Membrane attack complex (MAC)-mediated damage to spermatozoa: protection of the cells by the presence on their membranes of MAC inhibitory proteins.

Although antibody and complement are known to cause immobilization and killing of spermatozoa in vitro the components of the complement system mediating these effects remain undefined. Here we have examined the effects of the membrane attack complex (MAC) on spermatozoa and demonstrate that spermatotoxic effects are dependent on assembly of the complete MAC. We subsequently examined the presence and functional significance of the complement regulatory proteins decay accelerating factor (DAF), MAC-inhibiting protein (MIP) and CD59 antigen on spermatozoa. Both DAF and CD59 antigen were present on the membranes of these cells. Neutralization of CD59 antigen with specific antibodies increased the susceptibility of the cells to MAC-mediated damage, suggesting a role for this molecule in the protection of spermatozoa from complement-mediated damage in the female reproductive tract.


New monoclonal antibodies in CD59: use for the analysis of peripheral blood cells from paroxysmal nocturnal haemoglobinuria (PNH) patients and for the quantitation of CD59 on normal and decay accelerating factor (DAF)-deficient erythrocytes.

CD59 is a widely expressed cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein which acts as an inhibitor of the assembly of the membrane attack complex of autologous complement. Four new monoclonal antibodies to CD59 (2/24, 1B2, BRIC 229, BRIC 257) are described. Competitive binding experiments using these antibodies, two known CD59 antibodies (MEM-43, YTH 53.1) and a previously described antibody LICR-LON-Fib75.1 demonstrated that all seven antibodies see related epitopes on human erythrocyte CD59. In common with other GPI-linked proteins, CD59 (as defined by antibody 2/24) was sensitive to treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) on lymphocytes and monocytes but not on erythrocytes. Flow cytometric analysis using antibody 2/24 identified two populations (CD59 positive and CD59 deficient) of lymphocytes, monocytes and erythrocytes in peripheral blood from a patient with paroxysmal nocturnal haemoglobinuria (PNH). The abundance of CD59 on normal erythrocytes was determined as 21,000 copies/cell when radioiodinated BRIC 229 was used. Other CD59 antibodies gave values of 10,000 (IF5) and 15,000 (2/24) against the same target cells. Radioiodinated Fab fragments of BRIC 229 gave a value of 39,000 copies/cell. Erythrocytes from two individuals with a rare inherited deficiency of decay accelerating factor (DAF), known as the Inab phenotype, expressed normal levels of CD59.


KY nailing of impending and pathological fractures of the proximal femur.

Eight two patients underwent 86 Küntscher Y nailing for impending and pathological fractures of the proximal femur. All but five mobilised post-operatively, thus improving their quality of life. Eighteen patients died in hospital. Prophylactic nailing improved survival. Fifteen out of 86 nailings were followed by complications.


The results of radiotherapy for isolated elevation of serum PSA levels following radical prostatectomy.

Eleven patients were initially treated for localized prostate cancer with radical retropubic prostatectomy following negative pelvic lymph node dissection. Six or more months after surgery, these patients had elevated serum prostate specific antigen (PSA) levels. No patient had other clinical evidence of disease as determined by history, physical examination, bone scan, computed tomographic scan of the abdomen and pelvis, chest radiograph, complete blood cell count, and serum chemistry profile. These patients received prostate bed irradiation using 10-MV photons and a four-field technique. Doses ranged from 60.0 to 65.8 Gy in 1.8 to 2.0 Gy fractions. Levels of serum PSA were monitored and decreased initially in all treated patients. In two patients, levels of PSA increased after this initial decrease. In 7 of the 11 patients (64%), PSA levels decreased to less than or equal to 0.3 ng/mL at last measurement. Radiotherapy resulted in no severe toxicity. None of the patients had developed clinical evidence of disease at the time of this report. Isolated elevations of serum PSA after prostatectomy reflect residual disease, and radiotherapy appears to effectively decrease the PSA levels in most cases. This effect appears to be accomplished by killing locally residual or recurrent cancer in the postoperative tumor bed.


A report of RTOG 8206: a phase III study of whether the addition of single dose hemibody irradiation to standard fractionated local field irradiation is more effective than local field irradiation alone in the treatment of symptomatic osseous metastases.

Hemibody irradiation (HBI) in a single exposure is an effective and safe technique for palliation of symptoms due to widespread bony metastases (RTOG 78-10). The present study (82-06) sought to explore the possibility that HBI added to local-field irradiation might delay the onset of metastases in the hemibody effected, as assessed by bone scans and X rays, and decrease the frequency of further treatment. The results of this clinical trial establish that 800 cGy of HBI is indeed causes micro-metastases to regress, perhaps completely. A total of 499 patients were randomized to receive either HBI or no further treatment following completion of standard palliative local field irradiation (300 cGy x 10) to the symptomatic site. Improvement was seen in time-to-disease progression at one year, 35% for local + HBI versus 46% on the local-only control arm. Time-to-new disease in the targeted hemibody was also improved. At one year, 50% of patients on the local + HBI arm showed new disease compared to 68% on the local-only arm. Furthermore, the median time-to-new disease within the targeted HBI area was 12.6 months for the local + HBI arm versus 6.3 months for patients in the local-only arm. Time-to-new treatment within the hemibody segment was also delayed. At one year, 76% of the local only group had been retreated versus 60% in the local + HBI arm. There were no fatalities and no radiation pneumonitis was seen in the local + HBI arm. Overall, the incidence of toxicities was low (5-15%). The occurrence of severe hematopoetic toxicities were significantly different in the local + HBI arm, but they were transitory. One life-threatening thrombocytopenia occurred, for a limited time, indicating excellent tolerance to HBI. This clinical trial demonstrates that HBI has the potential to be used to treat systemic and occult metastases, particularly if both halves of the body can be treated.


A review of local radiotherapy in the treatment of bone metastases and cord compression.

Local radiotherapy plays an important role in the management of bone metastases. Because it is given with palliative intent to patients with limited, if variable, life expectancy, radiotherapy schedules need to be identified which give maximum patient benefit (short and long term) with minimum associated morbidity and minimum disruption of the patients' remaining life. For localized bone pain, a single fraction of radiotherapy, repeated if necessary, appears to fulfill these criteria in patients with a short life expectancy. There are, however, unanswered questions regarding fraction size and the adequacy of one fraction for long-term control and for all pathological tumor types. Only randomized trials can answer these questions. Uncertainties also exist regarding the precise indications for radiotherapy to prevent and treat pathological fractures and the optimal dose schedule which will provide adequate local tumor control without inhibiting bone healing or interfering with bone integrity. Because of the many variables, guidelines on selection of cases of spinal cord compression for decompression by surgery or radiotherapy are likely to be of more value than randomized prospective studies in this condition. Experimental work and clinical experience to date suggest an advantage for a few large fractions of radiotherapy, at least initially, to achieve a rapid response, but this too needs confirmation. Treatment decisions based on past teaching and local custom rather than on valid clinical trial data have led to considerable differences in clinical practice among radiotherapists. Bone metastases are common and warrant a great deal more experimental and clinical study than they have received to date.


The role of radiation therapy in the treatment of brain metastases.

Whole brain irradiation is the most effective means for treating the patient with brain metastases with symptom relief occurring in 70 to 90% of patients. However, 25-50% of patients with brain metastases will die due to eventual failure in the brain and therefore entry of patients into investigative trials is essential for continued progress in the management of this problem. For the patient who is not part of an investigative trial, short courses of radiation of 20 Gy in 1 week or 30 Gy in 2 weeks are generally as effective as more prolonged courses and even shorter courses of treatment could be considered, particularly for the patient with an estimated survival of only 5-6 weeks. The importance of the treatment of brain metastases on the practice of radiation oncology is significant and comparable to other major cancers treated with radiation. It is critical that radiation oncologists can apply this treatment modality in a cost effective manner with careful consideration for the patients' quality of life.


Prostate-specific antigen as a prognostic factor for prostate cancer treated by external beam radiotherapy.

The potential prognostic significance of prostate-specific antigen (PSA) serum concentrations was evaluated in 171 patients with stages A2 to C adenocarcinoma of the prostate treated with external beam radiotherapy. After a median follow-up of 17 months, 12 patients sustained relapse of disease and PSA levels were found to be prognostically significant in three ways. (a) Pretreatment PSA level: none of 59 patients with a pretreatment PSA level less than or equal to 4 ng/ml relapsed to date and only one developed a subsequently rising PSA profile; 7 of 102 patients (7%) with a pretreatment PSA level in the range 4-40 ng/ml relapsed and 17 (17%) showed a rising PSA profile; 5 of 10 patients (50%) with a pretreatment PSA level greater than or equal to 40 ng/ml relapsed and six (60%) developed rising PSA values. The differences were significant and were maintained when patients were stratified by stage or grade. (b) PSA level at 6 months: for patients with pretreatment PSA levels in the range 4-40 ng/ml, a 6-month value greater than 2 ng/ml predicted a significantly worse outcome than a 6-month value less than 2 ng/ml. (c) Rising post-treatment PSA values: following a radiation-related nadir in PSA levels, 24 patients experienced rising PSA values and 8 (33%) relapsed at a median time of 5 months after onset of the rising values--a significantly higher relapse rate than observed in patients with non-rising PSA values. Whether the majority, or all, of the patients with rising PSA levels relapse, requires further follow-up. In conclusion, serum PSA levels are strong prognostic determinants of outcome following radiotherapy for prostate cancer and appear to add prognostic information independently of tumor stage and grade.


Balbiani ring hnRNP substructure visualized by selective staining and electron spectroscopic imaging.

The Balbiani Rings (BR) in the polytene chromosomes of Chironomus salivary glands are intense sites of transcription. The nascent RNPs fold during transcription into 40-50-nm granules, containing in the mature transcript approximately 37-kb RNA. Using a new nucleic acid specific stain, osmium ammine B on Lowicryl sections, in combination with electron energy filtered imaging of sections containing BR granules, we demonstrate a RNA-rich particulate substructure (10-nm particle diameter; 10-12 particles per BR granule). Elemental imaging supports that these particles are enriched in phosphorus. The possible relationship of these RNA-rich particles to ribonucleosomes is discussed, as well as models for their arrangement in the mature BR granules.


PKC epsilon-related kinase associates with and phosphorylates cytokeratin 8 and 18.

A 40-kD protein kinase C (PKC)epsilon related activity was found to associate with human epithelial specific cytokeratin (CK) polypeptides 8 and 18. The kinase activity coimmunoprecipitated with CK8 and 18 and phosphorylated immunoprecipitates of the CK. Immunoblot analysis of CK8/18 immunoprecipitates using an anti-PKC epsilon specific antibody showed that the 40-kD species, and not native PKC epsilon (90 kD) associated with the cytokeratins. Reconstitution experiments demonstrated that purified CK8 or CK18 associated with a 40-kD tryptic fragment of purified PKC epsilon, or with a similar species obtained from cells that express the fragment constitutively but do not express CK8/18. A peptide pseudosubstrate specific for PKC epsilon inhibited phosphorylation of CK8/18 in intact cells or in a kinase assay with CK8/18 immunoprecipitates. Tryptic peptide map analysis of the cytokeratins that were phosphorylated by purified rat brain PKC epsilon or as immunoprecipitates by the associated kinase showed similar phosphopeptides. Furthermore, PKC epsilon immunoreactive species and CK8/18 colocalized using immunofluorescent double staining. We propose that a kinase related to the catalytic fragment of PKC epsilon physically associates with and phosphorylates cytokeratins 8 and 18.


Slow axonal transport mechanisms move neurofilaments relentlessly in mouse optic axons.

Pulse-labeling studies of slow axonal transport in many kinds of axons (spinal motor, sensory ganglion, oculomotor, hypoglossal, and olfactory) have led to the inference that axonal transport mechanisms move neurofilaments (NFs) unidirectionally as a single continuous kinetic population with a diversity of individual transport rates. One study in mouse optic axons (Nixon, R. A., and K. B. Logvinenko. 1986. J. Cell Biol. 102:647-659) has given rise to the different suggestion that a significant and distinct population of NFs may be entirely stationary within axons. In mouse optic axons, there are relatively few NFs and the NF proteins are more lightly labeled than other slowly transported slow component b (SCb) proteins (which, however, move faster than the NFs); thus, in mouse optic axons, the radiolabel of some of these faster-moving SCb proteins may confuse NF protein analyses that use one dimensional (1-D) SDS-PAGE, which separates proteins by size only. To test this possibility, we used a 2-mm "window" (at 3-5 mm from the posterior of the eye) to compare NF kinetics obtained by 1-D SDS-PAGE and by the higher resolution two-dimensional (2-D) isoelectric focusing/SDS-PAGE, which separates proteins both by their net charge and by their size. We found that 1-D SDS-PAGE is insufficient for definitive NF kinetics in the mouse optic system. By contrast, 2-D SDS-PAGE provides essentially pure NF kinetics, and these indicate that in the NF-poor mouse optic axons, most NFs advance as they do in other, NF-rich axons. In mice, greater than 97% of the radiolabeled NFs were distributed in a unimodal wave that moved at a continuum of rates, between 3.0 and 0.3 mm/d, and less than 0.1% of the NF population traveled at the very slowest rates of less than 0.005 mm/d. These results are inconsistent with the proposal (Nixon and Logvinenko, 1986) that 32% of the transported NFs remain within optic axons in an entirely stationary state. As has been found in other axons, the axonal transport system of mouse optic axons moves NFs and other cytoskeletal elements relentlessly from the cell body to the axon tip.


Regulation of the VLA integrin-ligand interactions through the beta 1 subunit.

Integrins from the very late activation antigen (VLA) subfamily are involved in cellular attachment to extracellular matrix (ECM) proteins and in intercellular adhesions. It is known that the interaction of integrin proteins with their ligands can be regulated during cellular activation. We have investigated the regulation of different VLA-mediated adhesive interactions through the common beta 1 chain. We have found that certain anti-beta 1 antibodies strongly enhance binding of myelomonocytic U-937 cells to fibronectin. This beta 1-mediated regulatory effect involved both VLA-4 and VLA-5 fibronectin receptors. Moreover, anti-beta 1 mAb also induced VLA-4-mediated binding to a recombinant soluble form of its endothelial cell ligand VCAM-1. Non-activated peripheral blood T lymphocytes, unable to mediate VLA-4 interactions with fibronectin or VCAM-1, acquired the ability to bind these ligands in the presence of anti-beta 1 mAb. The anti-beta 1-mediated changes in the affinities of beta 1 integrin for their ligands were comparable to those triggered by different lymphocyte activation agents such as anti-CD3 mAb or phorbol ester. Adhesion of melanoma cells to other ECM proteins such as laminin or collagen as well as that of alpha 2-transfected K-562 cells to collagen, was also strongly enhanced by anti-beta 1 mAb. These beta 1-mediated regulatory effects on different VLA-ligand interactions do not involve changes in cell surface membrane expression of different VLA heterodimers. The anti-beta 1-mediated functional effects required an active metabolism, cytoskeleton integrity and the existence of physiological levels of intracellular calcium as well as a functional Na+/H+ antiporter. Beta 1 antibodies not only increased cell attachment but also promoted spreading and cytoplasmic extension of endothelial cells on plates coated with either fibronectin, collagen, or laminin as well as induced the rapid appearance of microspikes in U-937 cells on fibronectin. Moreover, both beta 1 integrin and the cytoskeletal protein talin colocalized in the anti-beta 1 induced microspikes. These results emphasize the central role of the common beta 1 chain in regulating different adhesive functions mediated by VLA integrins as well as cellular morphology.


In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.

Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-I and IGF-II, and conditioned medium from 3T3-J2 cells, caused a dose-dependent increase of 3H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alpha IR-3, which is a specific antagonist of type-I IGF receptor. Fetal mouse-derived 3T3-J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non-IGF-producing BALB/c 3T3 cells were used as a feeder-layer, the keratinocytes number was similar to that observed with 3T3-J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.


Analysis of inter-serotypic structural relationships of infectious bursal disease virus using detergent solubilization and radioimmunoprecipitation with monoclonal antibodies.

Monoclonal antibodies (MAbs) neutralizing only the infectious bursal disease virus strains (IBDV) belonging to serotype 1 also immunoprecipitated the heterologous major antigenic proteins of serotype 2 IBDV. Detergent-solubilization followed by radioimmunoprecipitation assays (RIPA) using the MAbs revealed structural similarities between the conformation-dependent antigenic determinants of IBDV of the two existing serotypes. The presence of non-ionic detergent Triton X-100 determined the binding of altered proteins by MAbs in RIPA.


Comparison of hepatitis C virus markers in patients with NANB hepatitis.

10 different HCV-specific assays and RT-PCR of the 5' untranslated region of HCV RNA were used to analyze sixty-four patients with chronic NANB liver disease. Po, CP-9 and C22 antigens are located in the putative core; C33c in the putative NS3; C100-3 in the putative NS3/4; KCL in the putative NS4/5 and C825 is located in the putative NS5. GOR protein is not part of the HCV genome, but antibodies to it appear to be present in response to a hepatitis C infection. Positive rates were 91% for Po, 89% for CP-9, 94% for C22, 97% for C33c, 88% for C100-3 (Ortho, EIA), 86% for C100-3 (Abbott, EIA), 84% for C100-3 (Ohtsuka, RIA), 88% for KCL, 59% for C825, 58% for GOR, and 83% for RT-PCR. There were 8 cases which were negative by all anti-C100 tests. 7 of these cases were positive by other anti-HCV markers and/or PCR suggesting the need for improved blood screening assays. There is a variation in the relative reactivity for different markers with different samples. Of the tests employed, anti C33c shows the highest positivity rate.


Rapid identification of legionellae by a colony blot assay based on a genus-specific monoclonal antibody.

We recently developed a monoclonal antibody immunoglobulin G2a (2125) recognizing a genus-specific epitope on the 60-kDa heat shock protein of all Legionella species. In the current study, this antibody was used in a colony blot enzyme-linked immunosorbent assay for the rapid identification of Legionella cultures on agar plates. The whole protocol was completed in less than 2 h. All 59 Legionella species and serogroups that were tested gave a positive signal. No unspecific reactions with nonlegionellae were observed. This test is a rapid procedure for the identification of legionellae growing on agar medium to the genus level.


Chemiluminescent universal probe for bacterial ribotyping.

We describe a novel procedure for direct covalent coupling of horseradish peroxidase to rRNA and ribotyping by using enhanced chemiluminescence. Compared with their 32P-end-labeled counterparts, chemiluminescent rRNA probes are stable and easy to synthesize and provide results as good as or superior to those obtained with isotopic labeling. Direct chemiluminescent labeling of Escherichia coli rRNA produces a sensitive, universal probe suitable for clinical laboratory use in the investigation of nosocomial outbreaks.


Detection of the etiologic agent of human ehrlichiosis by polymerase chain reaction.

Polymerase chain reaction (PCR) primers derived from a variable region of the 16S rRNA gene sequence were used to amplify DNA specifically from Ehrlichia chaffeensis (the recently proposed name for the etiologic agent of human ehrlichiosis). The 389-bp product defined by the specific primers was not detected when DNA samples from any of the other recognized species of Ehrlichia were used as amplification templates. When the PCR was applied to five suitable blood specimens obtained from patients subsequently shown to be serologically positive for E. chaffeensis, all five were positive. The same technique was applied to a total of six control blood specimens, three from febrile patients who had no serologic evidence of infection with Ehrlichia or Rickettsia species and three from patients diagnosed with Rocky Mountain spotted fever, and all six were negative. A chemiluminescent, group-specific oligonucleotide probe was shown to hybridize only with the PCR products obtained upon amplification of the five blood specimens from patients serologically diagnosed as having human ehrlichiosis. The results indicate that PCR, coupled with a nonisotopic method of confirming the identity of the PCR product, is a highly specific and efficient method of detecting the agent of human ehrlichiosis in blood. The results also suggest that E. chaffeensis is the sole etiologic agent of human ehrlichiosis in the United States. The technique was also applied to four ticks that were positive by direct immunofluorescence for Ehrlichia species, and one tick was PCR positive, indicating that E. chaffeensis DNA can be detected in ticks harboring this organism, although the sensitivity may be low.


Identification of Chlamydia pneumoniae by DNA amplification of the 16S rRNA gene.

Chlamydia pneumoniae is an important cause of respiratory disease in humans, but diagnosis of C. pneumoniae is hindered by difficulties in the in vitro growth of the organism. In order to improve detection and identification, we recently developed a polymerase chain reaction (PCR) assay which uses oligonucleotide primers specific for C. pneumoniae. The nucleic acid sequence was determined for the 16S rRNA of C. pneumoniae, and regions in which C. pneumoniae differed from both Chlamydia psittaci and Chlamydia trachomatis were identified. Oligonucleotide primers corresponding to these unique regions were then synthesized and used in a PCR for the detection of C. pneumoniae. The C. pneumoniae-specific primers permitted the identification of six isolates of C. pneumoniae, but no reaction was observed with the 15 serovars of C. trachomatis or two strains of C. psittaci. PCR should prove to be valuable in confirming the identification of C. pneumoniae and in the diagnosis of C. pneumoniae infections.


Analysis of rRNA restriction fragment length polymorphisms from Bacteroides spp. and Bacteroides fragilis isolates associated with diarrhea in humans and animals.

The Escherichia coli rRNA operon rrnB was used as a 32P-labeled hybridization probe in Southern blots of genomic DNAs from representative strains of the saccharolytic, gram-negative, obligate anaerobes of the genus Bacteroides. Control experiments with the B. fragilis type strain ATCC 25285 established that nearly identical rRNA fragment patterns were produced when either the E. coli rrnB gene probe or homologous rRNA isolated from B. fragilis was used as the probe. In addition, it was shown that a specific 16S or 23S rrnB gene probe also could be used to produce fragment patterns suitable for analysis. Thirty-one strains from 8 of the 10 recognized Bacteroides species were then examined. The resulting autoradiographs revealed specific fragment patterns for all but one (B. ovatus) of the species tested. Restriction fragment length polymorphisms were observed for many of the strains tested, but these differences did not hinder species classification. The five B. ovatus strains examined did not form a distinct group, and their rRNA fragment patterns displayed a marked heterogeneity. The same approach was applied to a unique set of enterotoxin-producing B. fragilis strains isolated from animals and humans with diarrhea. The results demonstrated that these strains were in fact B. fragilis and that they produce rRNA fragment patterns closely related to those of the type strain ATCC 25285. This set of strains did not appear to form a separate subgroup or genotype within the B. fragilis species, and there were no distinguishable restriction fragment length polymorphisms that could be used to specifically separate enterotoxin-producing strains from nonenterotoxigenic strains.


Specific immunofluorescent staining of pathogenic treponemes with a monoclonal antibody.

Two hybrid cell lines which produced mouse monoclonal antibody to the DAL-1 street strain of Treponema pallidum subsp. pallidum were established. These monoclonal antibodies strongly reacted with T. pallidum subsp. pallidum (Nichols strain, DAL-1, and two other street strains, strains MN-1 and MN-3) and T. pallidum subsp. pertenue by indirect microimmunofluorescent antibody and enzyme-linked immunosorbent assay techniques, but they did not react with normal rabbit testicular tissue. These monoclonal antibodies did not react with nonpathogenic treponemes, such as T. phagedenis Reiter, T. denticola MRB, T. refringens Noguchi, or other spirochetes, such as Borrelia burgdorferi and Leptospira interrogans serovar pomona in microimmunofluorescent antibody smear slides or in Western blots (immunoblots). While unlabeled antibodies are useful for investigating the antigenic structures of T. pallidum, we labeled these monoclonal antibodies with fluorescein isothiocyanate and used them for diagnosing syphilis by direct staining of lesion exudate or T. pallidum subsp. pallidum in formalin-fixed tissues from patients suspected of having syphilis. Both monoclonal antibodies were directed against antigens of T. pallidum subsp. pallidum with a molecular weight of 37,000 as determined by the Western blotting technique.


Rapid detection of respiratory syncytial virus in nasopharyngeal aspirates by reverse transcription and polymerase chain reaction amplification.

A rapid method for detection of respiratory syncytial virus (RSV) in nasopharyngeal aspirates, involving a combination of reverse transcription and polymerase chain reaction amplification (RT-PCR), has been developed. The RT-PCR assay employs oligonucleotide primers specific for the region of the RSV genome which encodes the F1 subunit of the fusion (F) glycoprotein. Other respiratory viruses do not give a positive reaction. The RT-PCR assay was tested on 202 nasopharyngeal aspirates collected from children with clinical signs of respiratory infection, and the results from RT-PCR were compared with those obtained from virus culture and direct detection by enzyme immunoassay (EIA). RT-PCR results were positive in 118 of 125 samples from which RSV was cultured, as well as in 4 of 7 samples which were culture negative but EIA positive. RT-PCR results were negative in 68 of 70 culture-negative, EIA-negative samples, which included 11 samples from which other respiratory viruses were isolated. The speed, sensitivity (94.6%), and specificity (greater than 97%) of the RT-PCR assay suggest that this technique could be useful for rapid detection of RSV in clinical samples.


Nitric oxide synthetase (NOS)-containing sympathoadrenal cholinergic neurons of the rat IML-cell column: evidence from histochemistry, immunohistochemistry, and retrograde labeling.

Nitric oxide synthetase (NOS) can be selectively stained in neurons by either NADPH-diaphorase (i.e., NOS)-histochemistry or immunohistochemistry with antibodies raised against NOS, which apparently label identical reactive sites (Hope, B.T., G.J. Michael, K.M. Knigge, and S.R. Vincent, Proc. Natl. Acad. Sci. USA 88:2811-2814, '91). We provide histochemical evidence for the existence of a neuron-specific NOS-activity in autonomic neurons of the thoracic spinal cord. Among the four main preganglionic cell clusters investigated at mid-thoracic levels, Th7-10, the intermediolateral (IML)-cell column was the most prominently stained cell group. The histochemical staining was absent in other spinal cord neurons and non-neuronal cells, e.g., GFAP-positive glial cells. Staining was completely blocked by N omega-nitro-L-arginine (L-NNA), a potent NOS-inhibitor for brain and peripheral autonomic neurons, but was still observed in the presence of another NOS-inhibitor, N omega-monomethyl-L-arginine (MeArg). The NOS-activity co-localized with nearly half of the ChAT-immunostained neurons located in the mid-thoracic IML-cell column as quantified by cell counts in single and double-stained tissue sections. We conclude that NOS-activity-containing neurons represent a distinct group among cholinergic IML-neurons, which suggests a more general function of this newly defined subpopulation of the spinal cord autonomic system. In vivo Fast blue retrograde labeling combined with histochemical staining and immunostaining revealed that sympathoadrenal projection neurons belong to the distinct NOS and ChAT-positive IML-cell group.(ABSTRACT TRUNCATED AT 250 WORDS)


Premotor descending neurons responding selectively to local visual stimuli in flies.

The responses of dorsal descending neurons suggest great versatility of the visual system in detecting features of the visual world. Although wide-field motion-sensitive neurons respond to symmetric visual flow fields presented to both eyes, other neurons are known to respond selectively to asymmetric movement of the visual surround. The present account distinguishes yet a third class of descending neurons (DNs) that is selectively activated by local presentation of moving gratings or small contrasting objects. Excitation of these DNs in response to local motion contrasts with their inhibitory responses to wide-field motion. The described DNs invade dorsal neuropil of the pro- and mesothoracic ganglia where they converge with other morphologically and physiologically characterized descending elements. Axon collaterals of DNs visit thoracic neuropil containing the dendrites of motor neurons supplying indirect neck and flight muscles. The present results are discussed with respect to the organization of small-field retinotopic outputs from the lobula, and with respect to the parallel projection of many information channels from the brain to the neck and flight motors.


Organization of inferior olivary projections to the flocculus and ventral paraflocculus of the rat cerebellum.

The climbing fiber projection to the rat flocculus and adjacent ventral paraflocculus was investigated by using Phaseolus vulgaris-leucoagglutinin as an anterograde and horseradish peroxidase as a retrograde tracer. Large injections of horseradish peroxidase in the flocculus and ventral paraflocculus indicated that the climbing fibers to this region are derived exclusively from any of the following contralateral olivary regions: the dorsal cap of Kooy, the ventrolateral outgrowth, the caudal half of the ventral leaf of the principal olive near its lateral bend, and the rostral pole of the medial accessory olive. Subsequent anterograde and retrograde studies with small injections demonstrated that the latter area projects to the C2 zone, which runs caudally in the ventral paraflocculus and enters the caudal most aspect of the flocculus. The ventral leaf of the principal olive is connected to a D zone in the cerebellar hemisphere and paraflocculus, which, upon entering the ventral paraflocculus, divides into a caudal and rostral strip, termed FD and FD', respectively. The dorsal cap and the ventrolateral outgrowth each project to two distinct zones in the flocculus and part of the ventral paraflocculus. Two floccular zones, which are continuous with the parafloccular FD and FD' zones, receive their climbing fibers from the ventrolateral outgrowth. Two other zones, (FE and FE') receive their climbing fibers from the dorsal cap. The FE' zone is found at the rostral pole of the flocculus and is followed caudalwards by the FD', FE, FD, and C2 zones, respectively. The rostromedial part of the dorsal cap is connected to the continuation of the FE zone into the ventral paraflocculus. The observation that the dorsal cap and the ventrolateral outgrowth are both connected to a set of two alternating zones of floccular/ventral parafloccular Purkinje cells is in agreement with recent studies in the rabbit, and suggests that these zones reflect functionally distinct and discrete units related to specific aspects of visuomotor control.


Claustrum in the hedgehog (Erinaceus europaeus) brain: cytoarchitecture and connections with cortical and subcortical structures.

The cytoarchitecture of the claustrum in the hedgehog (Erinaceus europaeus) brain, the morphology of its neurons, and the efferent connections with cortical and subcortical structures were studied with the Nissl and Klüver-Barrera, the Golgi, and the horseradish peroxidase methods. It was found that the claustrum is a well developed nucleus in the hedgehog telencephalon and, as in other mammals, is divided into dorsal and ventral parts. In Golgi-stained sections, spiny multipolar cells are the predominant neurons of both the dorsal and the ventral claustrum and are projection neurons. Aspiny multipolar neurons with fewer, often beaded, dendrites constitute a minority in both divisions and are interneurons. Injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) in the prefrontal, motor, somatosensory, auditory and visual areas, and HRP or WGA-HRP injections in the thalamus showed that: (1) the claustroneocortical projections originate in the dorsal claustrum and are distributed to the entire neocortex; these projections are mainly ipsilateral but some also originate contralaterally; (2) the claustroneocortical projections show a rough topographic organization; there exists a substantial degree of overlap; and (3) the claustrothalamic projection, arising throughout the dorsal claustrum, is strictly ipsilateral. No evidence of a thalamoclaustral projection was found. The present results suggest that, although the hedgehog has been referred to as a "paleocortical mammal" owing to the great development of its rhinencephalic structures in comparison with its small neocortex, the dorsal claustrum is well developed and is connected with all neocortical areas as well as with the thalamus, establishing it as a key structure in the hedgehog forebrain.


Dorsal root ganglion neurons projecting to the dorsal column nuclei of rats.

Dorsal root ganglion (DRG) neurons may give origin to ascending branches that terminate in the dorsal column nuclei (DCN); uncertainties still exist with regard to the proportion of these neurons in different DRGs and to the type of these neurons. The percentage and size of neurons that project to the DCN were determined in a large number of DRGs by means of the retrograde transport of colloidal gold-labeled wheat germ agglutinin conjugated to enzymatically inactive horseradish peroxidase (WGAapoHRP-AU). A total of 16,239 neurons was tallied in 80 DRGs from nine rats; 3,240 (20%) of these were retrogradely labeled by the tracer injected in the DCN. Percentages of DCN projecting neurons vary considerably at different segmental levels: they are higher in cervical (up to 63%) than in thoracic (up to 31% for T1, up to 12% for thoracic DRGs below T1) or lumbar DRGs (up to 15%). At cervical levels highest percentages were encountered in C6, C7, and C8 and lowest percentages in C2-C4. At lumbar levels highest percentages were encountered in L4 and lowest in L1 and L6. When considering the soma size of DRG neurons it appears that: 1) there are more large cells, labeled and unlabeled, at cervical (38%) than at lumbar levels (30%) and more at lumbar than at thoracic levels (23%); 2) at every level, most labeled, i.e., projecting, neurons are large; and 3) DRGs with the highest proportions of large vs. small cells contain the highest percentages of DCN projecting neurons. These results represent the first attempt at establishing the percentages and soma size of DCN projecting neurons from a large number of DRGs and at comparing the contribution to these nuclei from cervical, thoracic, and lumbar DRGs. Some of the differences in the ratio of projecting neurons at different levels may be explained on the basis of well-known anatomical features, e.g., the projections to the Clarke's column of many DRG neurons in lumbar ganglia. The contribution of virtually exclusively large DRG neurons to the DCN, suggested by indirect or incomplete evidence, is demonstrated by the present retrograde labeling and soma size measurements. The results relate to the functional component of peripheral receptors that relay their input via the dorsal columns and do not seem to support a recent suggestion that a sizeable fraction of unmyelinated primary afferents ascend in the dorsal columns to terminate in the DCN.


Trigeminocerebellar projections to the posterior lobe in the cat, as studied by anterograde transport of wheat germ agglutinin-horseradish peroxidase.

Cerebellar projections of the nucleus interpolaris and oralis of the spinal trigeminal nucleus were studied in the cat by anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Injections of WGA-HRP into these nuclei labeled many mossy fiber terminals mainly ipsilaterally in the rostral folium of lobule IX (IXa or IXa + b), the simple lobule, the anterior part (sublobule A) of the paramedian lobule and the posterior part of crus II. Labeled terminals were also seen in the anterior lobe, lobules VI and VII, the anterior part of crus I, and the paraflocculus dorsalis. Projection fields in the horizontal plane of lobules were reconstructed from a series of transverse sections through each folium of lobule IX, the paramedian lobule, and the posterior part of crus II on the ipsilateral side. In sublobule IXa + b, labeled terminals were distributed in five longitudinal areas extending along the apicobasal axis of the sublobule. These five areas were located in the apical two-thirds of the ipsilateral half of the sublobule. Labeled terminals were distributed in five longitudinal areas in sublobule A (the rostral part) of the paramedian lobule. In the posterior part of crus II, four aggregations of labeled terminals were present in cross sections through a lobule. They were distributed in the apicobasal extent of the lobules. The present study indicates that the projection fields of trigeminocerebellar fibers are longitudinally arranged along the apicobasal axis of the cerebellar lobules.


Organization of parabrachial nucleus efferents to the thalamus and amygdala in the golden hamster.

While gustation in the hamster has been extensively studied at the behavioral and physiological level, very little is known about the central anatomy of the taste system. The purpose of this study was to trace the connections of the parabrachial nucleus (PBN) in the golden Syrian hamster (Mesocricetus auratus) using wheat germ agglutinin-conjugated horseradish peroxidase. The PBN is the site of the second central synapse for the ascending gustatory system and receives taste afferents from the nucleus of the solitary tract. Following large injections into the PBN, anterogradely transported label was seen in the lateral hypothalamus, dorsal thalamus, bed nucleus of the stria terminalis, and amygdala. The anatomy of the two primary targets, the ventral posteromedial thalamus and central nucleus of the amygdala, is described based on Nissl-stained material, and acetylcholinesterase and NADH dehydrogenase histochemistry. Injections into these two regions revealed different patterns of efferents within the PBN. Following injections into the thalamus, retrogradely labelled cell bodies were distributed throughout the PBN subdivisions bilaterally, but concentrated in the central medial (CM) and external lateral (EL) subdivisions. Following injections into the amygdala, retrogradely labelled cell bodies were primarily in the ipsilateral PBN EL, while anterogradely transported label was distributed throughout much of the ipsilateral PBN. The majority of CM efferents projecting to the thalamus were elongate cells, whereas the majority of CM efferents to the amygdala were round-oval cells. These results indicate that the ascending central gustatory system changes from a serial pathway (nucleus of the solitary tract-PBN) to a parallel organization consisting of two major projections, the parabrachio-thalamo-cortical and parabrachio-amygdaloid pathways.


[Massive fetal-maternal hemorrhage at term associated with a choriocarcinoma].

A case of malignant choriocarcinoma produced a major feto-maternal transfusion at term. This was proven using routine Kleihauer's test. The newborn died on the 5th day of life from haemorrhagic shock. The diagnosis of choriocarcinoma was made when acute post-partum haemorrhage occurred together with a high level of Beta HCG. The patient was cured by the use of Methotrexate followed by four courses of tri-chemotherapy (Methotrexate, Actinomycin and Cyclophosphamide). After treatment for secondary infertility, the patient had two normal pregnancies. This case makes possible to point out again the possible association of a positive Kleihauer test with choriocarcinoma. It also points out strongly the value of carrying out Beta HCG testing when bleeding occurs post-partum. Early diagnosis improves the maternal prognosis and allows conservative treatment to be carried out in cases of choriocarcinoma in young women.


Expression of alpha-lactalbumin, alpha-S1-casein, and lactoferrin genes is heterogeneous in sheep and cattle mammary tissue.

We used 35S-labeled cRNA probes to localize the sites of alpha-lactalbumin, alpha-S1-casein, and lactoferrin mRNA synthesis in sheep and forcibly weaned cattle mammary tissue. Expression of alpha-lactalbumin was absent in three of four "virgin" glands studied, present in some alveoli of "pregnant" glands but not in others, despite a similar histological appearance. In the early lactating gland, expression was high in those alveoli with few fat globules in their cells and lumen and was absent in alveoli with abundant fat globules. These observations suggest either that alpha-lactalbumin gene expression is linked to the long-term secretory activity of cells and falls once cells are resting or regressing, or that there are cyclical variations in expression, or that in the lactating gland some groups of epithelial cells are synthesizing alpha-lactalbumin and some are synthesizing fat. Expression patterns of alpha-S1-casein were similar to those of alpha-lactalbumin. Lactoferrin, in contrast, was expressed almost exclusively in the "fatty alveoli" of both species. Our results show that dramatic variations in milk gene expression can occur throughout the mammary gland of sheep and cattle and that at no stage of pregnancy, lactation, or involution can the gland be considered metabolically homogeneous.


Antigenic profile of the human lacrimal gland.

The antigenic profile of 13 normal formalin-fixed, paraffin-embedded human main and accessory lacrimal glands, biopsied from patients aged 11 to 78 years, was studied using a panel of 27 polyclonal and monoclonal antibodies. Secretory cells of lacrimal acini reacted with antibodies to S-100 protein and simple epithelium-type cytokeratins CK 7, CK 8, CK 18, and CK 19. Their luminal membranes were labeled with antibodies to carcinoembryonic antigen, epithelial membrane antigen, and epithelial glycoproteins recognized by Ber-EP4. Myoepithelial cells were often immunopositive for S-100 protein, vimentin, glial fibrillary acidic protein (GFAP), and alpha-smooth muscle actin. More rarely, they reacted with antibodies recognizing CK 5, CK 13, and CK 14, which consistently labeled the basal cells of lacrimal ducts. Unlike myoepithelial cells, basal ductal cells were immunopositive for CK 7, CK 8, CK 18, and CK 19. In main excretory ducts, dendritic melanocyte-like cells co-expressing vimentin and S-100 protein intermingled with ductal epithelial cells. The luminal cells of lacrimal ducts basically paralleled secretory cells in their antigenic profile, although they lacked Ber-EP4 and were immunopositive for CK 4. Antibodies to neuron-specific enolase and synaptophysin reacted with nerve fibers among negatively reacting secretory acini. This antigenic profile closely parallels that of salivary glands and provides a basis for studies of lacrimal gland pathology.


Characterization of an antigenic determinant preferentially expressed by type I epithelial cells in the murine thymus.

A hamster monoclonal antibody (MAb), designated 8.1.1, was raised against murine thymic stromal cell lines and was found to react with cell surface molecules expressed by a morphologically distinct population of epithelial cells of the murine thymus comprising the subcapsular environment, cells investing vascular structures throughout the thymus, and some of the cellular elements in the medulla. The epithelial nature of the labeled cells was confirmed with immunoelectron microscopy. Reactivity with MAb 8.1.1 was associated with thymic epithelial cells in contact with basal laminae. Ontological studies of thymic tissue demonstrated that the epitope recognized by this MAb was expressed before Day 14 of gestation, although the restricted subcapsular and medullar expression of 8.1.1 was not apparent until sometime after birth. MAb 8.1.1 also reacted with a number of extra-thymic tissues, including lamina propria of gut, glomeruli and tubules in the kidney, mesothelia covering a number of organs, and the dermis and epidermis of skin. Within the epidermis, reactivity of MAb 8.1.1 was largely restricted to basal epithelial cells. Immunochemical analysis of 8.1.1 reactivity with detergent-soluble extracts of thymic stromal cell lines and thymus tissue indicated that detergent-soluble extracts of thymic stromal cell lines and thymus tissue indicated that the epitope recognized by this MAb was associated with a glycoprotein bearing terminal N-acetylglucosamine residues and possessing an Mr of approximately 36-38 KD under reducing or non-reducing conditions.


Immunohistological demonstration of pyruvate kinase isoenzyme type L in rat with monoclonal antibodies.

Four monoclonal antibodies (MAb) specific for the L-type isoenzyme of rat pyruvate kinase (L-PK) were produced and characterized. They detect at least two different epitopes of the isoenzyme, as shown in interference binding assay and Western blot analysis after peptide mapping. The MAb were used in immunohistology to demonstrate the L-PK isoenzyme in paraffin-embedded normal rat tissues. L-PK was found only in hepatocytes, kidney proximal tubules, islet cells of pancreas, and epithelial cells of the villi of small intestine. The content of L-PK in hepatocytes was often lower in the periportal areas as compared with the periveneous zone. In kidneys a clearcut difference in L-PK content and distribution existed between male and female rats. Male animals possessed more L-PK in the kidney cortex than females. The L-PK content in the inner cortical zone (straight proximal tubules) was higher than in the outer cortical zone (convoluted proximal tubules) in kidneys of males. In contrast, female rats displayed less L-PK in the inner than in the outer cortical zone of the kidneys. Only some of them exhibited the same amount of the isoenzyme in both parts of the kidney proximal tubules.


Identification and analysis of a novel human surface CD5- B lymphocyte subset producing natural antibodies.

The production of "natural" autoantibodies or antibodies, i.e., Ig that bind a variety of self- and/or exogenous Ag and arise independently of known immunization, is though to be a feature of CD5+ B lymphocytes. To determine whether other lymphocyte subsets exist that might be committed to the production of natural antibodies, human peripheral blood B cells were sorted on the basis of surface CD5 expression and differential expression of surface CD45RA (CD5+CD45RAintermediate(int), CD5-CD45RAlow(lo), and CD5-CD45RAhigh(hi)), and analyzed for the type of Ig produced after EBV infection and culture. Like their CD5+ counterparts, most CD5-CD45RAlo B lymphocytes were precursors of cells producing IgM, a major proportion of which displayed the Ag-binding features of natural antibodies. In contrast, CD5-CD45RAhi B cells comprised a high frequency of IgG-producing cell precursors, possibly including memory B lymphocytes. Six of seven IgM mAb generated from sorted CD5-CD45RAlo B cells and three of four IgM mAb from sorted CD5+ B cells were polyreactive, binding with different affinities (Kd, 10(-5) to 10(-8) M) to two or more Ag; the remaining mAb from CD5-CD45RAlo and the mAb from CD5+ B cells each bound to a single Ag (Kd, 10(-7) to 10(-8) M), beta-galactosidase and ssDNA, respectively. CD5-CD45RAlo B cells account for 4.1 +/- 1.2% (mean +/- SD in 11 healthy subjects; CD5+ B cells, 23.3 +/- 6.9%) of total B lymphocytes and display the features of quiescent cells. In a given individual, the number of CD5-CD45RAlo B cells remains constant over time. CD5-CD45RAlo and CD5+ B cells bear surface CD11b and CD14, at densities and/or frequencies apparently higher than those of CD5-CD45RAhi B lymphocytes. Despite their surface CD5- phenotype, CD45RAlo B cells express CD5+ mRNA at levels comparable with those of CD5+ B lymphocytes, whereas CD5-CD45RAhi B cells express only trace amounts of CD5 mRNA. The commitment to natural antibody production and the degree of CD5 mRNA expression suggest that the newly defined CD5-CD45RAlo B cell subset is related to CD5+ B lymphocytes, and may constitute the human homologue of the mouse Ly-1-"sister" B cell population.


CD4 epitope masking by gp120/anti-gp120 antibody complexes. A potential mechanism for CD4+ cell function down-regulation in AIDS patients.

The in vitro suppressive effect of gp120 and gp120/anti-gp120 antibody is well known but not yet proven to operate in vivo. We report findings consistent with the presence of gp120/anti-gp120 antibody complexes on CD4+ lymphocytes from HIV-infected patients with advanced disease. PBMC from most AIDS patients showed selective masking of the CD4 epitope associated with the gp120 binding site; immunoprecipitation of PBMC with anti-CD4 mAb disclosed high amounts of IgG bound to CD4 receptors. Antibodies against HIV env proteins, but not other HIV products or CD4 Ag, were detected in purified CD4+ cell culture supernatants; in vitro culture was associated with normalization of both CD4 expression in PBMC and the lymphocyte proliferative response to anti-CD3. gp120 presence could not be directly demonstrated, but findings strongly suggested that CD4+ lymphocytes from most HIV-infected patients with advanced disease were covered with gp120/anti-gp120 antibody complexes, which are responsible for down-regulation of surface CD4 expression as well as functional lymphocyte impairment; this event may represent an important mechanism in the pathogenesis of HIV-associated immunodeficiency.


Induction of VCAM-1 and ICAM-1 on human neural cells and mechanisms of mononuclear leukocyte adherence.

We propose that leukocyte-derived cytokines induce the expression of adhesion molecules on the surface of neural cells that facilitates the subsequent attachment of leukocytes. Leukocyte adherence may contribute to some of the neural cell injury seen with various inflammatory diseases of the nervous system. With an in vitro model system, we have shown that mononuclear leukocytes bind to human neuroblastoma and cortical neuron cells only after the neural cells are stimulated with TNF-alpha. TNF-alpha stimulates expression of vascular cell adhesion molecule-1 (VCAM-1) in both of these neural cell lines. VCAM-1 mRNA is increased and VCAM-1 protein can be identified on the neural cell membranes with a new VCAM-1-specific mAb, CL40/2 F8. TNF-alpha also induces ICAM-1 in both of these neural cell lines. Leukocyte beta 1 (CD29) and beta 2 (CD18) integrins and their respective ligands, ICAM-1 and VCAM-1, on neural cells appear to be the dominant ligands mediating MNL:neural cell adhesive interactions. mAb to CD18 block 32 to 57% of the MNL binding to neural cells; similar inhibition is seen with mAb to ICAM-1. mAb to CD29 block 16 to 17% of the MNL binding to the neural cells suggesting that leukocyte beta 1 integrins and neural VCAM-1 may be a second route for MNL:neural cell interactions. Addition of both anti-CD18 and anti-CD29 mAb have an additive blocking effect; both ligand pairs may participate in MNL adhesion to neural cells, reminiscent of the multiplicity of ligands used by MNL when binding to endothelium.


Cytotoxic T lymphocytes from HIV-1 seropositive individuals recognize immunodominant epitopes in Gp160 and reverse transcriptase.

The CTL response to HIV-1 is more vigorous than for any known human pathogen and may be a significant factor in preventing the progression to symptomatic disease. T cell lines, generated by non-specific stimulation with PHA and IL-2, may be reproducibly used to identify HIV-1 isolate-invariant epitopes recognized by the CTL of infected individuals. The CTL response in each of 12 infected individuals to envelope and reverse transcriptase (RT) is dominated by the recognition of one or two viral isolate-invariant epitopes. Seven subjects respond to a single gp160 epitope; three subjects recognize 2 gp160 epitopes. There is a significant increase in recognition of epitopes in the C terminal 104 amino acids of gp41 (p less than 0.002); in fact 40% of the subjects that respond to gp160 recognize the C terminal 20-mer. The CTL-mediated lysis of gp160-expressing targets is MHC restricted, but not all individuals that share the same serologically defined class I-restricting element respond to the same epitope. Recognition of the terminal 20mer is restricted by both A30 and B8. The response to RT in six subjects is distributed over the RT protein. The six subjects recognize four separate regions defined by truncated RT-vaccinia recombinants, but none of the subjects' CTL demonstrate significant recognition of the RT epitope identified in H-2k mice and some humans.


Regulation of the effector stages of experimental autoimmune encephalomyelitis via neuroantigen-specific tolerance induction. II. Fine specificity of effector T cell inhibition.

Ag-specific tolerance induced by the i.v. administration of splenocytes coupled with mouse spinal cord homogenate, containing a mixture of myelin Ag, dramatically inhibits development and expression of clinical and histologic signs of both active and adoptive forms of relapsing experimental autoimmune encephalomyelitis (R-EAE) in the SJL/J host. Here we examined the dose-dependency, route of tolerogen administration, and fine neuroantigen specificity of inhibition of adoptive R-EAE. Expression of clinical R-EAE induced by a polyclonal population of bovine myelin basic protein (MBP)-specific effector T cells was dramatically inhibited in a dose-dependent manner following the i.v., but not s.c. or i.p., injection of MBP-coupled splenocytes. The exquisite Ag specificity of the inhibition was evident by the observation that splenocytes coupled with intact bovine MBP or species variants of MBP homologous with bovine MBP within the major encephalitogenic region (amino acids 84-104), but not with proteolipid protein or mouse kidney homogenate, were able to suppress disease expression. Splenocytes coupled with the MBP84-104 peptide, containing a nested set of the major SJL/J encephalitogenic epitopes, completely inhibited peptide-specific T cell responses, but only partially inhibited the expression of disease transferred by T cells specific for intact MBP, suggesting the participation of T cell responses specific for additional MBP determinants in disease pathogenesis. However, splenocytes coupled with previously identified minor SJL/J encephalitogenic epitopes (MBP91-104 or MBP17-27), or with the Lewis rat major encephalitogenic epitope (MBP68-86), did not suppress disease expression. Collectively, the results demonstrate that MBP84-104-specific T cells and T cells specific for an as yet unidentified MBP epitope(s) contribute to the pathology of R-EAE. In addition, the results demonstrate that peptide-specific tolerance induction appears to have potential for the treatment of T cell-mediated inflammatory diseases.


Expression of an exogenous tumor necrosis factor (TNF) gene in TNF-sensitive cell lines confers resistance to TNF-mediated cell lysis.

TNF, a cytokine with cytotoxic activity on a variety of tumor cells, is mainly produced by macrophages; however, some tumor cell types of non-macrophage origin, apparently resistant to TNF-mediated cell lysis, can also produce TNF. It is not clear whether these cells were TNF-resistant a priori or whether protective mechanisms against toxicity of autocrine TNF may be induced in TNF-producing cells. Murine L929sA fibrosarcoma cells, which are highly sensitive to TNF cytotoxicity, were transfected with the neomycin resistance (neor) gene, alone or in combination with the human (h) or the murine (m) TNF gene. All exogenous genes were under control of the constitutive SV40 early promoter. After cotransfection, the number of neor colonies was 10 to 100% as compared with the number of colonies upon transfection with the neor gene alone. An appreciable fraction of these colonies (50-100%) constitutively produced biologically active TNF. mTNF-producing L929 cells were fully TNF resistant, whereas hTNF-producing cells showed partial TNF resistance. Specific TNF binding could not be detected on mTNF-producing L929sA transfectants, whereas hTNF-producing cells showed reduced TNF binding. Apparently, TNF gene expression, even in a priori TNF-sensitive cells, can induce mechanisms to prevent toxicity by both autocrine and exogenous TNF. No TNF resistance was induced by expression of a gene sequence encoding the 9-kDa membrane-bound presequence part of the 26-kDa mTNF proform. Expression of a mutant 26-kDa TNF gene coding for a quasi-inactive mature mTNF induced only weak TNF resistance as compared with the complete resistance obtained after transfection with the wild-type gene. These findings show that the membrane-bound TNF presequence as such is not sufficient for induction of TNF resistance and imply that the active site of mature TNF is involved in modulation of TNF responsiveness upon autocrine TNF production.


H chain V region sequences of three human monoclonal IgM with anti-myelin-associated glycoprotein activity.

The amino acid sequence corresponding to the V region H chain gene used by three monoclonal IgM directed to the myelin-associated glycoprotein (MAG) is presented. They all belonged to the VHIII variability subgroup, but each may well represent a new member of this family inasmuch as their homology with previously sequenced VHIII genes was less than 80%. Strikingly, there was no greater homology between the H chain V regions of the anti-MAG IgM. Partial amino acid sequence data indicated that these V regions were joined to as yet unidentified DH segments; however, two H chains used very similar DH, possibly indicating that this sequence was involved in the fine specificity of the IgM for MAG. All H chains included a JHIV region. These data, together with results obtained from the sequence of the three kappa L chains of the same IgM molecules (Mihaesco, E., H. Ayadi, N. Congy, M. C. Gendron, J. P. Roy, H. Heyermann, B. Frangione, and J. C. Brouet. 1989. J. Biol. Chem. 264:21481), indicate that the repertoire of VL and VH gene segments used by anti-MAG IgM is quite diverse, in contrast to previous structural data obtained for other human monoclonal IgM autoantibodies. Possibly, these differences reflect distinct pathogenesis.


Murine common acute lymphoblastic leukemia antigen (CD10 neutral endopeptidase 24.11). Molecular characterization, chromosomal localization, and modeling of the active site.

To further analyze CD10/NEP function in lymphoid and nonlymphoid cells using well characterized murine systems, we isolated the murine CD10/NEP homologue, determined its chromosomal location, and modeled the enzyme's active site. The murine CD10/NEP cDNA predicts a 750-amino acid (aa) type II integral membrane protein with 90% identity to the human CD10 sequence and 100% conservation of critical aa and functional motifs. The latter include the pentapeptide consensus sequence required for zinc binding and catalytic activity, additional aa associated with substrate binding, and the extracellular cysteines that participate in disulfide bonds required for enzymatic activity. Like its human homologue, murine CD10/NEP has multiple alternative 5'-untranslated region sequences. The gene is localized on the proximal half of murine chromosome 3. In Northern analysis, murine CD10/NEP transcripts are abundant in bone marrow stromal cells that support pre-B cell differentiation but are undetectable in representative Abelson transformed pre-B cell lines. The murine CD10/NEP active site was modeled by aligning critical conserved CD10/NEP residues with comparable residues in the active site of thermolysin, a bacterial metalloprotease with similar substrate specificity. The model predicts that the two enzymes have similar clefts that comprise the active site and permit zinc-dependent substrate interactions.


Prostaglandin E2 and other cyclic AMP-elevating agents modulate IL-2 and IL-2R alpha gene expression at multiple levels.

cAMP is an intracellular second messenger that conveys inhibitory signals for T cell activation and clonal proliferation. cAMP also inhibits the production of IL-2 and IL-2R alpha-chain expression. To determine the mechanisms of this inhibition, human peripheral blood T lymphocytes were stimulated with anti-CD3 mAb, PHA, PMA, or ionomycin, alone or in combination. cAMP elevation by PGE2, cholera toxin, or the cell-permeable analogue 8-bromo-cAMP inhibited the tyrosine phosphorylation of a protein of 100 kDa. This inhibition was associated with decreased IL-2 production and IL-2R alpha expression at both the protein product and the mRNA levels. Nuclear run-off assays showed that the inhibitory effect of cAMP on IL-2 and IL-2R alpha gene expression is mediated at the transcriptional level. H-8, an inhibitor of protein kinase A, reversed the inhibitory effect of cAMP on nuclear transcription of the IL-2 gene, suggesting that this is mediated through activation of protein kinase A. Post-transcriptionally, cAMP elevation decreased the t1/2 of IL-2 mRNA by more than 50%. These data indicate that cAMP inhibits cell membrane, cytoplasmic, and nuclear events associated with T cell activation and highlight the complexities of its action of lymphocyte function.


IFN-gamma-producing ability as a possible marker for the protective T cells against Mycobacterium bovis BCG in mice.

We searched for a functional marker with which T cells mediating acquired cellular resistance (ACR) can be discriminated from those mediating delayed-type hypersensitivity (DTH) in mice immunized with Mycobacterium bovis BCG. Four wk after injection of the mice with 10(5) viable BCG (vBCG) cells emulsified in IFA, a passive transfer experiment revealed that T cells mediating DTH as well as ACR, which we designated TACR, were generated in the spleen. In contrast, T cells mediating only DTH (TDTH) were generated by immunization with 10(7) killed BCG cells along with IFA. The transferring ability of both TACR and TDTH was substantially reduced by treatment with anti-Thy-1.2 or anti-CD4 mAb plus complement, whereas anti-CD8 treatment had no effect. To determine the functional differences between TACR and TDTH, we assessed IL-2 and IFN-gamma production from TACR and TDTH after stimulation with PPD in vitro. Similar levels of enhanced IL-2 activity were detected in the culture supernatants of both groups of T cells. However, augmented production of IFN-gamma was observed only in TACR. This finding was confirmed by Northern blot analysis for detection of IFN-gamma-specific mRNA. In addition, a significant increase in the number of IFN-gamma-producing cells was observed only in T cells from mice immunized with vBCG. The production of IFN-gamma was also totally abolished by treatment with anti-Thy-1.2 and anti-CD4 mAb plus complement in vitro, whereas anti-CD8 mAb treatment had no effect. These results suggest that CD4+ protective T cells generated by immunization with vBCG are characterized by the ability to produce IFN-gamma after stimulation with specific Ag.


Expression of c-kit by mesenteric lymph node cells from Nippostrongylus brasiliensis-infected mice and by mast cell colonies developing from these cells in response to 3T3 fibroblast-conditioned medium.

Mast cell committed progenitors are nongranulated cells found in mesenteric lymph nodes of mice infected with Nippostrongylus brasiliensis (Nb-MLN) but not from normal mice. Mast cell committed progenitors can respond to either IL-3 or to a factor(s) present in 3T3 fibroblast conditioned media (F-CM) by formation of mast cell colonies. Previous studies from ours and other laboratories suggested that mast cell differentiation involved the W allele product, c-kit, as a receptor and Sl allele product, stem cell factor, as a growth factor. We report here that Nb-MLN cells, which can respond to F-CM by mast cell colony formation, also contain cells that express message for c-kit, and that c-kit message cannot be detected in naive mesenteric lymph node cells, which cannot respond to F-CM. Antisense oligonucleotides to c-kit inhibit mast cell colony formation by Nb-MLN cells in response to F-CM, but not to conditioned medium of PWM-stimulated spleen cells as a source of IL-3. The antisense oligonucleotides also inhibit the degree of granulation by mast cells derived from culture. The results suggest that c-kit and its ligand, stem cell factor, are necessary for mast cell-committed progenitors to proliferate and granulate in response to F-CM but not IL-3.


Regulation of lymphokine-activated killer cell induction by human recombinant IL-1 receptor antagonist. Obligate paracrine pathway of IL-1 during lymphokine-activated killer cell induction.

IL-2-stimulated human lymphocytes, referred to as lymphokine-activated killer (LAK) cells, can develop a broad range of lytic activity against fresh tumor cells and cultured tumor cell lines. IL-1, a pleiotropic cytokine shown to synergize with IL-2 on LAK induction, is endogenously synthesized and secreted by LAK cells. Immunoblot analysis demonstrated that IL-2-stimulated PBL produced the 31- to 34-kDa pro-molecules of IL-1 within 24 h and maintained their expression for at least 96 h. The role of secreted IL-1 has been examined using rIL-1R antagonist (IL-1ra). The addition of IL-1ra to LAK activation culture resulted in dose-dependent inhibited lytic activity, which was more apparent in LAK cells cultured with higher doses of IL-2. However, IL-1ra had no effect on proliferative responses elicited in LAK cells by IL-2. Moreover, when IL-1 binding was blocked by IL-1ra, the expression of the IL-2R p55 subunit was reduced compared with control LAK cells. The effect of IL-1 binding blockade on expression of other cytokine mRNA was further examined by polymerase chain reaction analysis, and, specifically, inhibition of both TNF-alpha and TNF-beta mRNA expression by IL-1ra was observed in PBL stimulated with IL-2. The reduced biologic activity of TNF in culture supernatants correlated well with the inhibition of mRNA expression. These findings suggest that autocrine/paracrine IL-1 is involved in the initial generation of LAK activity and, in particular, that TNF expression could be induced via an IL-1 autocrine pathway.


Expansion of murine T cells bearing a unique T cell receptor beta-chain in Friend virus-induced tumor in situ.

Heterogeneity of V alpha 1+ and V beta 10+ TCR alpha beta-chains, which are predominantly used in anti-FBL-3 CTL clones established in vitro, was investigated at a nucleotide level in FBL-3 tumor-infiltrating lymphocytes (TIL) in vivo. The majority (90%) of V beta 10+ beta-chains dominated in TIL used homogeneous V beta 10D beta 2.1 sequences identical to that used in the T cell clones with cytotoxic functions. The homogeneous TCR beta-chain expression was dominant and found to be about 10% of the total TCR beta-chains in the TIL population, which was a greater than 300-to 900-fold increase than in the regional lymph nodes. This is in good agreement with the in vitro data showing that about 11% CTL clones used the homogeneous V beta 10D beta 2.1+ beta-chain. However, the J beta segment does not seem to contribute greatly to the recognition and selection of this TCR because some of homogeneous VD+ beta-chains were associated with J beta segments other than J beta 2.7 of the CTL clones. The frequency of the V alpha 1J alpha 112-2+ alpha-chain expression of the CTL type was much less (3- to 80-fold increase compared to that of lymph node) and also varied in sample materials, indicating the lower contribution of the alpha-chain for the oligoclonality of the TCR. The results were also confirmed by quantitative PCR and RNase protection assays. This suggests that the dominant expression of the homogeneous TCR beta-chain is due to the expansion of the particular anti-FBL-3 CTL in the tumor in situ. Also, the TCR beta-chain, especially the V beta D beta region, rather than alpha-chain is more important for the recognition and selection of the anti-FBL-3 TIL with cytotoxic functions.


Genes for the trophoblast interferons in sheep, goat, and musk ox and distribution of related genes among mammals.

The trophoblast interferons (IFNs) are a family of Type 1 IFN found in domestic ruminants that are most closely related to the little-studied 172-amino-acid IFN-omega. They are produced in massive amounts by the preimplantation conceptus at a time coincident with maternal recognition of pregnancy, and are implicated in playing an important role in this process. Here we report the characterization of four distinct members of the ovine trophoblast IFN (oTP-1) gene family, and demonstrate that they, along with previously characterized bovine trophoblast (bTP-1) genes, possess distinctive promoter sequences when compared to ovine and bovine IFN-omega genes. Genomic Southern blot analysis of numerous mammalian species (zoo blots) indicate that, whereas the IFN-omega are widely distributed among mammals, genes for the trophoblast IFN appear to be limited to ruminant species within the Artiodactyla order. Further polymerase chain reaction (PCR) analysis of trophoblast IFN genes in these ruminant species has permitted isolation of genes for goat and musk ox trophoblast IFN. These data suggest that the trophoblast IFNs are a distinct family of IFN with a limited distribution among mammals.


Interferon-gamma receptor: mRNA half-life, receptor mass, and abundance on A431 human epidermoid carcinoma cells.

The human interferon-gamma (IFN-gamma) receptor protein and mRNA were identified in the epidermoid carcinoma cell line A431 by radioligand binding and Northern hybridization, respectively. Receptor affinity and receptor number per cell were calculated by Scatchard analysis of saturation binding data. The half-life of the receptor mRNA was determined by actinomycin D inhibition of de novo transcription. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of covalently cross-linked 32P-labeled IFN-gamma and its receptor is consistent with a molecular mass of approximately 99 kD. Radioligand binding analysis revealed 27,000 high affinity (KD = 7.1 x 10(-10) M) receptors per cell. Northern hybridization using an IFN-gamma receptor cDNA probe revealed a single mRNA of 2.2 kb. The receptor mRNA half-life in actinomycin D-treated cells was 4.7 h.


Specific interferon genes are expressed in individual cells in the peritoneum and bone marrow of normal mice.

The use of a highly sensitive method of in situ hybridization capable of detecting one copy of IFN mRNA per cell showed that from 20-50% of the cells from the peritoneum and bone marrow of both normal pathogen-free and axenic mice exhibited grain counts significantly greater than background levels following hybridization with riboprobes specific for the mouse interferon-alpha (IFN-alpha), IFN-beta, or IFN-gamma genes. Labeling was shown to be specific, as the labeled probe was displaced by a 200-fold excess of the specific unlabeled probe but not by a 200-fold excess of an unrelated probe. Grain counts were reduced to background levels when cells were pretreated with ribonuclease prior to in situ hybridization. The extent of labeling with either IFN-alpha or IFN-beta-specific probes increased following i.v. inoculation of mice with the IFN-inducer Newcastle disease virus (NDV) whereas the degree of labeling observed with a probe specific for beta-actin remained unchanged. No significant differences were observed in the number of bone marrow or peritoneal cells that expressed IFN-alpha or IFN-beta mRNA from either high (C57B1/6) or low (BALB/c) IFN-producing strains of mice. The majority of IFN-alpha and IFN-beta-containing cells from both the bone marrow and peritoneum of normal pathogen-free and axenic mice resembled monocytes morphologically, whereas the majority of IFN-gamma mRNA-containing cells resembled small lymphocytes. In addition, in the bone marrow a number of large cells which resembled megacaryocytes were found to express high levels of IFN-alpha mRNA. Nuclear run-on assays showed that IFN-alpha and IFN-beta genes were actively transcribed in both bone marrow and peritoneal cells from normal and axenic mice. Low levels of de novo IFN-gamma RNA synthesis were detected in the nuclei of peritoneal cells only. The expression of IFN genes in individual cells in the tissues of normal animals may constitute a basis for the regulation of both homeostasis and host defense against virus infection and neoplastic cells.


The shape of DNA elution dose-response curves under non-denaturing conditions: the contribution of the degree of chromatin condensation.

We have re-examined the effect of detergent type, pH and temperature of lysis on the shape of the DNA elution dose response under non-denaturing conditions using plateau-phase CHO cells. Results practically identical to those previously published (Okayasu and Iliakis, 1989) were obtained, with a 1 h incubation at 60 degrees C during lysis with sodium-N-laurylsarcosine (NLS) resulting in almost linear dose-response curves. We also examined chromatin decondensation as a contributing factor in the linearization observed in the elution dose-response curve under the above conditions. When nuclei with condensed chromatin were prepared from irradiated cells, applied on the filter and lysed with NLS at room temperature, a shoulder-type elution dose-response curve was obtained only slightly higher than that of cells lysed under the same conditions. However, when nuclei prepared from irradiated cells were applied on the filter after relaxation of chromatin by incubation in low ionic strength buffer and lysed with NLS at room temperature, an almost linear dose-response curve was obtained similar to that of cells lysed with NLS at 60 degrees C. Lysis with NLS at 60 degrees C of nuclei with relaxed chromatin did not further modify the DNA elution dose-response curve. Based on these results we propose that the linearization of the DNA elution dose-response curve observed after chromatin decondensation reflects a reduction in the degree of chromatin compactness in the nuclear complexes that leads to a relatively uniform distribution of the DNA on the filter and reduces trapping of elutable material in the compact nuclear structures otherwise present. Since high radiation doses dissolve compact nuclear structures, trapping of elutable material is expected to be highest at low doses of radiation, leading to the observed reduction in the fraction of DNA eluted per Gy at low versus high radiation doses and thus to the observed shoulder. Furthermore, we propose that the linearization of the DNA elution dose-response curves observed in cells lysed in NLS at 60 degrees C may also be due to a decondensation of the nuclear complexes on the filter as a result of the combined action of detergent and high temperature. The notion of a correlation between DNA elution dose response and cell radiosensitivity was examined in two human (SQ20B, SCC61) and two Chinese hamster (V-79, irs-2) cell lines with widely different radiosensitivities.(ABSTRACT TRUNCATED AT 400 WORDS)


Nuclear area measurement on viable cells, using confocal microscopy.

We describe a rapid procedure for the accurate measurements of nuclear areas on unperturbed living cells as used in radiobiological experiments, using the confocal laser scanning microscope. The microdosimetric interpretation of radiobiological data requires precise information on the nuclear area of cells as irradiated with high-LET radiation.


Humoral immune responses in human HIV-1 infection clearance of initial burst of virus replication and protection against disease progression.

An attempt is made to summarize the evidence that humoral immune responses in natural HIV-1 infection play a role in clearance of the initial burst of replication as well as in protection against rapid disease progression. Therefore, the so-called "asymptomatic carrier state" was defined on the basis of immunological characteristics, such as CD4+ cell number, CD45RA-CD29+ cell number, CD4+ proliferative responses to anti-CD3 mAbs and soluble activation markers, as well as virological characteristics such as the state of the viral genome in the cell, levels of genomic RNA production, antigenemia, viremia and virus phenotype. During natural infection two major classes HIV-1 neutralizing and cell-fusion inhibiting antibodies are elicited. One population directed against mostly continuous epitopes localized in the third variable domain (V3) of the envelope and one against discontinuous epitopes of the envelope. The last population blocks gp120-CD4 attachment, the first does not. The role of each of these populations of functional antibodies, in the clearance of viremia and the maintenance of the asymptomatic carrier state is discussed.


Prognostic assessment of fulminant hepatitis from indication criteria for liver transplantation.

The clinical outcome in 15 patients with fulminant hepatitis was predicted by the indication criteria for liver transplantation used in five different transplantation centers. Fifty-four to 100% of 13 fulminant hepatitis patients died who were fulfilled with these criteria for transplantation. Most of the patients died within five to nine days after the decision of liver transplantation was made. Takahashi's score of prognostic assessment for fulminant hepatitis decreased rapidly during the time when all the different criteria in transplantation centers were fulfilled. However, two survival cases with the acute type of fulminant hepatitis were incorrectly judged to be transplanted by the two criteria. The Cambridge's criterion for liver transplantation predicted the prognosis of fulminant hepatitis most correctly; sensitivity was 84.6% and specificity 100%.


A tau-related protein of 130 kDa is present in Alzheimer brain.

Two abnormal entities of 69 and 130 kDa, immunologically related to the microtubule-associated tau proteins, are present in the hippocampus and the frontal cortex of the Alzheimer brain, which contain a large number of neurofibrillary tangles (NFTs), but are absent in the cerebellum, which does not contain these structures. Epitope mapping with antibodies spanning domains present in the N-terminal, middle, and C-terminal tau sequence demonstrated that the 69- and 130-kDa entities belong to the tau family. Both the 69- and the 130-kDa proteins were found in an insoluble form and were the major tau species present in purified NFTs. A procedure was devised that allowed us to prepare from Alzheimer hippocampi two NFT fractions differing in size (20 and 3 microns), both of which contained the tau entities of 130 and 69 kDa.


Characterisation of an allosteric modulatory protein associated with alpha-[3H]amino-3-hydroxy-5-methylisoxazolepropionate binding sites in chick telencephalon: effects of high-energy radiation and detergent solubilisation.

alpha-[3H]Amino-3-hydroxy-5-methylisoxazolepropionate ([3H]AMPA) binds to 1-day-old chick telencephalon membranes with KD and Bmax values of 138 nM and 2.56 pmol/mg of protein, respectively. High-energy radiation bombardment of intact frozen telencephalon resulted in a biphasic inactivation curve for [3H]AMPA binding. At a 5.8-Mrad radiation dose, the affinity of [3H]AMPA binding was increased (54 nM), but there was no apparent alteration in the Bmax value (2.76 pmol/mg of protein). We attribute this phenomenon to the inactivation of a high molecular weight modulatory protein that down-regulates the affinity of [3H]AMPA binding. The estimated molecular masses of the AMPA binding site and of the modulatory component were 59 and 108 kDa, respectively. Solubilisation with n-octyl-beta-glucopyranoside resulted in an increase in the Bmax (4.7 pmol/mg of protein) with no pronounced alteration in the affinity (109 nM) of [3H]AMPA binding. However, the solubilisation-induced increase in Bmax did not occur in telencephalon irradiated before solubilisation. In contrast, the increase in affinity induced by radiation treatment was still detected in solubilised extracts. These results suggest that the number and affinity of [3H]AMPA sites in chick telencephalon are closely regulated and that the modulatory systems involved are affected by both irradiation and solubilisation.


Actin depolymerizing factor is a component of slow axonal transport.

We examined the low molecular weight proteins transported with actin in the chicken sciatic nerve after injection of [35S]methionine into the lumbar spinal cord. A prominent component of slow axonal transport with apparent molecular mass 19 kDa comigrated on two-dimensional gels with chicken actin depolymerizing factor (ADF), previously shown to be a major actin-binding protein in brain. There was comparatively little radioactivity associated with the actin monomer sequestering proteins, profilin or cofilin, and examination of the rapid component of axonal transport failed to reveal appreciable quantities of actin, ADF, profilin, or cofilin. These results show that both actin and ADF are carried by slow axonal transport and raise the possibility that actin travels within the axon in an unpolymerized form in a complex with ADF.


A dihydropyridine-resistant component in the rat adrenal secretory response to splanchnic nerve stimulation.

A study of the effects of dihydropyridine Ca2+ channel modulators on the release of catecholamines from perfused rat adrenal glands, evoked by electrical stimulation of their splanchnic nerves, is presented. Electrically mediated secretory responses were compared to chemically mediated responses (exogenous acetylcholine, nicotine, or high K+). Intensities of stimuli were selected to produce quantitatively similar secretory responses (between 100 and 200 ng per stimulus). The main finding of the study is that responses to transmural stimulation (300 pulses at 1 or 10 Hz) and to acetylcholine were inhibited only partially (about 50%) by isradipine, an L-type Ca2+ channel blocker. In contrast, responses to high K+ (17.5 mM for 2 min) were highly sensitive to isradipine (IC50 = 8.2 nM). Responses to nicotine were also fully inhibited by this drug. Bay K 8644 (an L-type Ca2+ channel activator) potentiated mildly the secretory responses to electrical stimulation at 10 Hz and to acetylcholine, but increased threefold the responses to K+ and nicotine. It is, therefore, likely that responses mediated by high K+ or nicotinic receptors are triggered by external Ca2+ gaining access to the internal secretory machinery through L-type, dihydropyridine-sensitive voltage-dependent Ca2+ channels. However, in addition to nicotinic receptors, the physiological stimulation of adrenal medulla chromaffin cells through splanchnic nerves has other components, i.e., muscarinic receptor stimulation or the release of cotransmitters such as vasoactive intestinal polypeptide. The poorer sensitivity to dihydropyridines of secretory responses triggered by electrical stimulation of splanchnic nerve terminals or exogenous acetylcholine speaks in favor of alternative Ca2+ pathways, probably some dihydropyridine-resistant Ca2+ channels, in modulating the physiological adrenal catecholamine secretory process.


Selective expression of DM-20, an alternatively spliced myelin proteolipid protein gene product, in developing nervous system and in nonglial cells.

Mutations within the gene for myelin proteolipid protein (PLP), a major myelin structural protein, result in abnormal glial differentiation, suggesting that the PLP gene products play some other functional roles. Transcripts from the PLP gene were analyzed in the developing mouse brain by a sensitive method using polymerase chain reaction. The mRNA for DM-20, an alternatively spliced transcript from the PLP gene, was detected in the embryonic mouse brain as early as embryonic day 11, long before the appearance of oligodendrocytes, which were considered to be responsible for PLP production. PLP gene expression was analyzed in various cell lines to determine whether synthesis of the DM-20 mRNA is restricted to those of glial cell lineage. All of the nervous system cell lines examined, including nonglial cell lines, produced DM-20 mRNA but no or very little PLP mRNA. Peripheral sciatic nerve from adult Wistar rats also produced mainly DM-20 mRNA. These results indicate that DM-20 is not only a myelin structural protein, but it also plays other roles in the nervous system that seem to relate, at least in part, to glial differentiation.


The NK-1 receptor and a calcium-phospholipid pathway: inositol trisphosphate production and calcium movements induced by selective agonists of neurokinin receptors in rat parotid glands.

In the rat parotid gland, substance P has been shown to induce a phosphatidylinositol bisphosphate breakdown resulting in an inositol trisphosphate production. These data suggested that substance P activated a phospholipase C and thus mediated its effects through the calcium-phospholipid pathway. To determine which neurokinin (NK) receptor was involved in the substance P response, we have used selective agonists of the different NK receptors and examined their effects on both inositol trisphosphate production and calcium movements. A selective NK-1 receptor agonist, [Sar9Met(O2)11]-substance P, evoked an [3H]inositol trisphosphate production and a rapid and transient 45Ca2+ efflux. On the other hand, selective NK-2 and NK-3 receptor agonists, [beta-Ala8]-NKA(4-10) and [MePhe7]-NKB, respectively, were without effect. We conclude that, in the rat parotid glands, only the NK-1 receptors are coupled to the calcium-phospholipid pathway. The C-terminal part of substance P appeared to be sufficient to stimulate this route because the C-terminal octapeptide, substance P(4-11), mimicked substance P effects on both inositol trisphosphate production and calcium movements. The NK-2 and NK-3 receptors, if present in the rat parotid glands, are not associated with the calcium-phospholipid pathway.


Autologous facial fat transplantation: improved graft maintenance by microbead bioactivation.

An alternative approach to the management of free fat transplantation resorption was evaluated in a rat facial model. Fat grafts obtained from the inguinal region were transferred to subcutaneous lateral facial sites in 20 animals. The grafts were mixed with either basic fibroblast growth factor (bFGF) alone or dextran beads that had been pretreated with bFGF. The grafts were then compared by weight and histology at 1 and 6 months postoperatively. Although graft weights were nearly comparable at 1 month, substantial differences were seen at 6 months, with the bead-containing grafts exhibiting near complete weight maintenance and better overall graft form. Histologically, the bead-containing grafts had extensive intercellular collagen formation and a heterogeneity of adipocyte cell sizes, particularly after 1 month. These findings suggest that the addition of cell-specific bioactive peptides that affect either the preadipocyte cell line and/or the fibroblastic components of the recipient site improve postoperative fat graft weight maintenance. Delivery of the biochemical agent appears to require a carrier system to exert its effects.


Macroamylasemia in a 5-year-old girl.

In macroamylasemia, a macromolecular complex consisting of amylase linked to immunoglobulins circulates in the plasma and usually causes benign hyperamylasemia with low or normal amylasuria. Macroamylasemia is extremely rare in pediatric patients as it has been described in only four patients. We report herein the case of a 5-year-old girl with abdominal pain and macroamylasemia. To recognize macroamylase, we used agar gel electrophoresis, PEG precipitation, and fast protein liquid chromatography (FPLC). In our case, FPLC was found to be the most reliable method for the identification of the macromolecular complex. Macroamylasemia is merely a biochemical abnormality that is not associated with any kind of pathology. Its identification is therefore essential in order to avoid a wrong diagnosis, i.e., pancreatitis, with consequent inappropriate therapies.


Intestinal uptake and transmission of macromolecules into the blood in the young guinea pig.

The developmental changes in the ability of the small intestinal epithelium to take up and transfer into blood the macromolecules bovine serum albumin (BSA), bovine immunoglobulin G (BIgG), and fluorescein isothiocyanate-labeled dextran 70,000 (FITC-dextran) were studied in guinea pigs 0-14 days of age. In addition, in the same animals, the activities of the proteases cathepsin B and D within the intestinal mucosa were measured. Four hours after gavage feeding, 0-day-old guinea pigs showed an uptake of the markers into the enterocytes throughout the small intestine. In 2-day-old guinea pigs, the markers were only detected in the enterocytes in the distal part. All three macromolecules passed into the blood in these young animals. In 7-day-old guinea pigs, no epithelial marker uptake was observed, but low levels of BSA and FITC-dextran could still be detected in serum. Neither epithelial uptake nor transfer of the markers to the blood could be found in the animals that were 14 days of age. The activity of cathepsin B and D in the intestinal mucosa showed a tendency to increase with age, and for all ages the activity in the distal part was higher than in the proximal part. The results showed that the small intestine in guinea pigs at birth is capable of macromolecular uptake and transfer into the circulation. This ability decreased with age eventually leading to intestinal closure after 1 week. The histological findings implied that intestinal closure was a consequence of a replacement of fetal absorptive cells with adult cells lacking this ability.


Infantile refsum disease: gastrointestinal presentation of a peroxisomal disorder.

This article describes two siblings with infantile Refsum disease (IRD) whose initial presentation was that of malabsorption and mimicked a-beta- or homozygous hypo-beta-lipoproteinemia. Failure to recognize IRD in the first-born child precluded proper genetic counseling and prenatal diagnosis in subsequent pregnancies and also caused considerable delay in diagnosing IRD in the second child. The clinical heterogeneity of peroxisomal disorders constitutes a diagnostic challenge, which demands a high degree of awareness from the part of the clinician. This is particularly the case with IRD, where protracted diarrhea with low serum cholesterol levels appears to be a frequently occurring initial feature during the 1st months of life.


The apical border plaque in chronic adult periodontitis. An ultrastructural study. II. Adhesion, matrix, and carbohydrate metabolism.

THE AIM OF THIS STUDY was to characterize the plaque matrix and relevant aspects of metabolism of the apical border plaque in relation to teeth affected by chronic adult periodontitis. The material comprised 56 teeth from 24 patients. Ruthenium red, alcian blue, lanthanum nitrate, and safranin 0 were used to label matrix polyanionic macromolecules and periodic acid-thiosemicarbazide-silver proteinate for intracellular polysaccharide (IPS). The matrix components were amorphous, fibrillar, or globular. Many intact bacteria exhibited extracellular polysaccharides or glycocalyces associated with their cell wall and cytoplasmic IPS granules. The latter varied in size and distribution and were evident even in the most apically-advanced intact microorganisms. The results indicate that the matrix and IPS features of the apical border plaque in chronic periodontitis in certain respects resemble those of subcontact area plaque on children's teeth, associated with chronic gingivitis and approximal caries. They also suggest the establishment of acidic regions in the microniches of the periodontal pocket.


Changes in brain serotonergic activity during hierarchic behavior in Arctic charr (Salvelinus alpinus L.) are socially induced.

The experiment was performed in two phases. During the first phase (phase 1) the dominance hierarchy was determined in 4 groups of Arctic charr (Salvelinus alpinus L.), each group consisting of 4 fish. Phase 2 was started by rearranging phase 1 fish into 4 new groups. Group 1 consisted of previously dominant fish and groups 2, 3 and 4 of fish that previously held rank 2, 3 and 4, respectively. After phase 2 telencephalon and brain stem were analyzed with regard to their contents of serotonin (5-hydroxytryptamine, 5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), the principle metabolite of 5-HT. No correlation was found between the social rank (measured as dominance index) during phase 1 and the brain serotonergic activity (measured as the ratio 5-HIAA/5-HT) determined after phase 2. However, most important, the 5-HIAA/5-HT ratio was significantly correlated with the last experienced social rank, i.e. that acquired during phase 2. These results shows that the difference in brain serotonergic activity between dominant and subordinate fish develops through social interactions. Further, we found that previous subordinate experience inhibited aggressive behavior, an effect which, in the light of available information on stress and 5-HT, could be related to the increase in brain serotonergic activity. We hypothesize that stress induces an increased serotonergic activity which in turn inhibits the neuronal circuitry which mediates aggressive behavior.