as already described in Alzheimer's disease. Unassigned abnormal but non specific or constant resonances have been detected on MR spectra of demented patients. CSF inositol concentration is also increased in the CSF of patients with Alzheimer's disease. In vitro high resolution proton MRS of the CSF constitutes a new and original way to explore CSF for the differential and/or early diagnosis of dementias, as a complement to in vivo proton cerebral MRS.

[Benign tumors and pseudotumor of the maxilla in children]. / Tumeurs bénignes et pseudo-tumeurs des maxillaires de l'enfant.

The underlying causes in series of benign tumours and pseudo-tumours and cysts of the maxillary bone observed in children over a ten-year period in the Saint-Vincent-de-Paul Hospital in Paris are reported. Besides the frequently encountered odontogenic cysts, most cases involved aneurysmal cysts and benign odontogenic tumours, as well as rare tumours including a hydatic cyst. Several lesions were discovered on a panoramic X ray performed for orthodontal survey.

Improved microplate immunoenzymatic assay of PCR products for rapid detection of Mycoplasma pneumoniae.

We developed a microtitre hybridization assay for the detection of polymerase chain reaction (PCR) amplified sequences. For this, cloned Mycoplasma pneumoniae DNA containing a sequence complementary to the PCR products is first covalently bound to microtitre wells. These coated microplates can be stored for several months. Then, an aliquot of the PCR product, labelled with digoxigenin-dUTP during its synthesis is hybridized to the immobilized DNA. The use of a rapid hybridization buffer makes this step very short (5 min). Finally, the hybridization signal is detected by an anti-digoxigenin antibody conjugated with alkaline phosphatase. Compared to Southern or other microplate hybridization techniques, this method is cheaper, involved fewer steps and allows easy handling of a large number of samples. This method was used for detection of M. pneumoniae in a series of clinical specimens.

Antipeptide polyclonal antibodies that recognize a substance P-binding site in mammalian tissues: a biochemical and immunocytochemical study.

We used complementary peptide methodology to obtain antibodies against the receptor for the neuropeptide substance P, specifically directed at the ligand-binding domain. Rabbits were immunized with two distinct peptides derived from the sequence of the RNA complementary to the mRNA for substance P. Binding experiments revealed that antipeptide polyclonal antibodies were able to recognize, through their paratope, a specific binding site on the rat parotid cell membranes. Substance P and antibodies competed for this binding site, because preincubation of membranes in the presence of substance P significantly reduced antibody binding, and conversely, preincubation of membranes in the presence of antibodies partly inhibited the binding of radioiodinated substance P. Immunocytochemical experiments performed on the rat cervical spinal cord show that the distribution of labeling by antibodies is similar to that observed by conventional autoradiography using 125I-substance P. Here again, control experiments demonstrated that antibodies and substance P were competing for the same binding site on the spinal cord. These biochemical and immunocytochemical data indicate that antipeptide antibodies recognize a substance P membrane binding site in nervous and nonnervous mammalian tissues. This site is likely to correspond to the NK1 specific receptor for substance P.

[The advantages and limits of the use of desmopressin (Minirin) in stomatology. Apropos 2 young female patients presenting with von Willebrand's disease]. / Intérêts et limites de l'utilisation de la desmopressine (Minirin) en stomatologie. A propos de deux jeunes patientes présentant une maladie de Willebrand.

The authors describe two cases of wisdom tooth germ removal in female adolescents presenting with von Willebrand's disease. The effect of the Minirin injection was studied during a preoperative test and allowed carrying out the operation with normalized hemostasis, without using substitution factors and with no postoperative complications. The authors specify the indications and limitations of the use of desmopressin before surgery.

Enzyme immunoassay for tachykinin-like immunoreactivity in the guinea pig spinal cord.

A solid-phase enzyme immunoassay for quantitation of tachykinin-like immunoreactivity (TK-LI) is presented. Because the antiserum K-12 recognizes various tachykinins, such as neurokinin A (100%), kassinin (103%), eledoisin (51%), neurokinin B (18%), physalaemin (0.7%), and substance P (0.7%), the immunoreactivity detected in this enzyme immunoassay has been termed TK-LI. The assay was performed on 96-well microtiter plates coated with a mouse monoclonal second antibody. After preincubation of soluble neurokinin A or samples and K-12 antiserum for 3 h at room temperature, acetylcholinesterase-labelled neurokinin A was allowed to react overnight at 4 degrees C. Samples were finally incubated with Ellman's reagent for 2 h and the absorbance was measured at 414 nm. The threshold for detection of TK-LI was 2 fmol/well. TK-LI release from guinea pig dorsal spinal cord slices was evoked by capsaicin or high K+ medium. The capsaicin-evoked TK-LI release was increased in the presence of thiorphan, but not in that of captopril.

[Variation of substance P-like immunoreactivity in plasma and cerebrospinal fluid in the course of arthritis induced by Freund adjuvant in rats, a model for the study of chronic pain]. / Variation de l'immunoréactivité de type substance P dans le plasma et le liquide céphalo-rachidien au cours de l'évolution de l'arthrite induite par adjuvant de Freund chez le rat, un modèle d'étude de douleur chronique.

Parallel time courses of clinical and behavioural parameters and levels of plasma substance P-like immunoreactivity (SPLI-PI) were studied in arthritic rats (adjuvant induced arthritis, AIA, a chronic pain model). Acute (14 and 21 post-inoculation days,PI) and post-acute (42 days PI) phases of the syndrome were investigated. These data were compared with those obtained in a control situation (inoculation day). In a second experimental series, levels of substance P-like immunoreactivity in cerebrospinal fluid (SPLI-CSF) were determined at the same stages of AIA. In arthritic rats SPLI-PI was strongly enhanced (X4) as early as 14 days PI and remained increased (X4) at all stages studied, whereas SPLI-LCR was significantly increased (X2) only 21 days PI and returned to control levels at 42 days PI. These data suggest that SP could be distributed in two different pools, a peripheral one of inflammatory origin, and a central one which could be more specific to the chronic pain situation.

Anti-substance P anti-idiotypic antibodies modulate the secretory process in the rat parotid gland in vitro.

Anti-idiotypic antibodies (anti-Id) obtained in rabbits in response to immunization with polyclonal anti-substance P antibodies (anti-SP) were shown to bind specifically and with high affinity to membranes from rat parotid gland cells. Whereas substance P (SP) was unable to displace anti-Id from membrane binding sites, anti-Id partly inhibited the binding of radiolabelled substance P. Like substance P, anti-Id were able to trigger protein secretion by parotid cells i.e. to behave as physiological agonists of the neuropeptide. Under our experimental conditions, the biological effects of both ligands appear to be additive. Unlike substance P, however, anti-Id did not potentiate the secretory response induced by a beta-adrenoceptor agonist. Taken together, the present results might indicate that anti-Id interact with epitope(s) at or/and near the peptide-combining site on the substance P receptor. These data demonstrate further the possibility of raising pharmacologically active anti-receptor antibodies through the immunological anti-idiotypic approach.

Use of anti-idiotypic antibodies as probes for in vitro and in vivo identification of substance P receptor.

In order to develop immunological tools for studying the receptor of the neuropeptide substance P (SP), anti-idiotypic antibodies (anti-Id abs) were produced by immunization with anti-SP antibodies whose specificity was close to that of the SP receptor. Immunological studies revealed structural similarity between some anti-Id abs and SP itself. As a consequence, these anti-Id abs were able to bind to mammalian SP receptors. These antibodies were used in immunocytochemistry to label SP receptors both in the rat spinal cord and in rat and guinea pig peripheral tissues (parotid gland and trachea, respectively). Like SP, anti-Id abs were able to trigger protein secretion by isolated rat parotid gland cells. Finally, it was shown that anti-Id abs in vivo modulated reactivity to chemical stimuli. These antibodies therefore appear to be promising tools for further biochemical, cytochemical, and pharmacological characterization of SP receptors.

Pastoral care and bereavement.

Ministering to the bereaved is a demanding task. Pastoral caregivers have to learn to forget about themselves, to enter into the center of others' concerns. They have to be willing to deny themselves, stop concentrating on their problems to create the space where God can work. Their faith and insight into life must form the core of their pastoral work.

Increased reactivity to chemical but not to heat noxious stimuli in mice producing anti-idiotypic antibodies for substance P.

Mice immunized against anti-substance P (anti-SP) monoclonal antibodies produced anti-SP anti-idiotypic antibodies (SPAb2). In a previous report. SPAb2 antibodies were found to have in vitro biological activity i.e. to behave either as agonists or as antagonists for substance P (SP) depending on the biological test. In this study, the involvement of SPAb2 in in vivo biological activity has been tested. Because of the possible implication of SP in the generation and transmission of nociceptive information, we have tested the responsiveness of SPAb2 responding mice in behavioral nociceptive tests. SPAb2 mice showed very small behavioral variations in the hot plate test as compared with a control group of mice immunized against an unrelated monoclonal antibody. In the formalin test, however, SPAb2 mice displayed a significant increase in paw licking time, which was significantly correlated with SPAb2 serum concentration. These results are discussed in terms of the use of SPAb2 as pharmacological tools for studying the biological properties of SP receptors and more generally of auto anti-idiotypic antibodies in modulating behavioral responses.

ABO and Lewis blood group antigens of donor origin in the bile of patients after liver transplantation.

The authors prospectively studied the expression of blood group antigens and their corresponding glycosyltransferases in the bile of 11 patients who received orthotopic liver transplants. In six patients, the donor was of the same ABO/Lewis/secretor as the recipient, and the expected blood group antigens were found in the postoperative bile. Five patients who received transplants from donors discordant for either ABO, Lewis, or secretor status continued to produce blood group antigens of donor origin. This study provides evidence that the expression of soluble blood group antigens is under autonomous end-organ control.