[The regulation of protein kinase C activity in normal, immortalized and transformed rat fibroblasts]. In view of recent studies showing that cell proliferation of E1Aad5+c-Ha-ras-transformed fibroblasts cannot be regulated by growth factors and phorbol eaters in contrast to normal and E1Aad5-immortalized cell lines, the present work was undertaken to examine the role of protein kinase C (PKC) in the mitogenic signal transduction machinery in rat embryonal fibroblasts. It is shown that PKC is activated by acidic growth factor and phorbol esters in all the three cell lines. These findings suggest the existence of an additional, not associated with PKC-, growth-signaling pathway in E1Aad5-Ha-ras-transformed rat embryonal fibroblasts.


[The localization of DNA in the nucleoli of mammalian cells]. The localization of DNA within nucleoli of mammalian cells was studied in human diploid fibroblasts (strain MRC-5), stained with chromomycin A3--the fluorochrome specific for G-C DNA base pairs. To change the rate of rDNA transcription, actinomycin D (0.08 mkg/ml) was added to log phase cells for 30-90 min. The stained cells were analysed with a highly sensitive video camera and deblurring algorithm (Jordan, Rawlins, 1990). The pattern of staining was compared with the ultrastructural peculiarities of the cells fixed in the same conditions. As the result, it is inferred that the intranucleolar DNA (rDNA) is consistent with the fibrillar centres as well as with the dense fibrillar component. The localization of rDNA may alter due to the transcriptional activity of ribosomal genes. The data obtained support the idea on the transcription of ribosomal genes inside the dense fibrillar component of the nucleolus.


Obstructive nephropathy in the pig. Possible roles for insulin-like growth factor I. Kidney growth was investigated in 30-kg pigs after 72 h of unilateral ureteral obstruction. The data were compared to control kidneys from normal non-operated pigs at same weight. Kidney wet weight was determined. Cortex and medulla were separated, and from both regions RNA, DNA, protein and kidney tissue insulin-like growth factor I was determined. Unilateral obstruction caused a doubling of the wet hydronephrotic kidney weight and an ipsilateral 76% increase in total kidney protein content. RNA increased by 45% in the cortex and 76% in the medulla. Kidney protein in the contralateral cortex increased by 23% and RNA by 42%. In the hydronephrotic kidney DNA was reduced by 13% in the cortex and by 21% in the medulla. Contralaterally, DNA was the same as in the controls. Mean kidney insulin-like growth factor I increased sevenfold in the ipsilateral medulla but in the cortex it was the same as in the controls. Serum insulin-like growth factor I concentration was 1.7 +/- 1.1 micrograms/l in the hydronephrotic animals and 1.2 +/- 0.8 micrograms/l in controls. At this stage of obstruction, our data demonstrate (1) hydronephrotic growth that is most probably hyperplastic in the medulla, associated with an increase in medullary insulin-like growth factor I, (2) hyperplastic growth in the cortex, and (3) contralateral kidney growth that is mainly hypertrophic after 72 h of contralateral ureteral obstruction.


Effects of cytokines on growth in vitro of primary human renal cell carcinoma. In clinical trials different haematopoietic active cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) have been proven to alleviate myelosuppressive side effects of intensive chemotherapy in different non-urological malignancies. On the other hand, these cytokines can directly stimulate the proliferation of cells originating from some non-urological tumours. To clarify the impact of these cytokines on the proliferative behaviour of human renal cell carcinoma (RCC), 29 previously untreated RCC tumours were prepared for culturing in vitro using the cell cluster technique. The success rate for growth in vitro was 82.8% (24/29). The malignant renal cells were treated with different cytokines (GM-CSF, G-CSF and interleukin-3) in different dosages. Cell number and proliferation rates detected by immunostaining were used for treatment evaluation. A dosage-dependent stimulation of cell growth could not be observed compared to untreated cells. From the data presented in this study, proliferative stimulation of RCC by administering colony-stimulating factors in clinical trials cannot be assumed.


Development of a control strategy for Leptospira hardjo infection in a closed beef herd. Serological evidence of infection with a leptospire belonging to the Sejroe serogroup was identified in a closed population of Luing cattle in the west of Scotland, and the geographical isolation of the population presented an opportunity to control and possibly eradicate the infection in a large beef herd farmed under extensive conditions. Serological and bacteriological studies revealed that infection was present at a high level throughout the herd, and that the infecting serovar was hardjo. Unlike endemic hardjo infection in dairy herds, new infections were still occurring in older age-groups. Investigations of other domestic and free-living species sharing the habitat demonstrated that the maintenance of an endemic focus of hardjo was restricted to the cattle. Changes in management to prevent the transmission of infection to successive cohorts of young animals were impractical and risky, and antibiotic treatment followed by removal to clean pasture failed to prevent new cases. Thus vaccination offered the only means of control and possible eradication, and the epidemiological characteristics of the infection dictated that the programme be applied to the whole herd.


Immune responses of specific pathogen free foals to EHV-1 infection. Four foals were raised under specific pathogen free (SPF) conditions. At 3 to 4 months of age, SPF foals and 1 other non-SPF foal were intranasally inoculated with equine herpes virus type 1 (EHV-1). Clinical signs included depression, fever, inappetence and intermittent coughing. Clinical recovery was complete by seven days but high titres of virus were detected in nasal mucus for at least 10 days after inoculation. Clinical illness was less severe in the non-SPF foal. Interferon was detected in the nasal mucus of all foals from 2 days post infection (dpi), persisting until 8 or 10 dpi. ELISA antibody was detected in serum from 6 dpi. Titres continued to rise throughout the period of observation, and were slightly stimulated by re-inoculation. EHV antibody, identified as belonging to the IgM class by the double sandwich ELISA, was detected from 6 dpi. Peak IgM titres were observed between day 10 and 18, declining to base levels by day 42. Virus neutralizing antibody was detectable in serum from day 14 and rises in titre were parallel to that of total ELISA antibody. Cellular immunity in EHV-1 infected SPF horses was examined by the antibody dependent cytotoxicity (ADCC) test and the specific lymphocyte transformation test. The ability of foal neutrophils to effect ADCC decreased significantly between 3 to 10 days after inoculation. Peripheral blood mononuclear cells (PBMC) displayed reactivity towards EHV-1 antigens from about day 14, with maximum stimulation indices being obtained between 28 and 42 dpi.


A characterization of monoclonal antibodies prepared against Pasteurella haemolytica serotype 1 surface antigens. The production and characterization of monoclonal antibodies against Pasteurella haemolytica serotype 1 is described. Ten monoclonal antibodies were produced and divided, on the basis of their properties, into six different groups. One produced bacteria agglutination only of P. haemolytica serotype 1. Three antibodies bound with P. haemolytica serotypes 1, 5-8 and 12 and the antigen was identified in immunoblots as lipopolysaccharide. Two antibodies bound P. haemolytica serotypes 1, 2, 5-8 and 12 and P. multocida serotypes 1-7, 9, 12, 15 and 16, recognizing an epitope present on a 29 kDa outer membrane protein. One antibody bound all P. haemolytica and P. multocida serotypes. The antigen was a hexosamine less than 30 kDa which contained a formalin sensitive epitope. One antibody bound only to P. haemolytica serotype 1 and the antigen was identified as a 66 kDa outer membrane protein. Two antibodies bound P. haemolytica serotypes 1, 2, 5-9 and 12 and the antigen, while not identified, was localized on the outer membrane. This study identified antigens which contribute to the cross-reactions among P. haemolytica and P. multocida serotypes and the antibodies may be useful in investigating the pathogenesis of pneumonic pasteurellosis.


Common and stage-specific antigens of Theileria annulata. Western blot analysis of Theileria annulata antigens was carried out using sera collected from cattle which had been immunised and challenged with either T. annulata sporozoites or schizont-infected cells. Three antigens between 71 and 73 kDa proved to be common to the three stages of parasite studied: sporozoites, schizonts and piroplasms. An antigen was found at 32 kDa which was specific to T. annulata piroplasms. Results were reproducible using sera from Morocco and the UK. At least one of the proteins at 71-73 kDa, but not that at 32 kDa were also recognised by sera from animals infected with Babesia species.


Immunostimulants added to injected Aeromonas salmonicida bacterin enhance the defense mechanisms and protection in rainbow trout (Oncorhynchus mykiss). Immunostimulants were given to rainbow trout for assaying effects on modulating non-specific defense mechanisms, specific immune response, and protection levels against pathogen challenge with Aeromonas salmonicida. Three drugs, levamisole (an approved veterinary drug in the USA), a quaternary ammonium compound (QAC), and a short-chain polypeptide (ISK) were found to affect the non-specific defense mechanism activities, which were measured by changes in circulatory neutrophil and phagocytic activity levels, and the specific immune response factors, which were measured by numbers of plaque-forming cells, and circulatory antibody levels. When given alone, the immunostimulants elevated the non-specific factors. When injected in combination with an A. salmonicida O-antigen bacterin, the non-specific factors were further elevated, and the specific response was raised over samples taken from fish given the bacterin without the immunostimulants. Challenge tests with the virulent pathogen, A. salmonicida, showed a 5-6 day delay in the onset of mortalities in the fish given the immunostimulants alone, and a 12-14 day delay when immunostimulants given were combined with the bacterin. In the groups given the QAC or ISK with the bacterin, there was a 20% and 40% survival rate, respectively.


Multicystic mesothelial proliferation. Immunohistochemical, ultrastructural and DNA analysis of five cases. We investigated the clinicopathological findings in five cases of multicystic mesothelial proliferation (MMP). All masses consisted of multiloculated cysts attached to pelvic organs and sometimes growing into the upper abdominal cavity. The cystic spaces were lined by flattened or cuboidal cells. The stroma showed fibrosis, oedema and chronic inflammation. Immunohistochemistry revealed strong positive staining for cytokeratin and epithelial membrane antigen, and focal positivity for vimentin and carcinoembryonic antigen. The endothelial markers were negative. Electron microscopy showed abundant surface microvilli and well-developed basal lamina. DNA analysis identified euploid cell populations in all cases. All but one case had a previous history of abdominal surgery. Despite the worrying appearance the clinical outcome was favourable in all cases; there was one recurrence. Clinical and pathological data support the hypothesis that MMP represent a reactive mesothelial proliferation and not a neoplastic process.


Epithelioid sarcoma: an electron-microscopic, immunohistochemical and DNA flow cytometric analysis. Eight epithelioid sarcomas (ES) were studied by electron microscopy, immunohistochemistry, and DNA flow cytometry. Ultrastructurally, the tumour cells showed desmosome-like intercellular junctions and numerous microvilli, in addition to whorled arrangements of intermediate filaments. Tumour cells were positive for epithelial membrane antigen, cytokeratin, and vimentin, and negative for carcinoembryonic antigen and desmin. All seven cases examined by flow cytometry showed diploid or hyperploid (near diploid) DNA content. This seems to correspond to the relatively long clinical course and low-grade malignant nature of ES. Although the histogenesis of ES is still uncertain, the results of this study suggest that it is a tumour of primitive mesenchymal cells with the capacity to show epithelial differentiation.


Immunohistochemical and ultrastructural studies of intraluminal crystalloids in human prostatic carcinomas. Intraluminal crystalloids (ICr) observed in 19 cases of incidental or invasive human prostatic carcinoma (PCa) and in a case of benign prostatic hyperplasia were examined extensively by immunohistochemistry and electron microscopy. They were brilliantly eosinophilic with haematoxylin and eosin, manifesting needle-like, triangular, rectangular, hexagonal and irregular lump-like in shape. They were strongly positive, dark blue, with phosphotungstic acid -haematoxylin (PTAH) stain in all cases examined. Among the human antibodies tested, epithelial membrane antigen (EMA) gave specifically positive immunostainability with ICr in all cases. Annual ring-like lamellar or concentric structures were detected by electron microscopy. Positive staining of ICr with PTAH and anti-EMA antibody is very useful as a diagnostic marker for PCa in human prostatic tissues.


Histopathology of myoepithelial (basocellular) hyperplasias in adenosis and epitheliosis of the breast demonstrated by the reactivity of cytokeratins and S100 protein. An analysis of heterogenic cell proliferations in 90 cases of benign and malignant breast diseases. This study on the different types of epithelial hyperplasia in fibrocystic disease was inspired by the observation of myoepithelial (basocellular) hyperplasia identified by strong expression of S100 protein and a weak reaction with antibodies against cytokeratin (KL1) in cells forming solid and acinar buds. The cells do not contain immunohistochemically detectable actin or desmin. Glandular transformation and proliferation give rise to basocellular circumductal adenosis. Normal breast tissue, 51 cases of fibrocystic disease with mild, florid and atypical hyperplasias, 7 fibroadenomas and 20 cases of carcinoma in situ were studied and a semiquantitative analysis revealed basal buds and adenosis in less than 40% of cases of mild hyperplasia and up to 73% in florid hyperplasia. Epitheliosis is characterized by a heterogeneous cell pattern with cells positive for S100 protein in 30-60%, but in small ducts up to 100% with an immediate connection to the basal cell layer were positive. Carcinoma in situ contained very rare tumour cells positive for S100 protein. The cells expressing S100 protein in terminal ducts, in adenosis and epitheliosis showed only some of the characteristics of myoepithelial cells, since they lack immunoreactivity with antibodies against actin. These basal clear cells are interpreted as transitional or indeterminate cells with features of myoepithelial precursor cells, but with the ability to develop basocellular nodular and glandular hyperplasia in the ductulo-lobular units in cases of adenosis and juvenile fibroadenoma.


Activation of collagen type II expression in osteoarthritic and rheumatoid cartilage. In situ hybridization and immunohistochemical techniques were applied to investigate gene expression and extracellular deposition of collagen type II in normal, osteoarthritic and rheumatoid human articular cartilage. Normal cartilage showed an essentially even extracellular distribution of type II collagen with poly- and monoclonal antibodies, while only a few cells were positive for alpha 1(II) collagen mRNA. In situ hybridization of osteoarthritic and rheumatoid cartilage, however, showed strong enhancement of type II collagen gene expression; transcripts were observed predominantly in the upper middle zone of the articular cartilage while the upper layer was mostly negative and correlated with a zone of reduced proteoglycan staining. The elevated mRNA levels frequently coincided with pericellular immunostaining for type II collagen, indicative for enhanced synthesis of the protein. In two samples, however, pericellular loss of collagen type II staining was found despite positive cytoplasmic signals with the alpha 1(II) RNA probe, suggesting enhanced collagen destruction. Control hybridization with a probe for 18S rRNA revealed very few negative cells throughout both normal and arthritic cartilage samples, ruling out major cell necrosis in the specimens investigated. Thus, our observations identify sites of activated type II collagen synthesis in osteoarthritic cartilage that were predicted by previous biochemical studies and support the notion that damaged cartilage attempts to restore matrix by enhanced synthesis of its components.


Dendritic cells in glomerulonephritis.

Dendritic cells in glomerulonephritis. Renal biopsies (n = 45) from patients with various forms of glomerulonephritis (GN), comprising mesangial IgA-GN (n = 25), focal glomerular sclerosis (n = 13) and acute GN (n = 7), were examined by double staining immunocytochemistry (APAAP, streptavidin-peroxidase) using unconjugated monoclonal antibodies (Ab) against--(i) the CD1b antigen expressed on dendritic cells (DCs), (ii) the invariant chain (Ii), and (iii) biotin-conjugated Ab against HLA-DR. In normal control kidneys (n = 7) without interstitial inflammation, CD1b-positive DCs were not detected. Glomerular endothelial cells and a few cells in mesangial areas showed double staining with the Ab against HLA-DR in Ii. In GN without active interstitial inflammation (n = 9), CD1b-positive DCs were not found. In biopsies with interstitial inflammation (n = 36) CD1b-positive DCs were found interspersed among other inflammatory cells. In seven of the biopsies showing IgA-GN DCs were seen in the vicinity of those glomeruli that exhibited either crescents or glomerular sclerosis with splitting of Bowman's capsule. In proximal tubular epithelial cells de novo expression of HLA-DR/Ii-chain was only seen when DCs were present. We conclude that in different forms of GN: (i) CD1b-positive DCs play an important role in the development of interstitial inflammation, and (ii) their presence may be related to the de novo coexpression of HLA-DR/Ii in tubular epithelial cells, possibly mediated through the production of interferon gamma and other cytokines.


[Specific immune preparations for the prevention and treatment of suppurative-inflammatory diseases of Proteus etiology (a review of the literature)]. Literature data are analyzed on the possibility of designing vaccines Proteus agents for obtaining specific blood agents. Perspective are designs directed to investigation of the raw material base for obtaining allogenous anti-Proteus blood agents.


[A method for the individual choice of anti-arrhythmia preparations in extrasystole]. Pharmacological analysis of arrhythmogenesis was approved in 60 patients with ventricular extrasystolic arrhythmia (EA). Individual choice of anti-arrhythmia agents (AAA) was realized by successive drug-induced block of slow calcium cancels by isoptin and rapid sodium channel by class I AAA. Changes of arrhythmogenesis were registered electrocardiographically. Acute tests with isoptin revealed a good antiarrhythmic effect in 46.67%, with AAA--in 30%. It is concluded that methods of pharmacological analysis of arrhythmogenesis widely used in experimental cardiology may be used in the clinic for individual choice of AAA.


Comparison of immune responses to a native viral antigen and a synthetic peptide derived from it: implications for vaccine development. Murine immune responses to the hepatitis B surface antigen (HBsAg) and a synthetic peptide derived from it were compared at the humoral level. Six of nine strains used responded to either peptide or HBsAg, though restriction profiles were not superimposable. Two of three strains non-responsive to HBsAg produced an antibody response on immunization with peptide which was cross-reactive with both peptide and HBsAg. In in vitro lymphocyte stimulation assays, lymphocyte from all six peptide-immunized mouse strains could be induced to proliferate on challenge with HBsAg. However, of the HBsAg-immunized groups, lymphocytes from only three of six responder strains proliferated on in vitro HBsAg challenge. Cumulatively, these results suggest that a vaccine formulation that includes both protein antigens and synthetic peptides derived from these proteins may be more effective at eliciting an immune response in a broader cross-section of target population.


Influence of substance P on gastrointestinal myoelectrical activity in the conscious piglet. In conscious piglets with electrodes implanted in the wall of the antrum pylori, the small intestine and the caecum, the influence of intravenous infusion of substance P (SP) (0.1 and 1 micrograms/kg/min for 2 hours) on gastrointestinal myoelectrical activity and arterial pressure was studied. SP was without significant influence on electrical activity of the antrum. However, intestinal myoelectrical activities were significantly stimulated. In the small intestine SP infusion resulted in an increase in frequency of migrating myoelectrical complexes and in irregular spiking activity, resulting in an augmentation of the integrated total activity. In the caecum SP provoked a prolongation of complexes of long spike bursts (LSB) as well as an increase in number of LSB/complex. In the studied doses SP had no significant effect on arterial pressure.


Laser therapy of gastrointestinal tumors. Surgical resection remains the therapy of choice for the treatment of potentially curable gastrointestinal tract (GI) malignancies. Many of these tumors are incurable at the time of diagnosis and therapy should be directed towards palliation with the intent of minimizing pain, bleeding, obstruction, and potential morbidity. Endoscopic laser therapy is uniquely applicable for the palliation of GI tumors and in selective instances may be appropriate for the treatment of early lesions. Eighty-six patients with GI malignancy have been treated at our institution since 1985. Thirty-one patients had advanced upper GI lesions (esophagus: 26, gastric: 3, duodenal: 1, and pancreatic: 1) and 55 patients had lower GI tumors (colon: 37 and rectal: 18). Pre-resectional recanalization for obstructing colorectal carcinomas obviating initial operative diversion was performed in 31 (56%) of 55 patients. Twenty-four patients had palliative laser therapy (obstruction: 17 and hemorrhage: 7) with resolution of their symptoms. There was 1 laser related perforation in the pre-resectional group and the overall complication rate was 1.2%. Endoscopic Nd:YAG and currently photodynamic laser therapy for GI tumors has proven to be an effective mode of therapy for advanced GI neoplasms with minimal morbidity. The utility of photodynamic therapy for the treatment of early stage esophageal and gastric cancers remains controversial.


[The demonstration of recurrent motor axon collaterals in the chick embryo by using horseradish peroxidase axonal transport]. Motoneurons were labelled by retrograde axonal transport of HRP applied to transected spinal nerves in 9-11-day chick embryos in the in vitro spinal cord preparation. Recurrent motor axon collaterals were revealed in 17 of 48 motor axons which could be followed in the edge regions of labelled motoneuronal pools. The results, coupled with author's earlier electrophysiological data, provide further evidence for the presence of the Renshaw inhibition in the avian spinal cord.


[A light microscopic study of the axonal collaterals of the lumbar motoneurons in the spinal cord of the frog Rana ridibunda]. By using intracellular injection of horseradish peroxidase into the lumbar motoneurones of the isolated spinal cord of the frog Rana ridibunda the structure of axon collaterals was studied. It was shown that about 50% of the HRP-stained cells had mainly one axon collateral of the 1st order. The subsequent branching patterns of the collaterals showed considerable variations. The number of swellings in various collaterals was from 10 up 100. The mean diameter of swellings varied from 0.8 up 10.0 microns. It is believed that the axon collateral swellings from contacts on the dendrites of the nerve cells mainly. Apparent axosomatic contacts were revealed on the motoneurons and small nerve cells. As in the cat, collateral swellings were found on the dendrites of the parent motoneuron. Obtained morphological data the structure of motor axon collaterals in the frog are compared with those in the cat. Functional significance of the axon collateral in the frog is discussed.


[The quantitative morphological characteristics of the neurons of the anterior horns of the spinal cord in the kitten]. Golgi preparations of cervical part of the spinal cord of 30-day kittens were used to study sparely and densely branching neurons of lamina VII, sparely and densely branching neurons of lamina VIII and big densely branching motor neurons (as classified by Leontovich) of medial and lateral regions of lamina IX. Qualitative morphological characteristics of geometry of each cell type were obtained by the method of computerized morphometry. The details of the structure of neurons belonging to different laminae of grey matter are discussed.


Personality of patients with Sudeck's atrophy following tibial fracture. Patients with reflex sympathetic dystrophy are often considered by physicians and allied health personnel as having a peculiar personality. In medical literature they are frequently described as anxious and depressive, emotional, nervous and irritable patients with neurovegetative instability. A review of the literature on psychological research in this field is not always illuminating. Hypochondria and hysteria, whether or not accompanied by depression, are frequently reported to be typical traits, whereas other findings point more in the direction of psychosis. Increased anxiety, emotional lability and lowered self-esteem are psychological entities that are regularly encountered. The present study includes 42 cases of severe reflex sympathetic dystrophy. Except for the 7 cases of Sudeck atrophy of the hand and wrist, the localization was always in the foot or ankle. The majority of patients had a history of fractures or orthopedic procedures on the lower limbs as a causative factor. In addition to an interview, two questionnaires and a projective test (Rorschach) were used in the personality assessment. While the Rorschach test did not reveal any findings that could be considered as typical of our study population, we did observe different frequency distributions for the personality traits "self-satisfaction", "rigidity" and "somatization".


[The quantitative morphological characteristics of the spinal neurons in the kitten developing under conditions of limited afferent input]. Golgi preparations of cervical part of the spinal cord were used to study sparely and densely branching neurons of lamina VII, sparely and densely branching neurons of lamina VIII and large densely branching motor neurons of lamina IX in 30-day kittens developing under the conditions of limited reticulospinal input. Limitation was induced by electrocoagulation of bulbar magnocellular nucleus and caudal reticular pontine nucleus performed at the 7th day. Computerized morphometry revealed that partial deafferentation affects the geometry in all studied cell types except densely branching neurons of lamina VIII. Adaptive nature of structural reorganization of spinal neurons and possible relationship between morphological properties and functional profile of cells are discussed.


Mercury deposition and its relationship to inner ear function in methylmercury-poisoned rats. A histological and immunohistochemical study. Mercury deposition and its relationship to the inner ear function in methylmercury-poisoned rats were examined. Rats were poisoned with methylmercury chloride. After the appearance of signs of poisoning, the cerebellar and inner ear tissues were examined histochemically using an autoradiographic procedure. Mercury deposits were typically found in the cerebellum and appeared in parts of the vestibular nerves, cochlear nerves, spiral ganglion and stria vascularis. In the vestibule, a slight mercury deposition was observed in the acoustic maculae and in the cochlea; only one instance of mercury deposit in the organ of Corti was detected. The effect of mercury on the inner ear function was also assessed on the basis of immunohistochemical localization of substance P which is believed to be a neurotransmitter in the inner ear. However, no pathological changes were detected. These findings suggest that methylmercury deposition has little effect on the inner ear function.


Inhibitory effects of tetramethylpyrazine on platelets during cardiopulmonary bypass and arterial thrombus formation in dogs. The corrected platelet count at the end of cardiopulmonary bypass (CPB) in nontreated dogs fell to 56.7% of the pre-operative level, while in tetramethylpyrazine (TMP, 10 mg.kg-1, iv)-treated dogs it fell to 82.8%. The number of alpha-granule membrane protein (GMP-140) which was expressed on the surface of activated platelets increased from the outset of CPB and reached the peak at the end of CPB in control dogs. But the number of GMP-140 molecules did not increase significantly throughout the operative procedure in TMP-treated dogs. In experimental thrombosis in dogs, the weight of the thrombus reduced to 23% of that in controls and the radioactivity ratio between the thrombus and blood was only 29% of that in controls at 4 h after iv injection of 125I-SZ-51 (a monoclonal antibody against GMP-140). These results indicate that TMP inhibits the platelet activation during CPB and arterial thrombosis.


Expression of helix-loop-helix regulatory genes during differentiation of mouse osteoblastic cells. Although much is known about the hormonal regulation of osteoblastic cell differentiation, much less is known about the nuclear regulatory molecules that affect this process. We analyzed the expression of several regulatory molecules of the helix-loop-helix (H-L-H) group in primary mouse calvarial cells and in MC3T3-E1 mouse osteoblastic cells in situations representing different degrees of cellular differentiation. H-L-H class regulators are known to participate directly in directing cell fate and differentiation decisions in other mesodermal lineages. Two of the molecules that we studied, Id and E12, have well-established roles in this process. The other, mTwi, the murine homolog of the Drosophila twist gene, is a newly cloned mammalian H-L-H gene. Levels of E12 RNA remained unchanged during differentiation. On the other hand, in both primary osteoblastic cells and MC3T3-E1 cells, the abundance of Id and mTwi declined with cell maturation; mTwi less dramatically than Id. That Id expression is causally related to differentiation is suggested by the finding that MC3T3-E1 cells transfected with an Id-expression plasmid fail to undergo differentiation. We conclude that helix-loop-helix regulatory genes are expressed in mouse osteoblastic cells, where they are likely to participate in differentiation. The E12 gene product is likely to function as a positive modulating factor. In contrast, Id inhibits differentiation, probably by sequestering other H-L-H gene regulators, including E12, in inactive complexes. The precise role of mTwi is more speculative at this time, but the observed pattern of expression is consistent with a role in early and midmesodermal specification that is terminated as cells differentiate.


Release of oxygen radicals by articular chondrocytes: a study of luminol-dependent chemiluminescence and hydrogen peroxide secretion. We previously established that normal articular chondrocytes, like macrophages, express class II major histocompatibility antigens, present antigen, and induce mixed and autologous lymphocyte stimulation. In a recent study using the trapped indicator 2',7'-dichlorofluorescein diacetate, we were able to measure levels of intracellular hydrogen peroxide within normal articular chondrocytes (J Immunol 245:690-696, 1990). In the present study, we utilized the technique of chemiluminescence and the biochemical method of quantitating hydrogen peroxide release to measure the production of reactive oxygen intermediates by articular chondrocytes. Chondrocytes, in suspension or adherent to coverslips, showed luminol-dependent chemiluminescence that was dependent on the number and viability of cells. There was a dose-dependent increase in chemiluminescence in response to soluble stimuli, such as phorbol myristate acetate (PMA), concanavalin A (ConA), and f-Met-Leu-Phe (FMLP). Azide inhibited chemiluminescence, suggesting that the light emission in chondrocytes is myeloperoxidase dependent. The antioxidant, catalase, inhibited chemiluminescence but superoxide dismutase had no effect, suggesting that luminol-dependent chemiluminescence in chondrocytes mostly measured hydrogen peroxide. Chemiluminescence was also observed in fragments of live cartilage tissue, indicating that chondrocytes that are cartilage matrix bound can generate the respiratory burst response. Using the scopoletin oxidation assay, we confirmed the release of increasing amounts of hydrogen peroxide by chondrocytes exposed to interleukin-1, rabbit interferon, and tumor necrosis factor alpha. Tumor necrosis factor alpha had both priming and enhancing effects on reactive oxygen intermediate production by chondrocytes. Reactive oxygen intermediates have been shown to play a significant role in matrix degradation. We suggest that reactive oxygen intermediates produced by chondrocytes play an important role in the degradation of matrix in arthritis.


Prostaglandin E2 stimulates synthesis of insulin-like growth factor binding protein-3 in rat bone cells in vitro. Prostaglandin E2 is produced by bone cells and increases cyclic AMP in these cells. Like PTH and dibutyryl cyclic AMP, PGE2 is a potent stimulator of IGF-I synthesis in cultured rat osteoblasts and inhibits DNA synthesis and type I procollagen gene expression. In addition, PGE2 inhibits the response of the cells toward IGF-I after 1 day but not after 4 days of incubation. Rat calvaria osteoblasts constitutively release IGFBPs into the culture medium, in particular IGFBP-2 and IGFBP-3. Like growth hormone, PGE2 stimulates the accumulation of IGFBP-3. PGE2 rapidly increases IGF-I and IGFBP-3 mRNA expression in calvaria cells, with a time course clearly different from that observed in response to growth hormone. Thus, PGE2 modifies not only the synthesis of IGF-I but also that of IGFBP-3 in skeletal tissue.


Influence of protein kinase C, cAMP and phosphatase activity on histamine release produced by compound 48/80 and sodium fluoride on rat mast cells. We have studied the effect of protein kinase C and protein kinase A activation, and phosphatase inhibition on two different stimuli with distinct mechanisms of action. The first stimulus is compound 48/80, and its action is mediated probably by a Gi-protein, while the other is sodium fluoride, which unspecifically activates G-proteins. We established a comparative study because the action of compound 48/80 is calcium-independent, while fluoride is strictly calcium-dependent. The activation of protein kinase C was attained with the phorbol esther 12-O-tetradecanoylphorbol-13-acetate, protein kinase A was activated by increasing cAMP levels with forskolin or rolipram, and the phosphatase activity was inhibited with okadaic acid (OA), which inhibits phosphatases type 1 and 2A. Our results show that OA enhances the response to fluoride and compound 48/80 in the absence of calcium, and we conclude that calcium has a negative feedback role on the cell response. Protein kinase A activation strongly inhibits the response to fluoride, and the results show a positive regulation of protein kinase C and a negative regulation of protein kinase A over fluoride response. As previously reported by other authors for the ionophore A23187, TPA notably potentiates the response to fluoride, which supports its possible modulatory role on extracellular calcium-dependent stimuli.


Ionic regulation of human basophil releasability. I. Inhibitory effect of copper. The effects of copper (CuSO4 and CuCl2) on in vitro histamine release from human basophils stimulated by anti-IgE and Ca2+ ionophore A23187 were evaluated. Both CuSO4 and CuCl2 caused a dose-related inhibition of histamine release, which was more pronounced on anti-IgE- than on Ca2+ ionophore-induced histamine release. The concentration which produced 50% inhibition of anti-IgE-induced histamine release was 1.3 microM for CuSO4 and 1.5 microM for CuCl2; the maximal inhibition of Ca2+ ionophore-induced histamine release was 33% for CuCl2 (4 microM) and 51% for CuSO4 (16 microM). The inhibitory effect on anti-IgE-induced histamine release persisted also when extracellular Cu2+ was removed by cell washing before stimulation, whereas no inhibition of Ca2+ ionophore-induced histamine release was found when extracellular Cu2+ was removed. The activity of Cu2+ was independent of any effects of deuterium oxide and colchicine, two agents known to interact with microtubules. Increased extracellular Ca2+ concentrations reduced the inhibitory effect of CuCl2 on Ca2+ ionophore-induced histamine release, and Schild plot analysis demonstrated that Cu2+ ions are competitive antagonists of Ca2+ ions. These results indicate that Cu2+ ions in the micromolar range down-regulate anti-IgE- and Ca2+ ionophore-induced histamine release. Since Cu2+ concentration in human plasma is in the micromolar range (30 microM with 10-30% of free Cu2+), it is conceivable that Cu2+ ions contribute to the in vivo regulation of histamine release from human basophils.


Actions and cross-reactivity of antiallergic agents and a calcium channel antagonist on rat peritoneal mast cells. Difference in the action mechanisms and cross-reactivity among the agents. The actions of the antiallergic agents, disodium chromoglycate (DSCG), tranilast and ketotifen, and of a calcium channel antagonist, nicardipine, and cross-reactivity among the agents were examined by observing the inhibition of 45Ca uptake and histamine release in rat mast cells stimulated by antigen and compound 48/80 (comp. 48/80). 1) All agents inhibited 45Ca uptake and histamine release in mast cells stimulated by antigen. The inhibition of 45Ca uptake by the antiallergic agents paralleled the inhibition of histamine release, while nicardipine inhibition of 45Ca uptake was stronger than its inhibition of histamine release. 2) The action of DSCG on 45Ca uptake and histamine release was significantly decreased in cells stimulated with antigen and phosphatidylserine (PS), while tranilast inhibition of histamine release was not affected by the addition of PS despite a significant decrease in the inhibition of 45Ca uptake. 3) The inhibitory effect of DSCG and tranilast was significantly lower in mast cells stimulated by comp. 48/80 than in the cells stimulated by antigen. 4) Tachyphylaxis was observed in cells re-exposed to DSCG and tranilast following previous exposure to the agents. 5) Cross-reactivity was found between DSCG and tranilast.


Case report: granulocyte colony-stimulating factor overcomes severe neutropenia of large granular lymphocytosis. Large granular lymphocytosis (LGL) is characterized by enhanced proliferation of T lymphocytes that have antibody-dependent cell-mediated cytotoxicity or natural killer cell activity and that often produce severe cytopenias, including neutropenia. When a 68-year-old man with seropositive rheumatoid arthritis and severe neutropenia was examined, he was found to have LGL with a T cell gene rearrangement, indicating the presence of a clonal population of T lymphocytes. The patient was admitted with a fever of 102 degrees F and a nonhealing ulcer over the right tibia. When the infection did not respond to intravenous antibiotics, granulocyte colony-stimulating factor (GCSF) therapy was started at 5 micrograms/kg subcutaneously each day. The neutrophil count promptly increased and the patient subsequently defervesced and was able to have a skin graft placed, which healed without difficulty. GCSF, which is known to be an effective therapeutic agent for neutropenia associated with chemotherapy and bone marrow transplantation, also was a very valuable treatment for the life-threatening neutropenia of LGL.


Deletion (14) (q24.3q32.1): evidence for a distinct clinical phenotype. We report on a 4-year-old girl with distinctive facial features (redundant skin, bushy eyebrows, narrow palpebral fissures, short, upturned nose, epicanthal folds, and a long upper lip with well-defined philtrum) who has an interstitial deletion of chromosome 14 including band 14q31, designated as 46,XX,del(14)(pter-->q24.3::q32.1-->qter). Comparison with previously reported patients with deletions of 14q involving band 14q31 suggests that there is a distinctive clinical phenotype associated with this deletion. Our patient had dental abnormalities (3 maxillary and 3 mandibular incisors) not described in the other patients.


A role of autoimmunity in the etiopathogenesis of Landau-Kleffner syndrome? Four children, 3 boys and 1 girl, with the syndrome of acquired aphasia Landau-Kleffner were followed up during the last 10 years. In 3 children an episodic form of the disease with a good response to corticosteroid treatment and with a favourable prognosis was observed. In the other 1 no improvement of speech function was observed and also epileptic fits were reduced only with difficulty. A positive autoimmune reaction to central (MOZAN) and peripheral (LISAN) myelin was observed repeatedly during the attacks of clinical worsening. On the contrary, during the periods of improvement of language disturbances during the treatment with corticosteroids this hypersensitivity to central as well as peripheral myelin disappeared. Possible changes in myelinization and the role of autoimmune reactions in the etiopathogenesis of the disease are discussed.


Microscopic histopathology of chronic refractory lateral epicondylitis. The histopathologic features from 11 patients who were treated surgically for lateral epicondylitis were graded and compared to similar tissue from 12 cadaveric specimens. All studies were done by a single pathologist who had no knowledge of the origin of the specimen. The surgical specimens were interpreted as abnormal in all 11 specimens, and all 12 of the control specimens were reported as being without histologic abnormality. Vascular proliferation was present in 10 of 11 and focal hyaline degeneration was recorded in all 11 of the surgical specimens. Neither feature was present in any of the control material (P < 0.001). These data suggest that chronic refractory lateral epicondylitis requiring surgery is a degenerative rather than inflammatory process. This may account for the lack of response to rest and antiinflammatory medication.


Growth and development in white patients with sickle cell diseases. We have evaluated height, weight, bone age, somatomedin-C levels, and pubertal development in 114 Sicilian patients affected by sickle cell diseases (SCDs). Thirty-one had homozygous sickle hemoglobin (SS), 55 S-beta 0 thalassemia, and 28 S-beta + thalassemia. In both children and adults, the mean height and weight were approximately 1 SD below the normal mean for age. The height was below the normal range only in a few subjects (8 children and 4 adult women). Somatomedin-C levels were within the normal range in most of the patients (37/44 children and 17/22 adults). Bone age revealed a slight delay in skeletal maturation (mean chronological age and bone age were 7.7 +/- 3 and 7.11 +/- 2.9 respectively; p < 0.05). Mean age at menarche was increased compared to normal subjects. Our findings show that Sicilian patients with SCD exhibit a moderate delay of growth and adolescence but attain a final height within the normal range.


A cystic partially differentiated nephroblastoma producing alpha-fetoprotein. A case of cystic partially differentiated nephroblastoma with immunohistochemical and serological demonstration of alpha-fetoprotein (AFP) production is described. To our knowledge, this is the first such case reported. The morphological heterogeneity of this rare tumor led us to describe the criteria of its nosological classification and of its differential diagnosis with intrarenal teratoma. AFP, besides being a marker of germline-derived tumor, was associated to this rare variant of Wilm's tumor. Although the tumor did not show regression either on clinical or pathologic assessments, serum AFP levels decreased after preoperative chemotherapy and returned to normal limits after nephrectomy.


Human basophil histamine release is differently affected by inhibitors of calmodulin, diacylglycerol kinase and peptidyl prolyl cis-trans isomerase in a secretagogue specific manner. To assess the role of calmodulin in human basophil histamine release, we triggered leukocytes with different secretagogues in the presence of putative inhibitors of calmodulin. Calcium ionophore-induced histamine release was reduced or blocked by calmidazolium, CGS 9343B, felodipine, metofenazate, and Ro 22-4839. H 186/86, a felodipine-related dihydropyridine derivative, blocked A23187-but not ionomycin-triggered histamine release, suggesting a difference in the mode of action of these ionophores. In contrast, leukocyte histamine release triggered by the purported protein kinase C (PKC) activator, 1,2-isopropylidene-3-decanoyl-sn-glycerol (IpOCOC9), was enhanced by calmidazolium, CGS 9349B and metofenazate but not affected by felodipine or Ro 22-4839, whereas the response triggered by 4 beta-phorbol 12-myristate 13-acetate (PMA) was reduced by metofenazate and Ro 22-4839 but not consistently affected by calmidazolium, CGS 9343B or felodipine. The PMA-induced histamine release was enhanced by H 186/86. Anti-IgE- and FMLP-induced responses were either unaffected or slightly enhanced by the examined calmodulin antagonists. In comparison with the calmodulin antagonists, R 59022, an inhibitor of diacylglycerol kinase, failed to reduce calcium ionophore-triggered histamine release, whereas FK506, an inhibitor of peptidyl prolyl cis-trans isomerase (PPI), reduced both anti-IgE- and ionophore-triggered responses. These results indicate that calmodulin constitutes an obligate link in signal transduction pathways leading to human leukocyte histamine release if the trigger is a calcium ionophore but not when responses are induced by anti-IgE, FMLP or PMA; a calmodulin-dependent component may rather balance responses induced by IpOCOC9.(ABSTRACT TRUNCATED AT 250 WORDS)


Cold urticaria as a model of mediator release: platelet factor 4, eosinophil cationic protein and histamine. Platelet factor 4 (PF4) has previously been linked to precipitation of cold urticaria (CU). The aim of the study was to assess the liberation of PF4, eosinophil cationic protein (ECP) and histamine after cold challenge in patients with CU. Ten controls and 8 patients with CU verified by clinical data and cold challenge test were investigated. Assessment of histamine, ECP and PF4 were done using radioimmunoassays. In patients histamine increased after 10 min on the challenged arm (NS), PF4 increase was statistically significant (p less than 0.05) both in patients and controls. ECP release showed no significant changes. Treatment with doxepin results in clinical improvement, but no changes in mediator release were seen. Thus, in contrast to previous reports an increase of PF4 was seen both in controls as well as in patients. An involvement of ECP was not ascertained. Our data suggest that neither basophils, nor eosinophils or platelets are directly involved in cold urticaria and that mast cell-dependent mediators may be of greater relevance.


Role of carbohydrate moieties in cross-reactivity between different components of Parietaria judaica pollen extract. Cross-reactivity between the different components in Parietaria judaica pollen extract has been investigated by polyclonal as well as monoclonal antibodies before and after chemical deglycosylation obtained by trifluoromethanesulphonic acid (TFMS) treatment of the extract. In western blotting a polyclonal rabbit antiserum, obtained by injecting purified Par j I, was able to recognise many components of the native extract. However, its reactivity was restricted, after chemical deglycosylation of the extract, to the major allergen alone, indicating that its cross-reactivity was due to sugar moieties. Moreover, out of several monoclonal antibodies raised by injecting the whole Parietaria judaica extract, one (1A4/2F8) was also able in western blotting to recognise an epitope shared by many components of the extract except the major allergen Par j I. However, in this case the broad reactivity of the antibody was not affected by the deglycosylating procedure. When the reactivity of Parietaria judaica extract was tested before and after sugar removal, against specific IgE from a pool of patient sera, no differences could be demonstrated, thus indicating that carbohydrates are not strongly involved in the binding of Parietaria judaica-specific IgE. The results indicate that both proteic and carbohydratic cross-reactive epitopes are shared by many components of Parietaria judaica pollen extract.


Use of [8-3H]guanine-labeled deoxyribonucleic acid to study alkylating agent reaction kinetics and stability. Alkylation at the N7 position of guanine in DNA renders the C8-hydrogen acidic. This serves as the basis for an assay of guanine N7 alkylation using [8-3H]-guanine-labeled DNA. I modified the assay by preparing a high specific activity substrate in vitro and by replacing the distillation step with charcoal adsorption of substrate. Using the appearance of noncharcoal-adsorbable label as a measure of guanine-N7 alkylation I examined the reaction of DNA with dimethyl sulfate and mechlorethamine. The rate of reaction of dimethyl sulfate with the N7 position of guanine in DNA was constant over time, i.e., loss of label from DNA proceeded linearly with time. On the other hand, the rate of reaction of mechlorethamine with DNA increased with time, consistent with the initial formation of the reactive aziridinium ion. The assay can also be used to compare the reaction rates of various alkylating agents with DNA. Thus, the acridine mustards ICR-170 and quinacrine mustard were far more potent alkylating agents than mechlorethamine. Furthermore the assay may be used to determine the alkylating potency and stability of various alkylating agent preparations: while frozen solutions of acridine mustards in organic solvents retained alkylating activity for several months, different commercial preparations of quinacrine mustard had little or no alkylating activity.


The zymogram method for detection of ribonucleases after isoelectric focusing: analysis of multiple forms of human, bovine, and microbial enzymes. A zymogram method for detection of in situ ribonuclease (RNase) activity, combined with isoelectric focusing in a thin layer of polyacrylamide gel (IEF-PAGE), has been developed. After incubation with a dried agarose film containing substrate RNA, ethidium bromide, and an appropriate reaction buffer, which was placed tightly on the top of the focused gel, sharp and distinct dark bands corresponding to RNase isoenzymes on a fluorescent background appeared under uv light. Addition of urea to the IEF-PAGE gel at a final concentration of 4.8 M permitted optimal focusing of the RNases. This method had not only a high sensitivity of less than 0.1 ng purified RNase A, but also a high band resolution compared with the immunostaining method. It was also useful for analysis of purified enzymes, including bovine pancreatic RNases and two types of human urine RNase as mammalian enzymes, and RNases T1 and T2 as microbial enzymes, as well as for detection of RNases present in crude tissue extracts, resulting in more detailed elucidation of the multiplicity of these enzymes.


Analysis of glycoform of O-glycan from human myeloma immunoglobulin A1 by gas-phase hydrazinolysis following pyridylamination of oligosaccharides. A comparative study was made on the glycoform of O-glycan from human myeloma immunoglobulin A1. By gas-phase hydrazinolysis, O-glycan was released from its hinge portion. The released oligosaccharide was pyridylaminated and separated by a two-dimensional analytical method of gel filtration and reverse-phase HPLC. Four major pyridylamino derivatives (P1-P4) were obtained. The neutral component (P4) among them was identified as Gal beta 1,3GalNAc-PA by cochromatography with an authentic standard pyridylamino sugar. The desialylation of the other components indicated the largest P1 and middle size P2 components possibly corresponded to a disialylated structure, NeuAc alpha 2,3Gal beta 1,3(NeuAc alpha 2,6)GalNAc-PA, and a monosialylated component, NeuAc alpha 2,3Gal beta 1,3GalNAc-PA, respectively. The structural assignment of P3 is still incomplete. Four similar components were also detected in bovine fetuin whose relative content (P1:P2: P3:P:4) was 16:43:19:22. The relative content (%) of P1-P4 (glycoform) in IgA1 from the healthy control was 10.1 +/- 3.3, 48.2 +/- 4.6, 7.0 +/- 2.6, and 34.7 +/- 4.5. The glycoform of O-glycan on IgA1 thus appears the same for any individual. Analysis of IgA1 myeloma protein indicated glycoforms distinct from those of the healthy controls. The relative content of these component could be classed as 2:8:0:90 (Type I, only one case designated as Kita), 5:24:3:68 (Type II, seven cases), and 9:41:5:45 (Type III, four cases). Thus, the results for IgA1 myeloma protein indicate that at least three glycoforms of O-glycan are possible for the IgA1 hinge structure. However, only one glycoform was found in the healthy controls.


Activity staining of endoglucanases in polyacrylamide gels. The endoglucanases of Penicillium funiculosum were analyzed for the presence of multiple forms using a modified version of the Congo red method. Postelectrophoretic slab gels were directly incubated in a solution of carboxymethylcellulose for a period as short as 15 min and then the activities were visualized by staining with Congo red. Ten distinct bands of clearances were obtained indicating the presence of at least as many multiple forms.


An extracellular matrix infrastructure provides support for murine secondary palatal shelf remodelling. A crucial part of secondary palate morphogenesis is the movement of the palatal shelves from an initial vertical position on either side of the tongue to a final horizontal one above it to achieve palate closure. The immunocytochemical localization of extracellular matrix (ECM) molecules in the palatal shelf during this remodelling and reorientation revealed the existence of an ECM infrastructure within the mesenchyme. The major components of this infrastructure were collagen III, fibronectin, and hyaluronate (HA). With remodelling, HA's domain within the mesenchyme was expanded, whereas those of fibronectin and collagen III became more circumscribed. The expansion of an HA-rich matrix within the mesenchyme is thought to be crucial for palatal reorientation. The results of this study suggest that, as this expansion occurs, it is modulated by collagen and fibronectin components of the ECM infrastructure. Prior to shelf remodelling, this infrastructure may be anchored by a specialized region of the midoral epithelial-mesenchymal interface and the subjacent mesenchyme which is characterized by the unique distribution of collagen III, fibronectin, and tenascin. The midoral palatal epithelium also may play a role in directing shelf expansion. This epithelial region undergoes changes in cell packing and epithelial cell layering that correlate with shelf remodelling. These changes occur concomitantly with changes in the expression of collagen III, collagen IV, and laminin within the underlying basement membrane. The localization and patterning of tenascin within the developing palate suggests that it not only contributes to the postulated anchoring structure of the midoral epithelial-mesenchymal region, but also plays a role in the determining the fate of the medial edge epithelial cells during the final stage of palate closure.


Expression of intermediate filaments and actins in human dental pulp and embryonic dental papilla. The localization of different cytoskeletal proteins (keratin, vimentin, desmin, actin, and alpha-smooth muscle actin) was examined by immunohistochemistry in normal human adult dental pulp and compared with dental papilla of tooth germs. Keratin and actin were localized in enamel organ. Vimentin and actin were observed in the dental papilla and in the adult dental pulp. Desmin and alpha-smooth muscle actin were present only in the vessel walls. These data are discussed paying particular attention to the origin and the peculiar functional characters of the dental papilla and pulp.


Single-step technique for staining Anaplasma marginale in bovine blood smears. Three available differential stains, Camco-Quik, Diff-Quik, and Wright-Giesma were compared for detection of intraerythrocytic Anaplasma marginale in bovine blood smears. In samples where < 1% to more than 51% of the RBC were infected, statistical analysis of the data indicated no significant difference in the detection of A marginale with Camco-Quik or Diff-Quik stains. However, a significantly lower percentage of infected RBC were detected when blood smears were stained with the Wright-Giemsa stain, compared with the other 2 methods.


Use of a biological extract of Serratia marcescens to decrease doxorubicin-induced myelosuppression in dogs. Fifteen dogs were given doxorubicin, IV, at a dosage of 30 mg/m2 of body surface. A commercially available biological extract of Serratia marcescens (BESM) was administered SC to 9 of these dogs (0.04 mg/kg of body weight every third day, n = 2; 0.08 mg/kg every other day, n = 2; and 0.08 mg/kg daily, n = 5), beginning the day after administration of doxorubicin, in an attempt to find an optimal dosage and schedule of administration of BESM to reduce the duration and severity of chemotherapy-induced myelosuppression. Nine additional dogs were randomized into 3 groups of 3 dogs to receive 1 of the following dosages of BESM SC: 0.08, 0.16, and 0.32 mg/kg. Serum was harvested immediately prior to treatment and at 2, 4, 6, 8, 12, 24, 48, and 72 hours from this latter group of dogs for subsequent analysis of canine granulocyte colony-stimulating factor (G-CSF) by enzyme immunoassay. Increasing the dosage and schedule of administration of BESM reduced the duration and severity of doxorubicin-induced myelosuppression. Neutrophil counts of the group of dogs given BESM daily at a dosage of 0.08 mg/kg and the controls were evaluated statistically. The neutrophil count increased significantly (P < 0.05) above pretreatment values in BESM-treated dogs after day 7. Median neutrophil counts of the BESM-treated dogs were never significantly lower than pretreatment values, whereas the median counts of the dogs treated with doxorubicin alone were significantly below normal for 6 days (days 7-12).(ABSTRACT TRUNCATED AT 250 WORDS)


Keratin and associated proteins of the equine hoof wall. In this study, we described water-insoluble proteins extracted from the germinative regions (stratum internum and coronary band epithelium) and the cornified outer surface (stratum medium) of the equine hoof wall. Two major types of polypeptides were identified: the intermediate filaments (IF) and the IF-associated proteins. The IF, including keratins, composed a major portion of this fraction, had electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the range of 40 to 80 kDa, and reacted with acidic or basic keratin-specific monoclonal antibodies. Differences in the composition of keratins between germinative layers and the stratum medium were seen. Another less well-characterized group of polypeptides associated with the IF also were extracted with the water-insoluble polypeptide fraction. These associated proteins had an apparent molecular weight between 10 and 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and contained a higher percentage of sulfur-containing amino acids than did the IF. Water-insoluble protein fractions compared favorably with those found in other less-specialized keratinizing tissue with respect to size, immunoreactivity with monoclonal antibody, and amino acid composition.


Mucus hypersecretion in bronchiectasis. The role of neutrophil proteases. To investigate the role of neutrophil proteases in the pathogenesis of mucus hypersecretion in bronchiectasis, we collected sputum samples from seven patients with bronchiectasis and measured their secretagogue activity by examining secretion of radiolabeled macromolecules by bovine airway submucosal gland cells incubated with sputum supernatants. There was marked secretagogue activity in bronchiectasis sputum, reaching a maximum of 1,963 +/- 292% (mean +/- SEM) above baseline at 1:15 dilution. Addition of ICI 200,355 (10(-5) M), a selective human neutrophil elastase inhibitor, decreased the secretory response markedly (72.53 +/- 5.89% reduction). The combination of aprotinin, an inhibitor of cathepsin G, and ICI 200,355 caused significantly more reduction in the secretory response than ICI 200,355 alone (89.12 +/- 3.8 versus 72.53 +/- 5.89% reduction, p < 0.05). We conclude that bronchiectasis sputum causes a large secretory response from tracheal submucosal glands due mostly to neutrophil proteases.


Detection of dysplasia and carcinoma in situ by ratio fluorometry. Fluorescence bronchoscopy was performed in 82 volunteers recruited from occupational groups at risk of exposure to asbestos and/or diesel fumes to determine whether differences in tissue autofluorescence between normal and malignant bronchial tissues can be used to improve the sensitivity of standard fiberoptic bronchoscopy in detecting dysplasic and carcinoma in situ (CIS). This study consisted of 25 nonsmokers, 40 exsmokers, and 17 current smokers with mean ages of 52, 55, and 49 yr, respectively. Tissue autofluorescence was induced by a blue light from an He-Cd laser coupled to the illumination channel of the bronchoscope and analyzed by a ratiofluorometer. One or more sites of moderate or severe dysplasia were found in 12% of the exsmokers and current smokers but in none of the nonsmoker volunteers. CIS was found in two of the exsmokers. The sensitivity of fluorescence bronchoscopy (86%) was found to be 50% better than that of conventional white-light bronchoscopy (52%) in detecting dysplasia and CIS. Pre- and post-bronchoscopy sputum cytology failed to detect these precancerous lesions. Our results suggest that fluorescence bronchoscopy may be an important new method that can improve the ability to detect and localize precancerous and/or CIS lesions.


Effect of inflammation of enteric nerves. Cytokine-induced changes in neurotransmitter content and release. The results of our previously published work provide evidence of inflammation-induced functional disturbances in the enteric nervous system. Data presented in this paper describe our preliminary results indicating that the altered function in enteric nerves in the nematode-infected rat model of intestinal inflammation is mediated by interleukin-1. This is based on the ability of the exogenous cytokine to mimic changes observed in the model, and on the ability of a specific IL-1 antagonist to attenuate these changes. In addition, we have identified mechanisms underlying the actions of IL-1 in the myenteric plexus. Our data are consistent with a direct interaction between the cytokine and neural membranes. In addition, the delayed effect of IL-1 beta on neurotransmitter release appears to be due to the release of endogenous IL-1, most likely from macrophage-like cells in the myenteric plexus (Fig. 3). If such cells possess receptors for neuropeptides, as has been found with macrophages elsewhere in the gut, a neuroimmune axis would exist in the myenteric plexus. Thus, the finding of a source of IL-1 in the plexus of the noninflamed intestine invites speculation on a neuromodulatory role of the cytokine within the enteric nervous system.


Analyzing mast cell development and function using mice carrying mutations at W/c-kit or Sl/MGF (SCF) loci. Mast cells have been implicated in a wide variety of biological responses, but identifying the nature and importance of the mast cell's specific contributions to these reactions has been difficult. W/Wv mice have mutations affecting the c-kit tyrosine kinase receptor which is encoded at the W locus and which is necessary for normal mast cell development. In W/Wv mice, the cells which ordinarily give rise to normal mast cell populations do not adequately respond to a major migration, survival, proliferation and maturation factor expressed in the microenvironments where mast cells ordinarily develop: the c-kit receptor ligand, SCF. As a result, W/Wv mice virtually lack tissue mast cells. However, adoptive transfer to W/Wv mice of immature mast cells derived in vitro from the bone marrow cells of the congenic normal (+/+) mice selectively repairs the mast cell deficiency of the W/Wv recipients. These "mast cell knock-in" mice can be used to analyze the expression of biological responses in tissues which differ only because they do or do not contain populations of mast cells. This approach permits identification and quantification of the specific contributions of the mast cell to biological responses expressed in the skin, gastrointestinal tract and other anatomical sites, and also greatly facilitates analysis of the mechanisms by which mast cells influence these responses.


The cortical lesion of Huntington's disease: further neurochemical characterization, and reproduction of some of the histological and neurochemical features by N-methyl-D-aspartate lesions of rat cortex. Huntington's disease is a progressive neurodegenerative disease in which the basal ganglia are preferentially affected. Recent evidence, however, suggests involvement of the cerebral cortex as well, with sparing of neurochemically defined subsets of gamma-aminobutyric acid (GABA)-ergic interneurons. In the present study, we examined changes in concentrations of the amino acid neurotransmitters GABA, glutamate, and aspartate in nine cortical regions from 23 patients with advanced Huntington's disease and 12 control brains. GABA concentrations were significantly increased in eight of the nine regions, consistent with a sparing of GABAergic local circuit neurons in the context of progressive cortical atrophy. Small but significant increases in glutamate were found in six of the nine regions, while aspartate levels were generally unaffected. Striate cortex (Brodmann's area 17) showed the most profound increases in GABA and glutamate. We also investigated the effects of powdering the excitotoxins N-methyl-D-aspartate (NMDA) or kainic acid onto the dura of rats. The resulting lesions were examined at 1 week and 6 months. The NMDA-induced lesions showed striking sparing of parvalbumin-positive neurons (a subset of GABAergic interneurons), and this sparing was reflected in neurochemical measurements of GABA; kainic acid lesions failed to display this selectivity. Somatostatin, cholecystokinin, and vasoactive intestinal polypeptide concentrations were spared by the NMDA-induced lesions, and substance P levels were significantly increased. These results provide evidence that NMDA excitotoxic lesions of cerebral cortex can produce a selective pattern of neuronal damage similar to that which occurs in Huntington's disease.


[Combined experimental use of vaccine against acute human encephalomyelitis and immunomodulators]. Combined use of vaccine and immunomodulators such as ridostin, inosiplex and polyribonate against acute encephalomyelitis of humans (AEMHs) was studied. It was shown that low immunogenic doses of the vaccine did not provide a protective action against the virus of AEMHs while after administration of the vaccine in combination with the immunomodulators there was protection in all the groups of the animals exposed to the low immunogenic doses of the vaccine during the first immunization. It was noted in regard to all the combinations of the immunomodulators and vaccine used in the low immunogenic doses that the level of the increase in the titer of the virus-specific antibodies, the proliferative activity to the specific antigen and mitogens and of interferon induction depended on the immunomodulator type. At the same time, it was found that the marked production of interferon within the first 24 hours observed after the use of the combination of inosiplex, ridostin and the vaccine resulted in increased activity of natural killer cells and lower proliferative activity of cells and production of virus-specific antibodies. This was indicative of the necessity of choosing the immunomodulators, their doses and time of the administration in relation to the immunization.


[Chemical modification of an antineoplastic antibiotic bleomycetin by the C-end fragment]. Bleomycetin, an antitumor antibiotic, was subjected to chemical modification by the C-end fragment i.e. the residue of 3-[(4-aminobutyl)amino]propylamine (spermidine++) with acylation, carbamoylation and reducing alkylation, which yielded its new semisynthetic derivatives. The use of physicochemical methods showed that the chemical modification involved the primary and secondary amino groups++ of spermidine++ and gave rise to N,N'-diacyl, N,N'-dicarbamoyl and N,N'-dialkyl bleomycetins. The biological properties of the derivatives, i.e. their cytotoxic activity, acute and pulmonary toxicities were studied. The transformation of bleomycetin by the C-end fragment lowered the antibiotic toxicity and was believed to be a promising approach to modifying its molecule.


Different distributions of lung and blood lymphocyte subsets in pediatric AIDS or tuberculosis. Pulmonary immunity has not been studied in children with acquired immunodeficiency syndrome (AIDS) or tuberculosis (TB), even though lungs of both children and adults infected with human immunodeficiency virus (HIV-1) or Mycobacterium tuberculosis are affected frequently and severely. In the present studies, the distributions of T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in bronchoalveolar lavage fluid (BALF) and blood of children with AIDS (N = 28) and children with pulmonary TB (N = 18) were determined using direct immunofluorescence (flow microfluorimetry). The distributions of lymphocyte subsets in BALF differed dramatically from those in blood. In pediatric AIDS, reduction of CD4/CD8 ratio was much more pronounced in BALF than in peripheral blood (0.15 +/- 0.04 vs. 0.43 +/- 0.11). This difference was due to selective depletion of BALF CD4+ lymphocytes, rather than to a great influx of CD8+ cells into the lung. In childhood TB, the CD4/CD8 ratio in BALF also was significantly decreased, despite its elevation in blood (1.02 +/- 0.26 vs. 1.96 +/- 0.32). The results show that (1) examination of peripheral blood lymphocytes does not reflect the kind and extent of changes observed in the distribution of pulmonary lymphocyte subsets, and (2) the profound decrease of the CD4/CD8 ratios in BALF of children with AIDS or TB is due to decreased percentages and absolute numbers of BALF CD4+ lymphocytes. The data suggest that analysis of BALF provides a more accurate evaluation of the patient pulmonary immune status than monitoring peripheral blood.


Identification of epitopes associated with different biological activities on the glycoprotein of vesicular stomatitis virus by use of monoclonal antibodies. Thirteen monoclonal antibodies (MAbs) to the glycoprotein (G) of vesicular stomatitis virus (VSV) serotype Indiana were prepared and examined for their effects on various biological activities of VSV, including in vitro infection, hemagglutination, adsorption to cells, and mediation of cell fusion. Competitive binding assays with these MAbs revealed the presence of at least seven distinct antigenic determinants (epitopes) on the G protein. In some cases, overlappings among epitopes to various degrees were observed as partial inhibition or binding enhancement. The MAbs to all the epitopes but one (epitopes 1-6) reacted with the denatured G protein in a Western immunoblot analysis. Four of the epitopes (epitopes 2, 4, 5, and 7) were involved in neutralization and two (epitopes 1 and 2) in hemagglutination inhibition. None of the MAbs inhibited the adsorption of radiolabeled VSV to BHK-21 cells; the MAbs to epitope 2 slightly enhanced the virus adsorption. All neutralization epitopes except epitope 2 (epitopes 4, 5, and 7) were associated with inhibition of VSV-mediated cell fusion. These results show a direct spatial relationship between the epitopes recognized by the MAbs and functional sites on G protein and further insights into the structure and function of G protein.


Production, characterization and use of monoclonal antibodies to grapevine virus A. Four stable hybridoma cell lines secreting monoclonal antibodies to grapevine closterovirus A (GVA) were obtained by fusing spleen cells of immunized BALB/c mice with mouse myeloma cell line Sp2/0-Ag 14. In ELISA all MAbs reacted with virus in leaf extracts from Nicotiana benthamiana, glass-house-, field-, or in vitro-grown grapevines, or with cortical shavings from mature grape canes. In IEM tests, only one of the MAbs (PA3.F5) decorated virus particles on the entire surface. This MAb was likely induced by a surface antigenic determinant, whereas the other three MAbs (PA3.D 11, PA3.C 6, and PA3.B 9) were originated by cryptotopes. Coupling polyclonal antibodies for coating protein A-sensitized plates, and monoclonal antibody conjugates for antigen detection, gave highly efficient and reproducible results for identification of GVA in field-grown grapevines.


Decondensation of human sperm nuclei and HP1 protamine degradation from normospermia and asthenospermia in Xenopus egg extracts. The process of human sperm decondensation has been studied in vitro in cytoplasmic extracts prepared from unfertilized Xenopus laevis eggs. The chromatin decondensation-recondensation cycle was divided into four stages according to chromatin appearance. Spermatozoa from normospermia and asthenospermia were evaluated according to their capacity to reach these stages, and their DNA integrity was assessed by acridine orange (AO) staining. We observed a significant difference between normospermia and asthenozoospermia in the ability to achieve the cycle of chromatin decondensation-recondensation. These results correlated with AO staining. The role of human protamine 1 degradation in the decondensation process was evaluated by immunostaining. It was found not to be a prerequisite for the earlier stage of chromatin decondensation and it was not implied in the latest stages of pronuclear development.


Neutralizing-enhancing monoclonal antibody recognizes the denatured glycoprotein of viral haemorrhagic septicaemia virus. Two monoclonal antibodies (MAbs), 1H10 and 1F10, have been selected against the denatured glycoprotein of the viral haemorrhagic septicaemia virus (VHSV) of salmonids by immunoblotting. Three reference VHSV serotypes (F1, F2, 23.75) and five VHSV isolates from either different host species (trout, salmon, barbel) or geographical locations in Spain reacted with both MAbs by ELISA. In vitro neutralization of all VHSV serotypes but not infectious haematopoietic necrosis virus (IHNV) was obtained only with MAb 1H10. However, when the MAb 1H10 (but not the non-neutralizing Mab 1F10) concentration was below 100 micrograms/ml rather than neutralizing VHSV infectivity, plaque counts increased 2-3-fold. MAb 1H10 is unique in that it has both, an in vitro enhancing infectivity effect (not described yet for VHSV) and it recognizes denatured G protein in contrast to other previously described neutralizing MAbs against VHSV.


Topographical analysis of the G virion of Aleutian mink disease parvovirus with monoclonal antibodies. The topography of the Aleutian mink disease parvovirus (ADV) G virion was analyzed with monoclonal antibodies and polyclonal antiserum. There was homology between the two major structural proteins as others have previously reported. Trypsin treatment of the virion with subsequent immunoblotting revealed that VP2 represents the main peptide on the exterior of virion and that VP1 is probably embedded within the capsid. Additional analyses of the trypsin-treated virions showed that VP2 is responsible for binding complement and that it also represents the structural part of the virion that binds to cellular receptors. A third protein, p34, was detected that might represent a third structural polypeptide because of its many unique epitopes relative to the other peptides detected.


Genetic characterization of an antigenic subtype of eastern equine encephalomyelitis virus. A 1983 human Mississippi isolate of eastern equine encephalomyelitis virus (EEEV), recently identified as an antigenic subtype of the North American variety, was genetically characterized using oligonucleotide fingerprinting and sequencing of viral RNA. This strain was found to be very closely related to other North American EEEV isolates from the same time period. Phylogenetic analysis suggested that this subtype belongs to a single EEEV lineage in North America. Two amino acid substitutions in the E2 envelope glycoprotein, not seen in either other isolates sequenced, probably contributed to the antigenic difference with respect to other EEEV strains. These substitutions include threonine for lysine at position 71, resulting in the addition of a potential N-linked glycosylation site, and lysine for glutamic acid at position 147.


Human rotavirus strain with unique VP4 neutralization epitopes as a result of natural reassortment between members of the AU-1 and Wa genogroups. Human rotavirus strain K8, which possesses unique VP4 neutralization epitopes, was examined by RNA-RNA hybridization to determine its genogroup. While it possessed four gene segments that formed hybrids with strain Wa (a prototype of the Wa genogroup), strain K8 possessed seven gene segments, including gene segment 4, that formed hybrids with strain AU-1 (a prototype of the AU-1 genogroup) which has been shown to share a unique gene 4 allele with feline rotaviruses. These results suggest that strain K8 is an intergenogroup reassortant formed in nature between a member of the Wa genogroup and a member of the AU-1 genogroup.


Objective evaluation of the quality of palliation in patients with oesophageal cancer comparing surgery, radiotherapy and intubation. The objective of this study was to prospectively evaluate the quality of palliation of 103 patients presenting to a joint oesophageal cancer clinic while recording the outcome in terms of treatment, morbidity, mortality and long-term survival. Twenty-five patients underwent surgical resection (S), 22 radical radiotherapy (RR), 30 palliative radiotherapy (PR), 13 intubation (I) and 13 had no treatment (NT). The quality of palliation was quantified by plotting a score out of 100 on a graph at each visit for Karnofsky performance, severity of pain and swallowing ability, then calculating the area under each curve created using an algorithm, Simpson's discrete approximation. Efficiency of palliation was estimated by comparing the area calculated to the maximum that could be achieved during the time frame being studied. The incidence of stricture (benign and malignant) was 16% after surgery and 50% after radical radiotherapy. Treatment mortality was as follows: RR, 0; S, 1 (4%); PR, 3 (7%); and I, 0. The median survival was 26 months after surgery and 16 months after radical radiotherapy. It was 6 months for palliative radiotherapy, 4 months for intubation and 4 months for no treatment. The difference in swallowing was the only statistical difference in the quality of palliation of patients having surgery and radical radiotherapy, there being no differences in patients having palliative measures.


Learning of a hippocampal-dependent conditioning task changes the binding properties of AMPA receptors in rabbit hippocampus. The N-methyl-D-asparate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subtypes of glutamate receptors have been shown to play critical roles in various forms of synaptic plasticity (i.e., learning and memory, long-term potentiation). We previously demonstrated that the binding of [3H]AMPA to the AMPA subtype of glutamate receptors was selectively increased in hippocampus following classical conditioning of the rabbit nictitating membrane response in a delay paradigm. We report here that the same effect was observed in a variant of this learning paradigm that requires the participation of the hippocampus, i.e., trace conditioning of the rabbit nictitating membrane. The binding of [3H]TCP (N-[1-(2-thienyl)cyclo-hexyl]-3,4-piperidine) to the NMDA receptor remained unchanged in all the experimental groups tested. Paired presentations of conditioned and unconditioned stimuli resulted in an increased binding of [3H]AMPA, an agonist of the AMPA receptors, in several hippocampal subfields while the binding of an antagonist, [3H]CNQX (6-nitro-7-cyanoquinoxaline-2,3-dione), was decreased. The results suggest that the learning-induced changes in binding of the ligands to the AMPA receptor reflect changes in affinity of the receptor rather than in the number of sites. These results support the hypothesis that changes in hippocampal glutamate receptors are a corollary of synaptic plasticity in certain forms of learning.


A rapid technique for visualizing cerebral arteries in rat. Intracardiac perfusion with cresyl violet acetate dissolved in 0.9% isotonic saline and dimethyl sulfoxide (DMSO) allows one to visualize the arterial blood supply to the central hemispheres. This technique is especially useful in demonstrating the ramifications of and areas supplied by the middle cerebral artery. Many individual differences in the course traversed by the artery and the distal areas supplied were observed. This technique could prove to be a useful adjunct to neurohistological techniques in studies investigating rodent models of stroke.


Anti-caldesmon monoclonal antibody reverses the inhibition of actomyosin Mg(2+)-ATPase activity by caldesmon. Five mouse monoclonal antibodies, CaD 1-5, against chicken gizzard caldesmon were prepared. One of them (CaD4) was characterized by means of immunoblotting and its effect on actomyosin Mg(2+)-ATPase activity. CaD4 recognized the tropomyosin-binding site of caldesmon. CaD4 reversed the caldesmon-induced inhibition of actomyosin Mg(2+)-ATPase activity in a dose-dependent manner. These results suggest that the epitope recognized by CaD4 is an important domain for the function of caldesmon on the actinmyosin interaction in the smooth muscle contraction-relaxation system.


Enhanced expression of type 2C protein phosphatase gene during myogenic differentiation of C3H10T1/2 cells. Type 2C phosphatase is one of the four major protein serine.threonine phosphatases (types 1, 2A, 2B and 2C). The mRNA level of the enzyme protein was 11.5 times higher in mouse skeletal muscle than in undifferentiated mesenchymal cells (C3H10T1/2). The mRNA level was enhanced 4.2-fold in accordance with the differentiation of C3H10T1/2 cells into myoblasts induced by transfection with a MyoD1 expression vector, and this high level was maintained in terminally differentiated myotubes. Furthermore, type 2C phosphatase activity in both myoblast and myotube fractions was slightly higher than in control C3H10T1/2 cells. These results indicate that the expression of type 2C phosphatase gene is enhanced during the course of myogenic differentiation.


Platelet cAMP and cGMP in essential hypertension. Vasodilator substances released in the blood vessel wall, such as the endothelium-derived relaxing factor (EDRF) and prostacyclin (PGI2), may participate in the regulation of arterial blood pressure. However, their role in the pathogenesis of human essential hypertension to date remains unclear. For some of these factors affecting vascular smooth muscle cells, blood platelets represent a second target tissue. Thus, EDRF and PGI2 inactivate platelets by stimulation of cyclic guanosine-5'-monophosphate (cGMP) and cyclic adenosine-5'-monophosphate (cAMP) synthesis, respectively. In the present study, platelet cAMP (n = 68) and cGMP (n = 60) were determined in a control group of healthy subjects (C) and in 12 patients with untreated essential hypertension (EH). In the control group, platelet cAMP and cGMP content averaged 13.52 +/- 0.38 and 1.48 +/- 0.06 pmol/10(9) platelets and no dependence of either variable on sex or age could be established. Furthermore, cGMP levels were similar in EH as compared to the control group (1.38 +/- 0.11 pmol/10(9) platelets). However, intracellular concentrations of cAMP were significantly lower in EH as compared to C (11.22 +/- 1.37 pmol/10(9) platelets; P < .01). In addition, we investigated the stimulatory effect on cAMP of the stable PGI2 analog iloprost (10(-9), 5 x 10(-9), 10(-8), 5 x 10(-8) mol/L) in the platelets of 12 control subjects (C12) and EH.(ABSTRACT TRUNCATED AT 250 WORDS)


Ultrastructural study of the choroid plexus of spontaneously hypertensive rats. We previously gave an account of an increased ion transport activity in choroid plexus from spontaneously hypertensive rats. We have since examined this organ in scanning and transmission electronic microscopy. In the choroid plexus from young spontaneously hypertensive rats, the epithelial cells showed the following: a partial loss of the brush border and infoldings of basolateral membranes, an increased number of Golgi apparatus, vesicles, and mitochondria, and an activated nucleus. In adult hypertensive rats, the mitochondria had increased in number and tended to fill the cytoplasma while the nuclei had returned to a resting level. These ultrastructural changes furthermore suggest an increased secretory activity in the choroid plexus in spontaneously hypertensive rats.


Three essential promoter elements mediate tumour necrosis factor and interleukin-1 activation of the granulocyte-colony stimulating factor gene. Granulocyte-colony stimulating factor (G-CSF) is a haemopoietic growth factor produced by mesenchymal cells but not T lymphocytes after stimulation with specific cytokines or mitogens. A 330 bp promoter fragment of the human G-CSF gene induced reporter gene expression in human embryonic lung fibroblasts in response to tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta). The same promoter fragment was not active in Jurkat T cells nor did it respond to phorbol ester in either cell type. At least three distinct elements, the CK-1 sequence, a decanucleotide present in haemopoietic growth factor genes, an NF-IL-6 consensus sequence and a consensus octamer sequence, were essential in the G-CSF promoter for TNF-alpha and IL-1 beta response. Mutation of any of these sequences abolished promoter function. In contrast, mutation of two other consensus protein binding sequences, i.e. a Pu-1 site and a CK-2-like sequence, did not eliminate promoter function. Both the CK-1 and octamer sequences acted independently as TNF-alpha and IL-1 beta responsive elements upstream of a heterologous promoter. The response of the octamer sequence and the 330 bp promoter but not the CK-1 sequence was greater with IL-1 beta than TNF-alpha reflecting a similar response of the endogenous gene.


Immunodominant epitopes of HIV-1 p17 and p24. Immunodominant antibody-binding sites were mapped using overlapping synthetic peptides of the structural proteins p17 and p24 of human immunodeficiency virus type 1 (HIV-1). Using sera from HIV-1-infected individuals at a variety of disease states, three major epitopes were identified within p17 and one within p24. Antibodies which recognized these epitopes were present in all risk groups throughout all stages of HIV infection, regardless of the presence of high levels of serum p24 antigen.


Mapping of linear B-cell epitopes on the major core protein p24 of human immunodeficiency virus type 1 (HIV-1). Three murine monoclonal antibodies (MAbs) F5-2, F5-4, and F5-16 defining three different epitopes on the major core protein p24 of the human immunodeficiency virus type 1 (HIV-1) were epitope mapped using a random fragment expression library representing the p17- and p24-encoding part of the gag open reading frame. F5-2 defined an epitope within amino acids (aa) 14-23 at the N-terminus of p24, and F5-4 defined an epitope within aa 153-174 in the C-terminus of p24. F5-16 did not recognize any of the fusion proteins produced by the expression library indicating that this MAb defines a true conformational epitope on p24. Since the N-terminus of p24 has been reported to be immunosilent in humans, 356 HIV-1 antibody-positive serum samples were tested for reactivity against the region of p24 defined by F5-2. More than one third of the samples recognized this region indicating that it is immunoreactive and, further, the presence of antibodies against this region was associated with a reduced CD4 cell count.


Direct observation of reverse transcriptases by scanning tunneling microscopy. First images on a nanometer scale of reverse transcriptases (RT) of the human immunodeficiency virus (HIV-1) and of the Moloney murine leukemia virus (MuLV) obtained by scanning tunneling microscopy (STM) are reported. The common feature of the observed molecules is a ring-type or horseshoe shape with hole diameters of approximately 30 A. The STM images are compared with high resolution transmission electron microscopy (TEM) and existing structure predictions. The similarities of the structural data obtained by STM and TEM and their agreement with the structure prediction for the RT of HIV-1 shows the principal possibility to image such biomolecules by STM.


[Multicentric study of 143 cases of resection of the proximal carpal bones]. Proximal row carpectomy is an old procedure but the good results published in the literature, it still has bad reputation. The authors conducted a retrospective multicentric study in 143 patients, 128 of whom were post-traumatic cases. And results were good in terms of pain relief. Increase in grip strength was observed in 80% of cases. Wrist range of motion was not improved by resection. The results were not predicted by preoperative status. No prognostic factor could be defined on the basis of study.


[Operative technique of arthroplastic resection of the three proximal bones]. Proximal row carpectomy is a controversial operative technique for difficult wrist problems. Based upon resection of bones, it represents in fact a reconstructive technique of a new joint. The majors steps of the technique are described in detail.


[Resection of the proximal carpal bones in the sequelae of scaphoid fractures]. Proximal row carpectomy has precise indications in cases of severe sequelae of scaphoid fractures. This procedure may be indicated either as first-line treatment in a case of advanced pseudarthrosis with osteoarthritis, particularly radio-scaphoid, or secondarily after scaphoid pseudarthrosis has been unsuccessfully treated by other methods. It is essential to make sure that the head of the capitatum and the radial surface of the lunate bone have a satisfactory appearance on plain X-rays, MRI and occasionally arthroscopy. The overall results of this operation which retains wrist movements and which also preserves a good grip strength are very encouraging, as demonstrated by the series of 48 cases of sequelae of scAphoid fractures treated by this technique.


[Resection of the proximal carpal bones versus partial arthrodesis in carpal instability]. Ten resections of the proximal row of carpal bones and 42 partial arthrodese were performed for carpal instability. Whenever possible, the authors prefer the second operation. This choice is often made during the operation and essentially depends on the presence of radio-scaphoid osteoarthritis and the condition of the cartilage of the head of the capitatum.


[Emergency resection of the proximal carpal bones]. Nine cases of emergency proximal row carpectomy are analysed. In 3 cases, the resection was performed in the context of transcarpal amputation in which the bone shortening obtained by resection allowed revascularisation without a flap. The functional result was poor because of the severity of the initial lesion. In the other 6 cases, consisting of proximal row bone defects or so-called intreatable fracture-dislocations, the results in terms of pain, range of movement and strength were similar to those obtained with elective operations.


[Wrist arthrodesis: alternative to resection of the proximal carpal bones?]. The results of a series of 36 post-traumatic radio-carpal arthrodeses were compared with those of the series of resection of the first row of carpal bones reported during the round table. The advantages and disadvantages of both procedures were discussed. Arthrodesis results in: 1) loss of grip strength in all cases, averaging 41%; 2) persistent pain in 78% of cases; 3) impairment of function resulting from blocking of the wrist. After the procedure, average time off work is 15 months, level of permanent disability was 30%, and 20% of patients returned to their previous jobs. These figures could be used as arguments against arthrodesis. However, they must be taken in context: final arthrodesis, usually performed on manual workers, is often chosen when resection of the first row of carpal bones would obviously be insufficient, for example, when severe arthritis of the head of the capitate is present.


[Denervation of the wrist].

[Denervation of the wrist]. Fifty patients with wrist arthritis mainly secondary to a scaphoid pseudarthrosis (44%) or a Kienböck disease (22%) were operated on by one surgeon by "total" denervation. With a post-operative follow up of 5 years, pain, strength and range of movement were reviewed. Four complications are by means of stressed: one painful neuroma and three radial nerve paresthesiae. 72% of the patients had pain improvement with an average of 72 points on a scale of 100 points. Strength and range of movement were not significantly improved.


[Experimental proximal carpectomy. Biodynamics]. Proximal carpectomy was performed in 10 fresh cadavre wrists. Dynamic x-rays were taken and the forces necessary to obtain different movements before and after the operation were measured. Comparison of these parameters clearly defines the advantages and limitations of carpectomy and indicates the reasons.


[Biomechanic considerations in wrist prostheses]. At the present time, in disorders of the wrist, avulsion of the first carpal row is the most commonly used technique as a last resort. However, there are many wrist prostheses, which are reviewed here. Roughly, they belong to two families: the three axis prostheses (spherical) that cannot transmit to the hand the pronation supination torque, because of their geometrical characteristics, and the two axis prostheses (universal joint) that are able to transmit this movement. The characteristics of future prostheses must include: based on the "universal joint" principle, occupy minimum space, isometric, maintain tendon tension, an axis identical to the true axis of the wrist, to maintain the hand in line with the forearm, fixed without cement but, not shortened with time, possibilities of mechanical flexibility immediately and lastingly stable, to be easily replaced modularly. This ideal prosthesis will certainly exist one day and will take the place of the first carpal row avulsion. In the meantime, this technique will still have a long use.


[Dorsal tenolysis and arthrolysis of the proximal interphalangeal joint. 19 cases]. 18 patients suffering from PIP stiffness in extension were treated by dorsal teno-arthrolysis. PIP joint stiffness was due to 3 cases of fracture of P1, 2 cases of fracture of P2, 2 cases of extensor tendons, 8 fractures of P1 associated with section of the extensor tendon, 2 cases of PIP closed trauma (1 sprain, 1 dislocation), and 2 crush injuries of the extensor tendon at the PIP joint. The surgical technique combined a dorsal sinuous approach, tenolysis of the extensor tendon on the dorsum of first phalanx dorsal capsulotomy, and more rarely (3 cases), section of collateral ligaments. The mean pre-operative active flexion was 44 degrees and the mean post operative active flexion was 78 degrees, which represents a gain of 34 degrees of active movement. Thanks to a classification which incorporates the range of active flexion in PIP joint, and the lock of active extension we rated 3 results as excellent, 4 as good, 8 as useful and 4 as insufficient. We therefore improved the range of movement in 15 out of 19 cases, which seams very encouraging.