Distribution of 72-kDa heat-shock protein in rat brain after hyperthermia.

The distribution of the 72-kDa heat-shock protein (hsp72) in rat brain, 24 h following in vivo transient hyperthermia (41.5 degrees C, 15 min), was studied using immunohistochemistry (n = 22). Tissue sections were also stained with hematoxylin and eosin, and with an anti-glial fibrillary acidic protein to evaluate neuronal and astrocytic response to transient hyperthermia, respectively. hsp72 was observed in glia and endothelial cells throughout brain. hsp72 was also found in neurons located in the: dentate gyrus, habenula, and hypothalamus, granular layer of the cerebellum and the olfactory area. Our data indicate, that hyperthermia causes neuronal expression of hsp72, particularly in cerebral neuronal populations which control the neuroendocrine stress response.


In vivo motor effects of loperamide on the rat urinary bladder.

Bladder motility recordings were performed in anaesthetized rats and the effect of the peripherally active opiate agonist loperamide on urinary bladder function was studied. Regional intra-arterial administration of loperamide (0.01-2 mg kg-1) induced weak bladder contraction per se. Loperamide caused an effective dose-dependent inhibition of bladder motility induced by regional injection of the receptor agonists acetylcholine (ACh) and substance P (SP), as well as by peripheral motor nerve stimulation (PNS). Pretreatment with naloxone (0.5 mg kg-1) partially antagonized the inhibitory action of loperamide on the nerve-mediated detrusor contraction. However, the depression of the motor responses induced by the receptor agonists ACh and SP was not influenced. It is suggested that the demonstrated inhibitory effect of loperamide on bladder motility is partially mediated by peripheral opioid receptors. The main non-opioid part of the inhibition might be a direct smooth muscle action.


[Can thermal therapy of ankylosing spondylitis induce an activation of the disease?].

A randomized, open, controlled study was carried out in eight patients with spondylitis ankylosans, in a cross-over procedure including total body cryotherapy and whole spine paraffin mud packs. The effect of both therapies on clinical status and different laboratory data was investigated. No clinical changes were seen after either therapy; in contrast, in answering the spine function questionnaire patients reported an improvement after cryotherapy and a worsening after thermotherapy, but because of the small number of patients no positive conclusion was possible. The acute phase proteins (alpha-1-AGP, alpha-1-ACT, alpha-2-haptoglobin and alpha-2-coeruloplasmin) did not show any changes, only CRP increased after thermotherapy and declined after cryotherapy. Interesting changes were seen in the glycosylation profile of alpha-1-AGP: after thermotherapy an increase of bi-antennary oligosaccharides occurred, observed as appearance of a third peak in the affinity immunoelectrophoresis with con A and an increase of reactivity-coefficient of alpha-1 and glycoprotein which was also seen in the exacerbation of AS. This shows, that an activation of the inflammatory process may be possible by thermotherapy (perhaps because of an amplification of Il-6 production), but not by cryotherapy. Similar studies should be conducted in larger numbers of patients.


Reduced expression of CD20 antigen as a characteristic marker for chronic lymphocytic leukemia.

The surface antigens expressed by the cells of chronic lymphocytic leukemia (CLL) are well known. Most CLL are monoclonal B-cell lymphoproliferative disorders characterized by the coexpression of B-cell antigens and CD5, an antigen present predominantly on T cells. Very little attention, however, has been paid to the quantitative characteristics of the expression of B-cell antigens in CLL. In this study, we used flow cytometry to analyze the expression of CD20, a well-known B-cell-associated antigen, in lymphocytes from 42 cases of CLL and its tissue counterpart, small lymphocytic lymphoma (SLL), and compared the results with results obtained from the analysis of 21 follicular lymphomas, 20 hyperplastic reactive nodes, and 26 samples of normal peripheral blood. The intensity of CD20 expression in the CLL/SLL cells was significantly lower than that of B cells in the other categories. This antigen expression abnormality does not appear to be a universal phenomenon in CLL/SLL, since CD19, another pan-B antigen, was expressed in CLL/SLL at levels higher than those in follicular lymphomas and comparable to those in reactive lymph nodes. These results indicate that the low CD20 expression can be used as a marker for CLL/SLL. The few cases exhibiting intense CD20 expression may represent a biologically different disease. CLL/SLL cells faintly expressing CD20 also show concomitant low CD5 expression in a manner not observed in normal CD5-expressing B cells.


Interleukin-5 in eosinophilic gastroenteritis.

During a 4-year period a 28-year-old female had 4 episodes of eosinophilia of over 10,000/mu 1; these episodes were associated with nausea, vomiting, diarrhea, and abdominal pain. On one occasion, she had ascites and pleural effusion which contained numerous mature eosinophils. On each occasion, these attacks disappeared within several weeks without any specific treatment. A diagnosis of eosinophilic gastroenteritis was made. A plasma sample obtained during the eosinophilia generated in vitro eosinophilic colonies when added to granulocyte/macrophage-progenitor (CFU-GM) cultures without exogenous growth factors. Colony formation was inhibited by anti-interleukin-5 (IL-5) antibody but not by antibodies toward IL-3, granulocyte colony-stimulating factor (G-CSF) or GM-CSF. A high plasma interleukin-5 (IL-5) level was noted when measured by enzyme-linked immunosorbent assay, while IL-3, G-CSF, and GM-CSF were undetectable. During remission the plasma gave negative results both for colony formation and IL-5 level. These results indicate that the eosinophilia of this disease is mediated by IL-5.


Beta-thalassemia intermedia with exceptionally high hemoglobin A2: relationship to mutations in the beta-gene promoter.

Small deletions of the 5' portion of the beta-globin gene that remove the promoters but stop 3' to the delta-globin gene are recognized as the sole cause of beta-thalassemia with exceptionally high hemoglobin A2 (HbA2) levels. Two patients with beta-thalassemia intermedia and exceptionally high levels of HbA2 (10.4 and 12.0%) were examined. One patient was a combined heterozygote for the -88 C----T and a novel -87 C----A mutation, while the other was homozygous for the -29 A----G beta(+)-thalassemia mutation. The remainder of the beta genes were normal. There was no evidence for deletions involving the 5' portion of the beta gene or the region between the beta and delta genes. Gene mapping studies excluded the possibility of a beta delta-anti-Lepore hemoglobin gene with beta promoters and delta coding sequences. There were no mutations in the promoters of the G gamma or A gamma-globin genes that have been associated with the hereditary persistence of HbF phenotype. The delta-globin gene promoters were normal from codon 17 to position -145 relative to the mRNA capping site. There appears to be considerable heterogeneity of HbA2 and HbF levels in patients who are homozygous or mixed heterozygotes for mutations in the TATA box and other promoter elements of the beta-globin gene. The capacity for proteolysis within the erythrocyte may vary among individuals. The authors hypothesize that in the exceptionally high HbA2 beta-thalassemia intermedia phenotype, proteolysis of superfluous alpha-globin chains is less efficient than in patients with customary levels of HbA2.(ABSTRACT TRUNCATED AT 250 WORDS)


Immunohistochemistry in the differential diagnosis of nodular hidradenoma and glomus tumor.

The histologic distinction between nodular hidradenoma and glomus tumor is an occasional difficult diagnostic problem. Both tumors may show circumscribed aggregates of uniform epithelioid cells, a myxoid stroma, and variable numbers of blood vessels. Especially troublesome are solid cellular hidradenomas without duct-like structures and glomus tumors without a vascular pattern. To develop an immunohistochemical profile useful in this differential diagnosis, 25 selected skin tumors and four normal glomus bodies were studied with antibodies against low molecular-weight cytokeratin (CAM 5.2), epithelial membrane antigen (EMA), carcino-embryonic antigen (CEA), S-100, and vimentin (VIM). The tumors included eight unequivocal hidradenomas, seven unequivocal glomus tumors, and 10 histologically equivocal cases, originally diagnosed as glomus tumors. In all unequivocal glomus tumors and glomus bodies, only VIM was positive. Of the eight unequivocal hidradenomas, three were positive for CAM 5.2, EMA, CEA, S-100, and VIM; two for CAM 5.2 only; one for CAM 5.2, EMA, and S-100; one for CAM 5.2, EMA, and CEA; and one for CEA only. In the histologically equivocal cases, eight were positive for VIM only, characteristic of glomus tumor; and two were positive for CAM 5.2, EMA, CEA, S-100, and VIM, and were reclassified as hidradenomas. The study suggests that morphologic criteria may not always accurately differentiate between hidradenoma and glomus tumor and that in equivocal cases immunohistochemistry may be useful in the differential diagnosis.


Porokeratotic eccrine ostial and dermal duct nevus. An abnormally keratinizing epidermal invagination or a dilated, porokeratotically plugged acrosyringium and dermal duct?

Porokeratotic eccrine ostial and dermal duct nevus (PEODDN) has been said to represent a widely dilated, keratin-plugged acrosyringium and dermal duct. We have observed in a case of congenital PEODDN a normal-appearing, acrosyringium-like duct that traverses vertically the entire length of the parakeratotic column. Also, in its lower course, it stained positively for carcinoembryonic antigen, while the inner borders of the invagination from which the parakeratotic column arose stained negatively. This leads us to suggest that the epithelial structure in PEODDN is an abnormally keratinizing epidermal invagination through which an acrosyringium-like duct traverses, rather than an abnormally dilated, parakeratotically plugged acrosyringium and dermal duct.


The nucleotide sequence of boar transition protein 2 (TNP2) cDNA and haploid expression of the gene during spermatogenesis.

A cDNA clone coding for boar transition protein 2 (TNP 2) was isolated from a randomly primed cDNA library of boar testis. Sequence analysis revealed an open reading frame of 414 bp (corresponding to 138 amino acids), 33 bp of the 5' untranslated and about 300 bp of the 3' untranslated region. As compared to TNP 2 of mouse and rat, similarity with TNP 2 of the boar is approximately 70% at the nucleotide level and only about 40% on the basis of amino acid sequence. The similarity between boar and bull TNP2 is 77% and 64%, respectively. Northern blot experiments with RNA of different boar tissues and in situ hybridization on mature boar testis sections revealed testis-specific expression of the TNP 2 gene which is restricted to haploid germ cells. Hybridization experiments of boar TNP2 cDNA with testicular RNA of boar, bull, rat and mouse revealed decreasing intensities of the hybridization signals. With human testicular RNA no hybridization could be obtained.


Effects of a DNA-specific fluorochrome, Hoechst 33258, on mouse sperm motility and fertilizing capacity.

DNA specific fluorochrome (Hoechst 33342 and 33258) as non-toxic stains, have been widely used to measure cell density and proliferation, detect sperm-egg fusion, and observe the development of pre-implantation embryos. It has been reported that Hoechst 33342 at a concentration of 10 micrograms ml-1 had significant inhibition on embryo cleavage. In this study, we incubated B6D2F1 mouse sperm and eggs with different concentrations of Hoechst 33258, 0, 1.0, 10.0, 20.0, 100 micrograms ml-1. We found that: (1) 100 micrograms ml-1 of H-33258 significantly decreased the sperm motility at 90 min and 4 h. (P less than 0.05), (2) 20 micrograms ml-1 and 100 micrograms ml-1 of Hoechst 33258 significantly inhibited mouse fertilization in vitro (P less than 0.05), and (3) 1.0 micrograms ml-1 and 10.0 micrograms ml-1 Hoechst 33258 had no effect on fertilization rate. But when we pre-incubated sperm at 10 micrograms ml-1 Hoechst 33258 for 90 min, and inseminated oocytes in the medium with same concentration of Hoechst 33258, the embryo cleavage was significantly inhibited.


Sperm plasma membrane integrity in fertile and infertile men.

Sperm plasma membrane characteristics were analysed by a combined method, the HOS-eosine test (HOS-E test), that consists of the hypoosmotic swelling test (HOS test), and the eosine-Y staining. Semen samples were categorized into four groups (Normo-, Oligo-, Astheno-, and Oligoasthenozoospermic) and subjected to the standard analysis (spermiogram), HOS test, eosine-nigrosine test (reflecting sperm viability); and HOS-E test. HOS-E test makes it possible to distinguish four groups of spermatozoa: type 1, HOS+/eosine-; type 2, HOS-/eosine-; type 3, HOS-/eosine+; and type 4, HOS+/eosine+. Normozoospermic samples showed 61.2 +/- 1.4% type 1, 9.2 +/- 0.8% type 2, 22.6 +/- 1.1% type 3, and 6.8 +/- 0.6% type 4 spermatozoa. Oligozoospermic samples showed no significant differences in these values, whereas asthenozoospermic samples showed a higher percentage of types 3 and 4 and a lower percentage of type 1. Oligoasthenozoospermic samples showed high percentages of types 2, 3, and 4 and a low percentage of type 1. Sperm plasma membrane integrity is a necessary condition for motility and fertilization. So, it is not surprising that semen samples with abnormal motility showed a HOS-E result indicative of a defective plasma membrane.


Treatment of angioimmunoblastic lymphadenopathy (AILD)-type T-cell lymphoma using prednisone with or without the COPBLAM/IMVP-16 regimen. A multicenter study. Kiel Lymphoma Study Group.

To describe the clinical course of patients with angioimmunoblastic lymphadenopathy (AILD)-type lymphoma with a sequential treatment with prednisone and COPBLAM/IMVP-16. A multicenter, prospective, nonrandomized trial. University medical centers and community hospitals. Sixty-seven patients were registered, 28 were excluded, and 39 patients were evaluable for response (median age, 59 years; range, 25 to 82 years) (stages I and II, 10%; stages III and IV, 90%; B symptoms, 74%). Response, survival, and relapse. Patients initially received prednisone and no further treatment if a complete remission was achieved. Relapsing or refractory patients were treated with COPBLAM/IMVP-16. Patients with life-threatening tumor progression or extension received COPBLAM/IMVP-16 initially. Treatments were chosen in accordance with tumor extension and response to prednisone. Treatment modalities were not compared. Twenty-eight patients received primary prednisone, 18 received secondary prednisone, and 11 received primary chemotherapy. The complete response rates (with 95% CIs) were 29% (CI, 12% to 46%), 56% (CI, 33% to 79%), and 64% (CI, 36% to 92%), respectively. The median observation time of surviving patients was 28 months (range, 7 to 53). The median overall survival time was 15 months. The probabilities (with 95% CIs) of overall survival, event-free survival, and relapse at 36 months were 40.5% (CI, 24% to 56%), 32.3% (CI, 17% to 47%), and 34.6% (CI, 14% to 56%), respectively. At the time of evaluation, 22 of 39 patients had died, 7 of noninfectious complications and 14 of infections. Prednisone with or without COPBLAM/IMVP-16 treatment in AILD-type lymphoma leads to complete remissions in about half of the patients and in long-term, disease-free survival for one third.


Placental intravillous accumulation of sulfated mucosubstances. A reevaluation of so-called hydropic degeneration of villi.

A histochemical study was performed on 46 placentas showing so-called hydropic degeneration of villi. The 46 cases included early abortuses (35 cases), incomplete moles (four cases), and complete hydatidiform moles (seven cases). All the specimens showed diffuse, positive staining of the distended villous stroma with alcian blue at pH 1.2 and 2.5 in the areas containing "hydropic changes." The alcian blue positivity was abolished following digestion with hyaluronidase. These findings indicate that so-called "hydropic degeneration of villi" represents an intravillous accumulation of strongly sulfated mucosubstances rather than the result of the accumulation of water. The intravillous accumulation of mucosubstances most likely represents a nonspecific stromal reaction of the connective tissue of the placenta to a variety of noxious stimuli. This finding may have some bearing on the interpretation of the physiologic mechanisms involved in placental villous distention, which has been largely centered in the past on the premise that the villous swelling was related to hemodynamic alterations caused by the accumulation of water.


Prostate specific antigen and prostatic acid phosphatase measurements for the follow-up of patients with prostate cancer.

The clinical application of 84 prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) measurements for the follow-up of 36 patients with treated prostate cancer was retrospectively examined by case study. Clinicians use several risk markers including serum levels of PSA and PAP to monitor prostate cancer progression or stability. These results of PAP and PSA tests were either utilized during the patient's clinical assessment or they were disregarded. In either case, the results would support or counter the physician's clinical decision for patient management. After predictive value analysis it was concluded that measurement of PSA alone is more useful than parallel measurements of PSA and PAP, when utilized together with standard criteria for assessing treated prostate cancer patients.


Peripheral blood stem cell transfusion for marrow replacement.

A patient is presented who was treated with ablative therapy for Hodgkin's disease and rescued by reinfusion of peripheral blood stem cells (PBSC). The PBSC were used because previous therapy (chemotherapy and radiation to the pelvis) had resulted in fatty hypocellular marrow which was inadequate for marrow transplantation. The PBSC were collected by leukapheresis before and after recovery of the marrow from suppression with cyclophosphamide to bring the stem cells into cohort cycle and to increase the proportion of stem cells in the peripheral blood for collection. The patient showed a successful recovery on a time scale somewhat longer cells administered, the absence of stimulation by granulocyte macrophage-colony stimulating factor or other cytokine, or potential damage done to stromal elements during previous radiation and chemotherapy. The patient remains in clinical complete remission, fully engrafted, more than one year since his autologous transplant.


Hyperthermic potentiation of bleomycin cytotoxicity in the presence of Bacillus thuringiensis subsp. israelenis delta-endotoxin.

We previously reported that a 25-kDa Bacillus thuringiensis subsp. israelensis (BTI) delta-endotoxin potentiated the antitumor activity of bleomycin (BLM) in both in vitro and in vivo systems. We report here a characteristic hyperthermic synergy toward the cytocidal activity of BLM in the presence of BTI endotoxin in cultured L1210 cells. Cytocidal gain, i.e., the ratio of the IC50 of BLM in the absence versus the presence of BTI delta-endotoxin, reached ca. 20 at 40 degrees C, suggesting that a more effective heat-BLM combination treatment may be achieved in the presence of BTI delta-endotoxin.


(-)-2'-deoxy-3'-thiacytidine is a potent, highly selective inhibitor of human immunodeficiency virus type 1 and type 2 replication in vitro.

The (-)-enantiomer of 2'-deoxy-3'-thiacytidine (3TC) was found to be a potent and selective inhibitor of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) in vitro. We determined its antiviral activity against a number of laboratory strains of HIV-1 and HIV-2 in a range of CD4-bearing lymphocyte cell lines (mean 50% inhibitory concentration [IC50] range, 4 nM to 0.67 microM). 3TC was also active against a range of HIV-1 strains in peripheral blood lymphocytes (mean IC50 range, 2.5 to 90 nM). The IC50 for cytotoxicity in seven lymphocyte cell cultures, including human peripheral blood lymphocytes, ranged from 0.5 to 6 mM. 3TC had no detectable antiviral activity against a range of other viruses or in cells chronically infected with HIV-1 or HIV-2. The effects of time of addition of the compound and varying the multiplicity of infection on the antiviral activity of 3TC were determined. The results showed that 3TC is a potent and selective inhibitor of HIV-1 and HIV-2 replication in vitro.


Heterogeneity and multiple expression of intermediate filament proteins, S-100 protein and neuron specific enolase in skin mixed tumor.

Intermediate filament proteins, keratin (KL1, PKK1, K8.12) and vimentin, S-100 protein alpha and beta subunits and neuron specific enolase were evaluated immunohistochemically to determine their distribution patterns in the tumor components of mixed tumor of skin. Keratin proteins were distributed widely in tumor epithelial cells or modified myoepithelial (MME) or neoplastic myoepithelial (NME) cells. Luminal cells of the tubulo-ductal structure of the tumor mass showed positive staining of KL1 and PKK1 keratins and an infrequently positive reaction of MoAb K8.12. The outer or basal tumor cells were characterized by coexpression of K8.12 keratin, vimentin, S-100 protein and infrequently neuron specific enolase reactivity. Heterogeneity of keratin distribution was seen in tumor epithelial cells. MME cells or NME cells of skin mixed tumor showed coexpression of keratin and vimentin, and multiple expression of intermediate filament proteins, S-100 protein and neuron specific enolase. Hyaline and chondroid changed cells stained intensely to vimentin and S-100 proteins, as well as to neuron specific enolase. The authors evaluate the histogenesis of skin mixed tumor in relation to epithelial and myoepithelial cells of the sweat gland and their immunohistochemical findings.


Macrophages in melanocytic naevi.

Whereas the inflammatory infiltrates of malignant melanoma have been widely investigated, little is known about the infiltrates accompanying benign melanocytic naevi. Using monoclonal antibodies directed against HLA-DR antigens, the CD1 antigen, the transferrin receptor and functionally divergent macrophage subpopulations, frozen fresh material of 87 melanocytic naevi (MN), ten primary cutaneous melanomas (PCM) and ten samples of normal skin were studied. Compared with normal skin, abundant HLA-DR+ cells were found in the stroma of MN equivalent to the quantity present in PCM. In MN we found higher numbers of dermal CD1+ dendritic cells compared with PCM and normal skin. There were more macrophages that expressed the transferrin receptor or the antigens 27E10, RM3/1 and 25F9 in MN than in normal skin but fewer than in PCM. No significant differences were found between congenital MN (n = 40), common acquired MN (n = 27) and dysplastic MN (n = 20) macrophage subpopulations. Also, no correlations were evident between macrophage infiltrates and naevus location or patients' age. Our data show that potential melanoma precursors among melanocytic naevi cannot be identified by the pattern of macrophage infiltrates.


Prognostic value of cell proliferation in breast cancer as determined by proliferating cell nuclear antigen (PCNA) immunostaining.

A series of 175 biopsies from primary tumours were subjected to immunostaining for proliferation cell nuclear antigen (PCNA) and histopathological analysis for prognostic factors in 175 women with breast cancer followed up for nine years. In normal breast epithelium, only occasional nuclei were positive for PCNA. In breast carcinomas, the fraction of nuclei positive for PCNA showed considerable intratumoural variation, usually being highest at the invasive tumour margins. The fraction of positive nuclei was significantly related to histological grade (p less than 0.001), histological type (p = 0.049) and tumor recurrence (p = 0.01). The fraction of positive nuclei predicted recurrence-free survival (p = 0.007) and overall survival (p = 0.0158) in univariate analysis. In the multivariate analysis including also the standard prognostic factors, the PCNA positivity predicted recurrence-free survival (p = 0.004) and overall survival (p = 0.030) independently. The results show that the light microscopic assessment of the growth fraction as determined by PCNA immunolabeling has independent prognostic value in breast carcinomas like some other (e.g. S phase fraction, mitotic index, Ki-67) previously characterized proliferation indices.


Expression of cytokeratins in granulosa cell tumors and ovarian carcinomas.

One of the problems in histopathology of ovarian tumors is differential diagnosis between the poorly differentiated carcinomas and granulosa cell tumors of the sarcomatoid type. Hence we evaluated the expression of various cytokeratins (CK 1-19; CK 10, 13, 14, 15, 19; CK 8, 18, 19; CK HMW 1, 5, 10, 11; CK 8; CK 1-19 Stähli; CK AE1/AE3) immunohistochemically in 53 ovarian malignancies (11 of them poorly differentiated carcinomas and 12 granulosa cell tumors). CK HMW was not detected in granulosa cell tumors, and in only half of the carcinomas. AE1/AE3 was expressed by more than 90% of ovarian carcinomas but by one granulosa cell tumor only. The other keratins we investigated showed higher expression rates in granulosa cell tumors and/or lower expression rates in ovarian carcinomas. We think that cytokeratin immunohistochemistry is of value in differentiating between granulosa cell tumors and ovarian carcinomas.


Distribution of peptide-containing nerve fibers in the gastric musculature of patients undergoing surgery for gastroesophageal reflux.

The distribution of nerve fibers and cell bodies reactive for the peptides enkephalin, neuropeptide Y, substance P, and vasoactive intestinal peptide were studied in biopsies of external muscle taken from the gastric body and antrum of 17 patients undergoing surgery for gastroesophageal reflux, and in regions of healthy stomach resected as part of gastric cancer operations. The results were correlated with preoperative measurements of liquid and solid emptying from the stomach in the patients with gastro-esophageal reflux. In three cases, no delay was detected in either liquid or solid emptying, and the distribution of peptide immunoreactive fibers in the external muscle was similar to that of healthy muscle. In 14 cases, the emptying of either liquids or solids or both was delayed, and in eight of these clearcut changes in the distribution of peptide-immunoreactive fibers occurred. In six cases, the number of enkephalin-immunoreactive fibers was fewer than normal in the biopsy from the gastric body (in one of these samples the number of substance P-immunoreactive fibers was also reduced). In another cae, the number of substance P-immunoreactive fibers in the antrum was reduced, and in another the number of vasoactive intestinal peptide and neuropeptide Y-immunoreactive fibers in the antral biopsy was reduced. It is concluded that in patients with gastroesophageal reflux who have delayed gastric emptying, a proportion demonstrate abnormalities of the peptide-immunoreactive fibers that innervate the gastric external muscle.


Determination of estrogen and progesterone receptors in endometrial adenocarcinomas. Comparison between dextran-coated charcoal and immunoenzymatic methods on curettage biopsies.

Estrogen receptors (ER) and progesterone receptors (PR) were determined on curettages from women with endometrial adenocarcinoma. The results obtained with the enzyme immunoassay (EIA) and the dextran-coated charcoal (DCC) assay were compared. A highly significant correlation was obtained between these methods for the ER measurement (Rs = 0.91). For PR determination, the Rs value between EIA and DCC assay was 0.57 and the mean value of PR-DCC is significantly higher than the mean value of PR-EIA. These results suggest that EIA is a suitable method for ER measurement. For PR determination on curettage material the DCC assay seems more accurate than EIA.


Analysis of urinary protein by immunoblot method using unconcentrated urine in preeclampsia.

In order to investigate the renal change in preeclampsia, molecular weight and specific protein analyses in unconcentrated urine were performed by the immunoblot method. Urine samples were taken from 34 preeclamptic cases (pure type), including 20 severe cases. Polypeptide profiles of urine consisted of four patterns: low MW (L) pattern (tubular damage), high MW (H) pattern (glomerular damage), high and low MW (HL) pattern, and middle MW (M) pattern. The incidences of the HL, H, L, and M patterns were 26.5%, 14.7%, 11.8%, and 47.1%, respectively. The HL pattern was found more frequently in severe proteinuria than in mild proteinuria. High incidences of the HL and H patterns were found in the hypertensive group. Larger amounts of IgM, fibronectin, IgG, and beta 2 microglobulin in urine were confirmed using specific antibodies. Our results suggest that the immunoblot method makes it possible to differentiate glomerular and tubular damages and to evaluate the severity of renal damage in preeclampsia using unconcentrated urine.


A binding profile of manganese to the nucleus of rat liver cells, and manganese-induced aberrations in thyroid hormone content and RNA synthesis in the nucleus.

Manganese (Mn) is accumulated in the nuclear and mitochondrial fractions when excess Mn is administered. However, little is known with respect to the behaviors of Mn in nuclei. In the present study, rats were given excess Mn and the nuclei were purified from liver cells by differential and sucrose gradient centrifugations. Being subjected to equilibrium dialysis with a radioactive 54Mn, the binding capacity of Mn in nuclei from the control rat was five-fold higher than BSA, which was used as a reference protein; and the capacity of 54Mn-binding rose in the nuclei from the Mn-treated animals in comparison with those from the control. On the analyses of nuclear materials with partial solubilization, sepharose column chromatography and HPLC, there were two major fractions which associated with a lot of Mn; one fraction was of large molecules of DNA, and the other fraction seemed to be peptides with small molecular weights. Therefore, Mn may open up a superhelical structure of DNA to provide more negatively charged phosphate moiety as a binding-site for a positively charged Mn. The results also disclosed a possible aberration in biological functions due to excess Mn in nuclei; the apparent association constant of triiodothyronine, a physiologically active thyroid hormone, to the nucleus was reduced by 75% and the uptake of 14C-labelled orotic acid to a newly synthesized RNA in the liver was severely inhibited.


[Catalytic activity of immune complexes of peroxidase and its conjugates with cortisol in Aerosol OT reverse micelles].

Conjugates containing 1, 6 or 11 cortisol molecules per peroxidase molecule were obtained by the reaction of the N-hydroxysuccinimide ester of cortisol 3-O-carboxymethyloxime (COR) with horse-radish peroxidase (HRP). Activities of the peroxidase conjugates and their immunocomplexes with antibodies against cortisol in the orthophenylenediamine oxidation in reversed Aerosol OT micelles in heptane at various hydration degrees of the micelles (omega 0) were studied. Catalytic activities of HRP and its conjugates, on one hand, and of the immunocomplexes of HRP with anti-HRP and the conjugates with antibodies against cortisol, on the other hand, differed significantly and depended in a different manner on the hydration degree of AOT micelles. These differences became the basis of a homogeneous enzyme-immunoassay of cortisol in the reversed micelles of AOT in heptane.


[The exposure of the population to toxic substances in the interior of motor vehicles--the example of benzene].

The exposure of the population to benzene is caused in the first place by emissions of the motor-vehicle traffic. The air pollution concentrations in main traffic routes and in the sphere of influence of industrial plants amount to 5-30 micrograms Benzene/m3 in the course of the year. In indoor air about 6-12 micrograms/m3 are detectable, in the interior of motor-vehicles between 50 and 200 micrograms/m3. Smoking raises the individual burden significantly; in contrast food and drinking water amount only for a small part of the total intake of benzene. In rural areas with low outdoor-air concentrations the main source of burden can be the intake of benzene during the use of motor-vehicles. Despite the relatively low carcinogenic potency of benzene but because of the unfavourable exposure conditions and the comparatively high concentrations measures for the reduction of benzene in fuel should be taken immediately.


Two-dimensional electrophoresis and densitometric analysis of solubilized bovine sperm plasma membrane proteins detected by silver staining and radioiodination.

Bovine sperm plasma membranes were extracted with deoxycholate and subjected to two-dimensional gel electrophoresis. Proteins were visualized either by silver staining or autoradiography using 125I. When 1.5-2.0 micrograms of protein extract was applied to the first-dimension get, 250 spots could be detected by autoradiography. Thirty micrograms of protein was required to obtain spot visualization using silver strain, revealing more than 500 spots on the gel. These data establish the limit under which two-dimensional electrophoresis can be used in conjunction with flow cytometric sorting of sperm for the analysis of possible sex-specific membrane differences. The necessity of orienting sperm as they past the laser in the cell sorting system reduces throughput; thus, the number of sperm sorted within a given time is limited. It is suggested that autoradiography will allow the flow cytometer to be used in a time-efficient manner. Longer sorting times would be required to obtain sufficient sample to analyze total protein composition.


MHC-binding peptides for immunotherapy of experimental autoimmune disease.

It is now well accepted that T helper cells play a central role in the induction and maintenance of autoimmune disease. Many experimental models have emphasized this fact and have illustrated the efficacy of therapeutic strategies aimed at disrupting T cell recognition of autoantigens. Antibodies directed at either class II proteins of the major histocompatibility complex (MHC) or CD4 accessory molecules have been universally successful. However, the potential use of antibodies for therapy in humans is complicated by host anti-globulin and anti-idiotype responses. An alternative approach to anti-MHC blockade with antibodies is peptide blockade of MHC molecules. In addition, peptides may be used as agonists of autoantigens in order to modulate the autoimmune response. The use of synthetic peptides for therapy is an innovative yet relatively unexplored approach and will be the subject for discussion in this article.


Immunocytochemical localization of bioregulatory peptides in marmoset testes.

Immunocytochemical localization of neuropeptides (beta-endorphin, substance P, arginine vasopressin, oxytocin), pituitary hormones (adrenocorticotropin, prolactin, growth hormone, follicle stimulating hormone (FSH), gonadal inhibin, gastrin, and human chorionic gonadotrophin (hCG)) was carried out in marmoset testis during development. Both intensity of immunostaining and distribution of these peptides in testicular compartments viz. seminiferous tubules and Leydig cells changed dramatically during development. In vitro biosynthesis of inhibin and FSH was increased by hCG, whereas prolactin (5 micrograms) and prostatic inhibin peptide suppressed the synthesis of these hormones.


The forces driving autoimmune disease.

There are two classes of autoimmune disease, organ-specific and non-organ specific or systemic. That cells producing autoantibodies are selected by antigen is strongly suggested by the presence of mutations and high affinity antibody. T-cells are pivotal in all forms of autoimmunity as evidenced by the therapeutic benefit of anti-T-cell monoclonals such as anti-CD4, and the frequent development of high affinity IgG autoantibodies. The production of anergic T-cells by the use of non-depleting anti-CD4 in the presence of antigen is discussed with particular reference to its potential for immunological intervention in autoimmune disease. It is possible to identify T-cell epitopes in organ-specific autoimmunity using pathogenic T-cell clones or hybridomas to identify the peptide sequences which are reactive. Antigen-specific therapy may ultimately be based on such peptide epitopes. The specificity of the T-cells in systemic autoimmunity is still obscure, but there is some evidence that reactivity with certain germ-line idiotypes can lead to the development of systemic autoimmunity. The possibility of stimulating B-cells specific for auto-antigens such as DNA becomes feasible if a complex of antibody and DNA is taken up by these specific B-cells and processed idiotype is presented to T-helpers specific for those idiotype epitopes. Evidence is presented that there may be pre-existing defects in the target organ in certain organ-specific disorders, and the evidence for a glycosylation defect in the IgG in patients with rheumatoid arthritis is explored. It is noted that the spouses of probands with rheumatoid arthritis is explored. It is noted that the spouses of probands with rheumatoid arthritis also tend to have this glycosylation defect and this raises the possibility of an effect due to an environmental factor, such as a microbial infection. Molecular mimicry of autoantigens by microbes can stimulate autoreactive cells by their cross-reactivity. It is emphasized that cross-reaction which gives rise to the priming of autoreactive T-cells could give rise to the establishment of a chronic autoimmune state. In animals with normal regulatory immune systems, such induced autoimmunity is ultimately corrected and it is only in animals where there are defects in regulation, that autoimmunity persists. Thus, there are many factors giving rise to autoimmunity, and the diseases are rightly regarded as multifactorial in origin.


Interleukin-6 and its receptor in autoimmunity.

Interleukin-6 (IL-6) is a multifunctional cytokine which acts on a wide variety of cells, regulating immune response, acute phase reaction and hematopoiesis. In accordance with its pleiotropic functions, IL-6 is indicated to be involved in the pathogenesis of several diseases including autoimmunities, lymphoid malignancies and inflammations. An elevated level of IL-6 is demonstrated in patients with rheumatoid arthritis and cardiac myxoma, which can explain symptoms of these diseases, such as autoantibody production and increase in acute phase proteins. Therefore, inhibitors of IL-6 production or IL-6 receptor-mediated signal transduction may be used for treatment of IL-6-related diseases. The IL-6 receptor system consists of two membrane proteins, a ligand-binding chain (IL-6R) and a non-ligand-binding signal transducer, gp130, both of which belong to the cytokine receptor family. Binding of IL-6 to IL-6R triggers the association of IL-6R and gp130, and gp130 in turn transduces the signal. A nuclear factor for controlling IL-6 gene expression (NF-IL6) is also involved in the transcriptional regulation of various acute-phase protein genes. IL-6-stimulation of hepatocytes, through modification of pre-existing NF-IL6 protein, leads to binding of NF-IL6 to IL-6-responsive elements and activation of acute-phase protein genes. NF-IL6 is shown to recognize the enhancer core sequence of several viruses, suggesting a possible relationship of virus infection and IL-6 expression.


SRC-related proto-oncogenes and transcription factors in primary human T cells: modulation by cyclosporin A and FK506.

Activation of T lymphocytes induces transcription of genes encoding for lymphokines. Interleukin-2 (IL-2) gene expression is controlled transcriptionally by the cooperative activity of specific trans-activating factors that bind to the IL-2 enhancer. Cyclosporin A (CsA) and FK506 inhibit the production of IL-2 in T lymphocytes at the level of gene transcription. A member of the src gene family, the lymphocyte-specific protein tyrosine kinase, p56lck, has been implicated in IL-2 production. CsA was found not to inhibit lck gene expression, nor the activity of the lck gene product. However, CsA and FK506 inhibit the appearance of DNA binding activity of factors that bind to the NF-AT and AP-1 sites in the IL-2 enhancer. Since the induction of NF-AT and AP-1 is induced by the same stimuli that stimulate IL-2 production, these results indicate that the immunosuppressant action of CsA and FK506 is exerted at the level of these trans-activating factors.


Molecular mode of action of cyclosporin and FK506 in human thymocytes.

The molecular mode of action of cyclosporin, three of its non-immunosuppressive analogs, N-methyl-l-alanyl cyclosporin, acetyl cyclosporin and cyclosporin S3, and of FK506 was studied in primary cultures of human thymocytes. Nuclear factors derived from thymocytes activated with phorbol myristate acetate and concanavalin A were tested for their ability to bind to a synthetic radiolabelled probe corresponding to the NF-AT region (-285 to -255) of the IL-2 gene. Binding was observed, and it was inhibited by CsA (100 ng/ml), while the analogs at ten-fold higher concentrations (1000 ng/ml) were only partially inhibitory. CsA in combination with FK506 inhibited binding of nuclear factors at the NF-AT site, and acted in concert.


Towards peptide immunotherapy in rheumatoid arthritis: competitor-modulator concept.

By the introduction of single-amino acid substitutions in well-defined T cell epitopes of autoimmunogenic proteins, e.g., mycobacterial heat shock protein (hsp60) in adjuvant arthritis (AA) and myelin basic protein (MBP) in experimental allergic encephalomyelitis (EAE), efficiently blocking MHC binding peptides were selected. Despite the finding that a substituted variant of epitope 180-188 of hsp60 was 'blocking' not only responses of the 180-188 specific arthritogenic T cell A2b, but also responses of the MBP specific encephalitogenic T cell Z1a, in vivo testing of this competitor peptide revealed a very prominent disease inhibitory activity in AA but not in MBP-induced EAE. The selectivity of this peptide in suppressing the disease in which native 180-188 appears to be of critical relevance, offers the possibility of achieving disease specific immunological intervention. Based on the results collected so far, it seems that, in vivo in addition to blocking activity, a variant peptide itself could trigger responses that confer protective activity in AA. Such combined activities may well be required for achieving full in vivo inhibition of a disease in which multiple distinct epitopes may play a role, possibly through presentation by more than one MHC product.


Immunointervention in autoimmune diseases from cellular selectivity to autoantigen specificity.

Immunosuppressive agents have proved to be remarkably successful in controlling the course of a growing number of autoimmune diseases. The usual occurrence of relapses when the treatment is stopped and the persisting risk of long term complications linked to over-immunosuppression have prompt the search for new approaches, aiming at more rapid and selective intervention and earlier onset of therapy. New chemicals and monoclonal antibodies presently represent the main clinical orientations, while at the experimental level major efforts are directed towards peptide therapy (autoantigen, T cell receptor) and tolerance induction.


Molecular mechanisms of immunosuppression.

The immunosuppressive drug cyclosporin A (CsA, Sandimmun, SIM) is currently being evaluated in a variety of autoimmune disorders with some remarkable successes. Despite the wide empiric application of CsA, the precise mechanism of action of this drug remains elusive. To identify the molecular mode of action of CsA in the process of T cell activation, we have compared the biological profile of cyclophilin-binding cyclosporin analogues (CBCA), which lack immunosuppressive properties, with CsA. We have found that CsA binding to its intracellular receptor (cyclophilin) is required but not sufficient for immunosuppression. Moreover, inhibition of the peptidyl-prolyl cis-trans isomerase activity of cyclophilin does not seem to be relevant for the inhibitory effects of CsA. In analogy to the immunosuppressants FK506 and rapamycin, a specific structure at the 'effector' domain of the CsA molecule different from the immunophilin 'binding' domain determines the biological activity. Overall, a significant understanding of the structure-activity relationship of CsA has emerged. This will have a major impact on the identification of the precise mechanism of action of CsA and its side effects in the process of immunosuppression.


Relationship between the expression of differentiation-specific keratins 1 and 10 and cell proliferation in epidermal tumors.

In normal epidermis, the expression of keratins 1 and 10 is associated with the loss of proliferative capacity and the onset of terminal differentiation. Keratins 1 (K1) and 10 (K10) are commonly expressed in the differentiating layer of benign tumors, but are lost during progression from the benign to the malignant state in skin carcinogenesis. Active gene constructs of mouse K1 and K10 were introduced into papilloma and carcinoma cell lines derived from keratinocytes to analyze the consequences of the expression of these keratins on the organization of the endogenous cytoskeletal network and on the mitotic activity of the recipient cells. Exogenous K1 integrated into the preexisting keratin K5/K14 network of both SLC-1 carcinoma and 308 papilloma cells. The formation of a recombinant cytoskeleton was more restricted for K10 than for K1 and appeared to be related to a requirement for cessation of cell division before K10 could integrate. The integration of exogenous K1 filaments into the endogenous keratin network was compatible with sustained proliferation of SLC-1 carcinoma cells in vitro. However, the exogenous gene was not expressed in tumor grafts in vivo. In contrast, stable K1 or K10 transfectants could not be selected in 308 cells, suggesting that benign tumor cells expressing suprabasal keratins cannot sustain proliferation.


Alloimmune thrombocytopenia: in utero treatment by high doses of intravenous gamma globulins.

The authors report successful in utero treatment by high doses of intravenous gamma globulins in a case of neonatal alloimmune thrombocytopenia. Early diagnosis allows an appropriate management: fetal blood sampling as early as 20 weeks of gestation; in case of fetal thrombocytopenia, treatment by intravenous gamma globulin (1.0 g/kg/b.w.) each week during 8 weeks or more; ultrasound screening of in utero hemorrhage, particularly intracranial hemorrhage; fetal blood sampling before delivery at term and in utero transfusion of platelet antigen negative in case of persistence of fetal thrombocytopenia.


Effect of gamma-interferon on the expression of major histocompatibility complex class I and II gene products in neural cells isolated from the developing human fetal peripheral nervous system.

The effect of gamma-interferon (gamma-IFN) on the expression of major histocompatibility complex (MHC) gene products was examined in the developing human fetal peripheral nervous system. RNA blot hybridization analysis of total RNA isolated from human fetal dorsal root ganglia (DRG) neural cell populations cultured in vitro for 5 days resulted in the detection of both MHC class I- and class II-specific RNAs. As determined by protein immunoblotting and fluorescence-activated flow cytometry, MHC class I and II proteins were also readily detectable in cultured human fetal DRG neural cell populations 5 days after isolation. In addition, treatment of 3-day human fetal DRG neural cells with gamma-IFN (100 U/ml; 48 h) resulted in a marked increase in the level of MHC class I- and class II-specific RNA and protein without inhibiting the proliferation of the neural cell population. These results suggest that changes in the levels of selected cytokines such as gamma-IFN may alter the ability of specific neural cell populations present in the developing human nervous system to participate in immune reactions by alteration of MHC class I and II antigen expression which may lead to perturbation in glial cell function and ultimately to nervous system dysfunction in general.


A simple procedure for recovering the denaturing effect of methylmercury in agarose gel electrophoresis.

A simple and rapid procedure for recovering the denaturing effect of methylmercuric hydroxide in agarose gel electrophoresis is described. The procedure consisted of the treatment of the commercial methylmercuric hydroxide solutions with Amberlite, a mixture of anion- and cation-exchange resins. This treatment greatly improved the resolution of RNA species when fractionated by electrophoresis through agarose-CH3HgOH slab gel.


Reverse staining of sodium dodecyl sulfate polyacrylamide gels by imidazole-zinc salts: sensitive detection of unmodified proteins.

We report here a modification to copper and zinc chloride staining methods. The introduction of a preincubation of the gels, prior to metal staining, with 0.2 M imidazole allows the formation of a homogeneous background for the subsequent precipitation of the metal chelate. The reported imidazole-zinc staining takes minutes, resulting in reproducible staining patterns with only slightly lower sensitivity than silver staining. The method allows efficient recovery of proteins from previously stained gels and is compatible with immunoidentification on Western blots and also with amino acid analysis and NH2-terminal sequence analysis of transferred proteins. A mechanism is proposed to explain the observed improvement in reproducibility and sensitivity of imidazole preincubation to zinc staining.


Use of in vitro-transcribed RNAs as immobilized targets during RNA:RNA hybridization.

We describe the use of in vitro-transcribed complementary RNAs as filter-bound targets during nuclear run-on analyses. Use of these single-stranded reagents in high-stringency RNA:RNA hybridizations increases signal-to-background hybridization seen using DNA targets and allows efficient measurement of transcriptional rates across genes in either direction.


Acidic fibroblast growth factor accelerates dermal wound healing.

Acidic fibroblast growth factor (aFGF) is a potent mitogen in vitro for many cells of ectodermal and mesodermal embryonic origin including skin-derived epidermal keratinocytes, dermal fibroblasts and vascular endothelial cells. Based on the mitogenic activity for these skin-derived cells, we tested the ability of topically applied aFGF to promote healing of full-thickness dermal wounds in healthy rodents. Low doses of aFGF can produce almost a two-fold maximum acceleration in the rate of closure of full-thickness dermal punch biopsy wounds in young healthy mice and rats. The mitogen also produces a 3 to 4 day acceleration in the time to complete closure in rats. Quantitative histomorphometric analysis of wound tissue shows that aFGF induces a marked stimulation of angiogenesis, granulation tissue formation and the growth of new epithelium, but does not promote dermal contraction. Application of aFGF to linear incisions in rat skin produces a transient increase in wound tensile strength accompanied by enhanced cellularity and deposition of collagen. Therefore, aFGF functions as a pharmacological agent that can accelerate dermal wound healing in rodents and could act therapeutically to promote dermal tissue repair in humans.


The vascular endothelial growth factor proteins: identification of biologically relevant regions by neutralizing monoclonal antibodies.

Angiogenesis plays critical roles in organ development during embryonic and fetal life, wound healing and in a variety of pathological conditions. Vascular endothelial growth factor (VEGF) is a secreted growth factor specific for vascular endothelial cells which induces angiogenesis in vivo. To gain a better understanding of the physiological role of VEGF, we have generated and characterized four murine monoclonal antibodies (mAbs) using the 165 amino acid species of recombinant human VEGF as immunogen. These mAbs (A3.13.1, A4.6.1, B4.3.1 and B2.6.2) belong to IgG1 isotype and have high affinities for VEGF (dissociation constants range from 2.2 x 10(-9) to 4 x 10(-10) M). Two different epitopes were detected with these mAbs. One epitope is recognized by mAbs A3.13.1 and B2.6.2, and the other recognized by mAbs A4.6.1 and B4.3.1. The epitope recognized by mAb A4.6.1 appears to be continuous while mAb B2.6.2 recognizes a discontinuous epitope. MAb A4.6.1 recognized three species of VEGF generated by alternative splicing, VEGF121, VEGF165 and VEGF189 while mAb B2.6.2 binds only VEGF165 and VEGF189. Results using an in vitro bovine adrenal cortex endothelial cell proliferation assay, in in vivo vascular permeability assay and an in vivo embryonic chicken angiogenesis assay showed that mAb A4.6.1 has potent VEGF neutralizing activities. MAb A4.6.1 was shown to block the binding of VEGF to its receptor(s) suggesting the inhibitory mechanism for VEGF activities. These well-defined mAbs should be very powerful tools to understand the structure-function relationship of various domains of VEGF and may have therapeutic potential.


Differentiation and activation associated expression of IL-6 and IL-6 receptors in U-937 monocytic cells: relationship to the expression of CD14.

Activated monocytes play an important role as producers of IL-6 during inflammation and immune response. We show that during monocytic differentiation of U-937 cells, induced by phorbolester (PMA), IL-6 mRNA expression was transiently up-regulated and IL-6 protein was secreted into the medium. In contrast, differentiation induced by VitD3 or Retinoic acid (RA) did not lead to an increase in the IL-6 expression. Thus, IL-6 expression does not seem to be associated with monocyte differentiation per se. However, U-937 cells terminally differentiated by VitD3, rapidly responded to bacterial lipopolysaccharide (LPS) induced activation by IL-6 expression and secretion. In cells, differentiated by PMA, the IL-6 expression was super-induced after activation by interferon-gamma (IFN-gamma) and LPS. The capacity of U-937 cells to respond to LPS activation by IL-6 expression was associated with the expression of CD14 and some serum components(s) were a prerequisite for a successful LPS induction. The IL-6R expression was down-regulated during monocytic differentiation of U-937 cells. In the terminally differentiated U-937 cells, the expression of IL-6R could be induced after activation by IFN-gamma and to a lesser extent by LPS, suggesting a mechanism by which activation positively regulates the response to IL-6 in macrophages.


Hematopoietic growth factors as adjuncts to antiretroviral therapy.

Anemia and neutropenia are common complications of HIV infection. Antiretroviral therapy with zidovudine exacerbates bone marrow suppression by inhibiting proliferation of blood cell progenitor cells. In addition, treatment for opportunistic infections or malignancies can involve the use of myelosuppressive drugs. As a consequence, severe anemia and neutropenia can result, thereby limiting the utilization of antiretroviral drugs. Since antiretroviral therapy can increase survival, drugs that ameliorate myelosuppression are important adjuncts in the treatment of HIV-infected patients. Three hematopoietic growth factors are effective in the treatment of anemia or neutropenia. In four placebo-controlled trials, erythropoietin (EPO) at doses up to 600 U/kg/wk decreased mean transfusion requirements by 37%, increased mean hematocrit by 4.5% and corrected anemia in the majority of patients receiving zidovudine over a 12-week period. In a separate study, granulocyte colony-stimulating factor (G-CSF) corrected leukopenia and isolated neutrophil defects in 22 patients with AIDS without altering HIV expression. When erythropoietin was added to the regimen, combined G-CSF and EPO corrected both anemia and leukopenia and lessened subsequent zidovudine toxicity. Similarly, granulocyte macrophage-colony-stimulating factor (GM-CSF) corrected leukopenia and pre-existing neutrophil defects in patients with HIV infection. In controlled and uncontrolled trials, GM-CSF also appears to reduce toxicity from zidovudine, ganciclovir, and antineoplastic therapy. New combinations of hematopoietic stimulants are being used to decrease the toxicity from combination antiretroviral therapy with alpha interferon and cytotoxic chemotherapy in the treatment of AIDS-related malignancies.(ABSTRACT TRUNCATED AT 250 WORDS)


Production of human monoclonal antibodies specific for conformational and linear non-V3 epitopes of gp120.

Three IgG1 human monoclonal antibodies (MAbs) directed against conformational epitopes of the gp120 envelope protein of HIV-1 were produced, as was a single human MAb to a linear epitope spanning amino acids 487-509 in the C-terminal portion of gp120. All three conformation-dependent MAbs reacted optimally with recombinant gp120 (rgp120) captured on plastic via its carbohydrate moieties with Concanavalin A. These MAbs were able to block the interaction between recombinant CD4 (rCD4) and rgp120; they were also able to achieve 50% neutralization of HTLV-IIIB and MN strains of HIV-1 in a concentration range of 0.5-12.8 micrograms/mL. The MAb to the linear determinant is the first reported human MAb specific for the immunodominant portion of gp120; this MAb was most reactive with rgp120 when it was coated directly on plastic. It could neither inhibit rCD4-rgp120 binding nor neutralize either HTLV-IIIB or MN. The binding affinities of the four human MAbs for rgp120 in solution, reflected by their dissociation constants (Kd), ranged from 0.5 x 10(-8) to 7.5 x 10(-8) M.


Characterization of a mouse/human chimeric monoclonal antibody (C beta 1) to a principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein.

A chimeric mouse-human antibody (C beta 1) was constructed that recognized the principal neutralizing domain (PND) of human immunodeficiency virus type 1 (HIV-1) gp120. The constant (C) immunoglobulin regions (C gamma 1 and C kappa) of a mouse monoclonal antibody, 0.5 beta, were substituted for the human C gamma 1 and C kappa by recombining the DNA modules encoding variable or C regions. The DNA constructs were then transfected into X63 Ag8.653 myeloma cells. A clone with a high production of the chimeric antibody (C beta 1) was selected. This antibody was tested for its biological activity against HIV-1. It bound to the surface of HTLV-IIIB-infected cells and reacted with gp120/160 with equal affinity and specificity to that of the parental 0.5 beta murine monoclonal antibody in a Western blot assay. Neutralization and/or enhancement of HIV infection were evaluated with C beta 1 and 0.5 beta. Both C beta 1 and 0.5 beta neutralized cell-to-cell infection and cell-free virus infection by HTLV-IIIB. Antibody-dependence enhancement of HIV infection was not observed with either C beta 1 or 0.5 beta in the presence or absence of human complement. Antibody-dependent cell-mediated cytolysis (ADCC) and antibody-dependent complement-mediated cytolysis (ACC) were observed with C beta 1 but not with the parental 0.5 beta. These findings suggest that the neutralizing antibodies to PND may neutralize but not enhance HIV infection. Furthermore, the high levels of ACC and ADCC shown against HIV-infected cells by C beta 1 indicate that the clinical application of such monoclonal antibodies may be possible.


Delineation of immunoreactive, conserved regions in the external glycoprotein of the human immunodeficiency virus type 1.

Immunization of mice and rats with purified external glycoprotein gp120 from two divergent human immunodeficiency virus type 1 (HIV-1) isolates resulted in the development of seven hybridomas secreting monoclonal antibodies able to recognize regions of gp120 which are common among divergent strains of HIV-1. These monoclonal antibodies cross-reacted with env glycoproteins from one African (Rutz), one Haitian (RF), and three North American viral isolates, namely IIIB, MN, and 451 by either immunoblot or radioimmunoprecipitation assays. All recognized denatured gp120 in immunoblots with the exception of one which required a conformationally intact glycoprotein for reactivity. The gp120 epitopes identified by these antibodies were mapped by screening of an env gene library in the lambda gt11 expression system. Three out of four epitopes were found to reside in the amino-terminal half of gp120 (Cys9 to Cys35, Thr44 to Glu72 and Val108 to Met130), the other was located in the middle region (Thr221 to Ser255). By virtue of their extent of cross-reactivity these reagents might provide a unique resource for the detection of new viral isolates related to HIV-1.


Study of viral replication in HIV-1-infected CEM T-cell subclones which are reduced in their ability to form syncytia.

The derivation of ethyl-methanesulfonate (EMS) mutagenized subclones from the CEM T-cell line has been described. These clones expressed CD4 and bound soluble gp120, however, two of the generated clones were markedly reduced in their ability to form syncytia after infection with either gp160-vaccinia vector or cell-free human immunodeficiency virus type 1 (HIV-1). Here, we asked at what stage(s) viral infection is blocked in these cells. Polymerase chain reaction (PCR) analysis revealed that at 6 and 72 h postinfection with HIV-1, cells of the syncytia-deficient clones expressed markedly reduced amounts of viral-specific DNA compared with cells of the parental line or the syncytia-positive clones. Long-term cultures revealed a marked delay in the appearance of reverse transcriptase (RT) activity in the supernatants of these subclones when compared with the parental line and viral replication did not lead to massive cell death. Syncytia formation in HIV-1-infected cultures of the syncytia-deficient subclones was enhanced by tumor necrosis factor alpha (TNF alpha) when added 24 h postinfection. In contrast, pretreatment with TNF alpha for 48 h followed by washing and infection of the cells with HIV-1 augmented syncytia formation of the syncytia-positive subclones, but not of the syncytia-negative subclones. Thus, the EMS-mutagenized subclones may provide a tool to study host cell factors required for the establishment of a productive HIV-1 infection and responsiveness to TNF alpha.


Identification of two neutralizing and 8 non-neutralizing epitopes on simian immunodeficiency virus envelope using monoclonal antibodies.

Ten new monoclonal antibodies (MAbs) to SIV envelope were produced and characterized. Using a panel of 28 MAbs, 10 antibody binding sites on SIV envelope protein were identified. Seven sites were located in gp120 and three in gp41. Five sites in gp120 and two in gp41 were defined by overlapping peptides. The remaining two sites on gp120 and one on gp41 were distinguished by competition binding assays but could not be defined by overlapping peptides, suggesting that they were discontinuous or conformational epitopes. Five of the 28 MAbs consistently and reliably neutralized the infectivity of SIVmac251. Two of these bound to a peptide (aa171-190) in the V2 region. The remaining three MAbs bound to a conformational epitope on gp120. These two neutralizing epitopes on SIV are analogous to similar epitopes recently described in HIV-1. In contrast, three MAbs binding to the V3 region of SIV failed to neutralize infectivity, suggesting that this region in SIV may by functionally different from the V3 loop in HIV-1.


Identification of broadly reactive continuous antigenic determinants of simian immunodeficiency virus glycoproteins.

The env polyprotein sequences of several simian immunodeficiency virus (SIV) isolates were analyzed using computer algorithms designed to predict immunologically reactive protein segments. Peptides corresponding to predicted epitopes were synthesized and employed in peptide enzyme-linked immunosorbent assay (ELISA) screening of serum panels from experimentally infected macaques, as well as naturally infected, asymptomatic mangabeys and African green monkeys. Several of the peptides are recognized by a high percentage of antisera from each panel of monkeys indicating that they represent group-specific antigenic determinants of SIV. Several type-specific determinants also were identified. These peptides may be a useful tool for studying the kinetics of SIV glycoprotein-specific immune responses produced by infected and vaccine-protected monkeys at the epitope level.


Identification of a neutralizing domain in the external envelope glycoprotein of simian immunodeficiency virus.

Two murine monoclonal antibodies (MAbs), designated MATG2014 and MATG2033, were generated. They are reactive with the external envelope glycoprotein gp130 of the simian immunodeficiency virus of macaque monkey (SIVmac251), and display a cell-free virus neutralizing activity in vitro. In addition, MATG2014 cross-reacts with HIV-2Rod gp140. Epitope mapping of these MAbs was performed by screening and SIVmac peptide library expressed in yeast and confirmed using synthetic peptides. MATG2014 and MATG2033 recognize two overlapping epitopes localized in an 18 residue domain between amino acid 171 and 188 of the SIVmac251 gp130. Sera from experimentally SIV-infected macaques are immunoreactive with this neutralizing domain. Sequence comparison with related SIV and HIV-2 viral strains indicates a low variability of this region, consistent with the cross-reactivity of MATG2014 with HIV-2Rod gp140. This domain should then be considered in designing experimental vaccines.


[Chronic, post-traumatic scaphoid-lunate instability treated by scaphoid-lunate arthrodesis].

The authors after a review of certain elements of the physiology and pathophysiology of the scapho-lunate couple, report a series of 10 patients presenting a post-traumatic scapho-lunate instability stabilized by scapho-lunate bone graft in order to obtain scapho-lunate arthrodesis. The bone fusion was obtained 5 times out of 10, 3 times complete and 2 times by an incomplete bony bridge. In 5 cases, bone fusion was not evident, a fibrous non-union probably occurred which maintained the correction. Nevertheless, the overall results were considered good in 9 out of 10 cases with only one poor result. The outcome with a mean follow-up of 4 years did not show any arthritic changes. The authors consider that scapho-lunate stabilization with an interposed bone graft is a good method which can ensure good stabilization and good clinical results.


[Lesions of the ligaments associated with distal fractures of the radius. 58 intraoperative arthrographies].

Intracarpal ligamentous tears and fractures of the radius often have a similar mechanism. For instance, no prospective studies have defined the real incidence of such associations, which are not antagonistic. The authors performed a systematic operative wrist arthrogram during distal radius fractures in a group of 58 patients with a mean age less than 50 years. Such a population was at low risk of degenerative ligamentous tears. Triangular fibrocartilage complex was torn in two-thirds of all types fractures. Extra-articular radius fractures were associated with an intracarpal ligamentous tear in 25% and always a luno-triquetral lesion type. In contrast, intra-articular and radius styloid fractures were frequently associated with a scapho-lunate lesion.


[Distal ischemia of the upper limb due to obliteration of the ulnar artery at the wrist: surgical treatment].

So-called "Raynaud's distal ischaemic phenomena of the upper limb may be responsible for disabling functional impairement and even lesions requiring amputation of one or several fingers. The ulnar artery is usually involved in these phenomena because of its anatomical position and its importance in the blood supply of the hand and the fingers. The authors present three clinical cases of distal ischaemia of the upper limb due to obliteration of the ulnar artery at the wrist, treated by microsurgical vein graft. A review of the literature revealed only 23 well documented cases of this type of treatment, i.e. 10% of all treatment options, which gave 95 to 100% of favourable results. The three patients in our series, reviewed after 2, 3 and 5 years, were considered to be cured. Although the severity of the disease can be evaluated by the clinical course, arteriography appears to be the key examination for diagnosis, prognosis and choice of treatment. However, two practical attitudes are recommended to avoid the risk of progression towards digital necrosis: when the symptoms are chronic, arteriography is indicated in the case of resistance to medical treatment or immediately in the case of unstable symptoms. The diagnosis of occlusion of the ulnar artery at the wrist then justifies treatment by microsurgical vein graft; when the symptoms are acute, treatment should preserve as much as possible the palmar and digital ulnar arterial blood supply.


[Chemical burns of the hand and upper limb: therapeutic approach].

Clinical burns of the hand and the upper limb induce various lesions, depending on the degree of tissue penetration and their aggressive properties; strong acids, particularly sulfuric acid and strong bases such as lime are the most dangerous. The clinical attitude should be surgical. Early skin excision, as economic as possible, will be indicated, taking into account the difficulties of precisely determining the limit between normal and necrotic tissues; fluorhydric acid, rapidly dissociates into H+ and Fl- which, because of its small size, will rapidly penetrate deeply into the blood circulation. Moreover this molecule has the peculiar power to provoke marked hypocalcemia able to cause death, due to its avidity for Ca++, with a subsequent cellular lysis. There is currently an increase in domestic accidents, as fluorhydric acid is present in several scouring solutions; in every case of chemical burns, the main treatment remains emergency washing with water possibly, followed by a specific neutralization; skin excision must be the rule when the power of neutralization is limited, because of the risk of progressively deepening lesions due to the presence of residual scouring solutions.


[Techniques of continuous nerve block at the level of the wrist].

Functional treatment without pain is a condition for good results after operations at gliding structures (tendons) or periarticular structures. Good analgesia of the arm and hand is obtained with continuous axillary nerve blocks. Often there is concomitant paralysis and active motion is not possible. We therefore use continuous nerve blocks at the wrist. The technique of these blocks is described in the paper. We use a prefabricated catheter set to introduce the polyethylene catheter and to place it near the nerve as in single shot wrist block. This technique for continuous analgesia of the hand can be used for functional treatment after tenolyses, arthrolyses and stable osteosyntheses of fingers. Median, radial and ulnar nerves can be blocked all at the same time or alone.


The pathophysiology of pain.

The two most common types of pain presented to dentists are acute orofacial pain and chronic craniofacial pain. This article presents some newer concepts in the understanding of the pathophysiology of pain. Pain perception, modulation and transmission are briefly described, followed by a discussion of the physiological and biochemical aspects of chronic and recurrent facial pain.


[Three rare fractures of the radius associated with an ulnar slipped epiphyses].

Over a period of 7 years, three adolescent boys had an associated diaphyseal fracture of the distal third of the radius with separation of the distal ulnar epiphysis. The fracture involved towards early epiphysiodesis of the growth plate of the distal ulna leading to a shortened ulna without any severe functional disability. The mechanisms of injury and complication are described. The treatment of this associated lesion is classical emergency, but the radiological follow-up must be extended in time because the growth potential of the forearm bones is important even during adolescence. The onset of a growth disturbance requires rapid surgical epiphysiodesis of the radius to prevent the development of deformity.


["Congenital" dislocation of the radial head. A bilateral case developing around the age of ten].

Dislocation of the head of the radius has important repercussions on pronation-supination, elbow stability and on the wrist joint. In the absence of a history of trauma, this anomaly is generally considered to be "congenital". Few authors have described the progressive acquisition of this dislocation during growth. The case of a 20 year old man is presented, corresponding to initial subluxation which gradually evolved towards dislocation of the head of the radius at about the age of 11 years. This type of dislocation appears to be related to abnormal growth of the proximal radial epiphysis, responsible for repercussions on the humero-radial articular relations and on the anatomy of the upper extremity of the radius. The patient was treated by ulnar osteotomy at the age of 17 years according to Hirayama's technique, when he was probably already too old. Surgical correction of the subluxation of the head of the radius at an earlier age would probably have been preferable.


[A case of primary intraneural ossification of the ulnar nerve].

The authors report a case of ossification of the ulnar nerve at the elbow. The dense bone tissue spread into the interfascicular space while the epineurium and the fasciculi were undamaged. The pathological tissue was removed and the patient recovered. No similar report has been found in the literature.


[Traumatic avulsion of the triceps brachialis tendon. A case report].

We report a triceps tendon avulsion (less than 40 cases reported in the medical literature). Clinical and radiological signs are: 1) Impossible elbow extension against gravity, 2) Flecks of avulsed osseous materiel from the olecranon on lateral elbow radiograph. Immediate surgical repair always produces good results.


[Pseudoaneurysm of the common digital artery].

Palmar aneurysms are very rare. The authors report a case of pseudoaneurysm in an 18 year old waiter. The mechanism was puncture by a chip of porcelain. The diagnostic errors, clinical and pathological criteria are recalled and the literature concerning this problem is reviewed.


[Primary malignant lymphoma localized in the trunk of the ulnar nerve at the elbow. A case report].

The subject of this report is the exceptional observation of an ulnar nerve syndrome in the right elbow induced by the intra and extra neural development of a primary malignant lymphoma. The patient is a man of sixty-eight. The syndrome evolved over one year. The electromyogram showed marked slowing of the conduction speed on the ulnar nerve. The operation unabled us only to carry out a partial biopsy resection. Complementary chemotherapy was then carried out making a survival of two years and seven months possible without any sign of recurrence or other sites. We have only been able to locate two other published cases of primary malignant lymphoma developing on a nerve trunk and more precisely in on the level of the sciatic nerve. No other case has been described in a nerve trunk in the upper limb.


[Lesions of the distal radio-ulnar joint in compression-extension fractures of the distal extremity of the radius. Six cases with associated anterior dislocation of the ulnar head].

A particular course of markedly displaced wrist fracture associated with complete dislocation of the distal radio-ulnar joint and a real anterior dislocation of the ulnar head is related. In 5 other identical cases, treatment with external fixation achieved a more favourable result. These 6 cases concerning a particular type of wrist fracture, and a review of the literature, led us to suggest a potential pattern of TFCC lesions. Finally, from an anatomical classification of the ulnar compartment, we propose some therapeutic guidelines concerning the ulnar side for more common fractures.


Whipple's disease, familial Mediterranean fever, adult-onset Still's disease, and enteropathic arthritis.

Whipple's disease is a rare multisystem disorder of infectious etiology. Efforts to culture the responsible organism have been unsuccessful. Nucleotide sequencing and amplification of bacterial 16S ribosomal DNA revealed the organism to be most similar to bacteria of the Rhodococcus, Streptomyces, and Arthrobacter genera. Several clinical studies of the long-term use of colchicine for the treatment of familial Mediterranean fever demonstrate its utility for symptom control and prevention of complications by amyloidosis in both adults and children. Normal growth, development, and subsequent fertility were seen in children treated with colchicine. Adult-onset Still's disease has previously been thought to have a generally good outcome, although some patients develop chronic arthritis and disability. No markers have been available for prognosis. A study of 62 patients revealed the presence of polyarthritis, root joint involvement, and rash at initial presentation to be associated with a poorer outcome. Enteropathic arthritis may be seen as a complication of both Crohn's disease and ulcerative colitis. The onset of peripheral arthritis coincides with or follows the onset of bowel symptoms in most cases, whereas spondylitis may precede the onset of inflammatory bowel disease by years. HLA-B27 is present in 50% to 75% of cases of spondylitis. No HLA association with inflammatory bowel disease or peripheral arthritis has been consistently found.


Quantitative determination of mdr1 gene expression in leukaemic cells from patients with acute leukaemia.

By using a quantitative RNA-RNA solution hybridisation method, the average number of mdr1 RNA transcripts per cell was measured in total nucleic acid extracts of leukaemic cells from patients with acute leukaemia. The results in different types of leukaemia were (number of patients with detectable mdr1 RNA/total number of patients; median number of transcripts per cell in samples with detectable mdr1 RNA); de novo untreated acute myelocytic leukaemia (AML): 20/44; 0.7, secondary acute myelocytic leukaemia: 8/13; 1.1, acute lymphocytic (ALL) and undifferentiated leukaemia: 5/14; 0.6, relapsed leukaemia: 7/15; 0.7. Forty-six patients with de novo untreated acute leukaemia (AML: n = 34, ALL: n = 12) were evaluable for response to induction chemotherapy. Twelve of 18 patients (67%) with detectable mdr1 RNA levels achieved complete remission compared to 23 of 28 (82%) with undetectable levels (P = 0.40). The remission duration tended to be longer among patients with undetectable mdr1 RNA (P = 0.08). Leukaemic cells were analysed on consecutive occasions in 12 patients. The level of expression increased in four and decreased in two. In conclusion, expression of mdr1 RNA is common in acute untreated leukaemia. However, treatment with cytostatic drugs seems only rarely to increase the proportion of leukaemic cells that express mdr1 RNA. Expression of the mdr1 gene could be one of several equally important factors contributing to drug resistance in acute leukaemia.


Expression of basic fibroblast growth factor, FGFR1 and FGFR2 in normal and malignant human breast, and comparison with other normal tissues.

The expression of basic fibroblast growth factor (bFGF) and two of its receptors, FGFR1 and FGFR2, was detected using the polymerase chain reaction, and quantified by comparison to the relative amount of product obtained following co-amplification of the ubiquitous glyceraldehyde phosphate dehydrogenase transcript. Varying levels were found in the vast majority of both cancer and non-malignant breast biopsies as well as in samples of several other normal human tissues. Significantly less bFGF was present in cancers (P less than 0.0001). Similarly, FGFR2 product was also much less in cancer tissues (P = 0.0078), as was FGFR1 (P = 0.002). FGFR1 levels in cancers tended to be higher in those which were oestrogen receptor positive (P less than 0.06). Amplification of different coding regions showed evidence of variant forms of FGFR1 RNA. Cancers appeared to have a significantly greater proportion of PCR product corresponding to the region between the third immunoglobulin like domain and the tyrosine kinase domain (P = 0.046). Differential expression was observed in breast cell lines, with bFGF in the normal derived HBL100, HBR SV1.6.1 and 184A1 but little or none in ZR-75-1, MCF-7, T47D and MDA-MB-231. FGFR1 was present in most of these but FGFR2 was absent from T47D, MDA-MB-231 and HBL100. ZR-75-1 cells had a marked preponderance of FGFR1 variants lacking part of the coding sequence. Aberrant receptor processing may provide clues concerning the role of FGF's and their potential involvement in malignancy.


The cytokine-like action of substance P upon B cell differentiation.

B cells respond to a variety of effector molecules that can induce these cells to differentiate. One such molecule is the neuropeptide, substance P (SP). Previous studies have demonstrated the presence of SP receptors on lymphocytes while limited studies have been able to demonstrate the biological significance of their expression. SP has been shown to enhance IgA and IgM responses by Peyer's patch and splenic B cells. A limitation of these studies was that the direct effect of SP upon B cells was not ascertained, suggesting these B cells were stimulated via alternate mechanisms. To this end, evidence here will be discussed that SP can directly interact with clonal B lymphoma cells and highly purified splenic B cells. The data implicate SP as a late-acting B cell differentiation factor that requires an additional triggering mechanism to initiate the B cell differentiation process.


Suramin prevents neovascularisation and tumour growth through blocking of basic fibroblast growth factor activity.

Inhibition of angiogenesis through blocking of growth factors involved in this process could be a novel therapeutic approach in several important pathologies, neoplasia among them. Suramin has recently been described to possess antineoplastic activity in animals and humans, and it has been proposed that an important role in this activity is played by antagonism of growth factors and especially bFGF. To investigate this hypothesis in vivo, we used gelatin sponges loaded with bFGF and implanted subcutaneously in mice. Suramin showed an inhibitory activity on bFGF-induced angiogenesis, whereas it was inactive in the case of heparin-complexed bFGF. Suramin was also studied in an in vivo model of tumour-induced angiogenesis using the murine M5076 reticulosarcoma, a tumour producing significant levels of bFGF. Suramin was able to reduce tumour growth and tumour induced angiogenesis, and exogenous administration of bFGF countered suramin effects.


Prognostic factors in juvenile chronic granulocytic leukaemia.

A retrospective analysis of the clinical and haematological characteristics of patients diagnosed as having juvenile chronic granulocytic leukaemia between 1971 and 1986 was carried out. Thirty-three children were identified who were between the ages of 18 weeks and 8.8 years at diagnosis. The disease was more frequent in boys than girls (23:10). The most common presenting symptoms were skin rash (58%) and bleeding manifestations (45%). All patients had some degree of splenomegaly and in 88% this was more than 3 centimetres below the costal margin. Hepatomegaly and lymphadenopathy were also frequent findings. Anaemia was common and leucocytosis an invariable finding with a white cell count above 50 x 10(9) 1-1 in 42%. Monocytosis was found in 78%. Haemoglobin F measurements were available in 31 children and above 10% in 22 (67%). No child had the Philadelphia chromosome or monosomy 7. Thirty children were treated with chemotherapy, with a variable degree of symptomatic improvement. Twenty-nine patients had died with a median survival time of 5 months. The commonest cause of death was complications of bone marrow failure and no child developed acute leukaemia. Presenting characteristics associated with a longer survival period were age less than 6 months (P = 0.02), female sex (P = 0.02), HbF less than 10% (P = 0.0004) and the absence of bleeding manifestations (P = 0.03). We conclude that the prognosis for children aged over 6 months, with a raised HbF level is very poor, and that, in the absence of possible bone marrow transplantation, consideration should be given to novel treatment approaches for these patients.


Modulation of the mRNAs encoding substance P and its receptor in rat macrophages by LPS.

Numerous soluble factors and their receptors contribute to the regulation of immune responses. An important area of investigation concerns defining the regulation of expression of such receptor/ligand pairs, since understanding such events are central in the quest to manipulate immune responses. Receptors for the neuropeptide, substance P, are present on a variety of leukocytes, and these receptor positive cells respond to in vitro stimulation with substance P in a variety of ways. Unfortunately, little is known about the regulation of expression of substance P or its receptor in leukocytes. Here we begin to address this question by examining the ability of macrophages to express mRNAs which encode substance P and its receptor. A radiolabeled oligonucleotide probe complementary to the mRNA which encodes substance P (i.e., preprotachykinin mRNA) hybridized to a 1.3 kb RNA species present in rat macrophages. In addition, the expression of this RNA could be upregulated 6 to 8 fold when macrophages were stimulated with LPS. The ability of macrophages to synthesize and secrete immunoreactive-substance P was demonstrated by incorporation of L-[35S]methionine into material from macrophage cultures which could be recognized by a monoclonal antisubstance antibody. Macrophage RNA of approximately 3.1 kb in size was capable of hybridizing with an oligonucleotide probe complementary to rat brain substance P receptors. In addition, this RNA could be upregulated when cells were exposed to LPS. Taken together, these studies suggest that the genes used by neuronal cells and macrophages to encode substance P and its receptor are similar if not identical.(ABSTRACT TRUNCATED AT 250 WORDS)


Phylogeny of rapidly growing members of the genus Mycobacterium.

The 16S rRNAs from nine rapidly growing Mycobacterium species were partially sequenced by using the dideoxynucleotide-terminated, primer extension method with cDNA generated by reverse transcriptase. The sequences were aligned with 47 16S rRNA or DNA sequences that represented 30 previously described and 5 undescribed species of the genus Mycobacterium, and a dendrogram was constructed by using equally weighted distance values. Our results confirmed the phylogenetic separation of the rapidly and slowly growing mycobacteria and showed that the majority of the slowly growing members of the genus represent the most recently evolved organisms. The 24 strains which represented 21 rapidly growing species constituted several sublines, which were defined by the following taxa: (i) Mycobacterium neoaurum and M. diernhoferi, (ii) M. gadium, (iii) the M. chubuense cluster, (iv) the M. fortuitum cluster, (v) M. kommossense, (vi) M. sphagni, (vii) M. fallax and M. chitae, (viii) M. aurum and M. vaccae, (ix) the M. flavescens cluster, and (x) M. chelonae subsp. abscessus. Our phylogenetic analysis confirmed the validity of the phenotypically defined species mentioned above, but our conclusions disagree with most of the conclusions about intrageneric relationships derived from numerical phenetic analyses.


Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis.

We studied 48 Borrelia isolates that were associated with Lyme borreliosis or were isolated from ticks and identified three DNA relatedness groups by using the S1 nuclease method. The three DNA groups (genospecies) were associated with specific rRNA gene restriction patterns, protein electrophoresis patterns, and patterns of reactivity with murine monoclonal antibodies. Genospecies I corresponded to Borrelia burgdorferi sensu stricto since it contained the type strain of this species (strain ATCC 35210); this genospecies included 28 isolates from Europe and the United States. Genospecies II was named Borrelia garinii sp. nov. and included 13 isolates from Europe and Japan. Genospecies III (group VS461) included seven isolates from Europe and Japan.


rRNA gene restriction patterns as taxonomic tools for the genus Aeromonas.

In the genus Aeromonas there are at least 13 DNA hybridization groups, which are difficult to differentiate biochemically. We investigated the usefulness of rRNA gene restriction patterns for characterization and identification of the various groups. Genomic DNA was digested with restriction endonuclease SmaI, transferred to a nylon membrane, and hybridized with biotinylated plasmid pKK3535 containing the rrnB operon of Escherichia coli. The SmaI bands at 0.8 to 4 kb but not those at positions corresponding to sizes larger than 4 kb showed a good correlation with hybridization groups, allowing identification of strains to the level of genetic species. We demonstrated that the 567-bp fragment localized between positions 80 and 647 of the 16S ribosomal gene of E. coli was essential for hybridization to the low-molecular-weight fragments, whereas the remainder of the operon did not hybridize to these fragments. On the basis of these results, we concluded that the Aeromonas chromosome contains multiple rRNA operons which may be used for species identification.


Desulfovibrio longus sp. nov., a sulfate-reducing bacterium isolated from an oil-producing well.

A novel type of sulfate-reducing bacteria with unusual morphology was isolated from an oil-producing well in the Paris Basin. The cells of this bacterium, strain SEBR 2582T (T = type strain), are long, thin, flexible rods, contain desulfoviridin, and are physiologically similar to members of the genus Desulfovibrio. On the basis of 16S rRNA sequence data, this strain should be included in the genus Desulfovibrio. However, strain SEBR 2582T differs from other members of this genus morphologically, physiologically, and phylogenetically. Thus, a new species, Desulfovibrio longus sp. nov., is proposed for this organism.


DNA relatedness among some thermophilic members of the genus Methanobacterium: emendation of the species Methanobacterium thermoautotrophicum and rejection of Methanobacterium thermoformicicum as a synonym of Methanobacterium thermoautotrophicum.

DNA reassociation was used to determine levels of relatedness among four thermophilic Methanobacterium strains that are able to use formate and between these organisms and two representative strains of Methanobacterium thermoautotrophicum, strain delta HT (= DSM 1053T = ATCC 29096T) (T = type strain) and strain Marburg (= DSM 2133). Three homology groups were delineated, and these groups coincided with the clusters identified by antigenic fingerprinting. The first group, which had levels of cross hybridization that ranged from 73 to 99%, included M. thermoautotrophicum delta HT, Methanobacterium thermoformicicum Z-245, Methanobacterium sp. strain THF, and Methanobacterium sp. strain FTF. The second and third groups were each represented by only one strain, Methanobacterium sp. strain CB-12 and M. thermoautotrophicum Marburg, respectively (cross-hybridization levels, 13 to 30 and 29 to 33%, respectively). Our results indicate that the name M. thermoformicicum should be rejected as it is a synonym of M. thermoautotrophicum. The taxonomic positions of strains Marburg and CB-12 need further investigation.


Phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas as determined by 16S ribosomal DNA sequencing: lack of congruence with results of DNA-DNA hybridizations.

The phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas were investigated by using small-subunit ribosomal DNA (rDNA) sequencing. Members of the genus Aeromonas formed a distinct line within the gamma subclass of the Proteobacteria. Plesiomonas shigelloides also clustered within the confines of the gamma subclass of the Proteobacteria but exhibited a closer association with members of the family Enterobacteriaceae than with members of the family Aeromonadaceae. Species of the genus Aeromonas exhibited very high levels of overall sequence similarity (ca. 98 to 100%) with each other. Several of the relationships derived from an analysis of the rDNA sequence data were in marked disagreement with the results of chromosomal DNA-DNA pairing experiments. Diagnostic rDNA signatures that have possible value for differentiating most Aeromonas species were discerned.


Bacillus methanolicus sp. nov., a new species of thermotolerant, methanol-utilizing, endospore-forming bacteria.

The generic position of 14 strains of gram-positive bacteria able to use methanol as a growth substrate was determined. All are obligately aerobic, thermotolerant organisms that are able to grow at temperatures of 35 to 60 degrees C. Nine of the strains produce oval spores at a subterminal-to-central position in slightly swollen rod-shaped cells. DNA-DNA hybridization studies, 5S rRNA sequence analysis, and physiological characteristics revealed that all 14 strains cluster as a well-defined group and form a distinct new genospecies. Analysis of the 16S and 5S rRNA sequences indicated that this new species is distinct from Bacillus brevis but closely related to B. firmus and B. azotoformans. The name proposed for this new species is B. methanolicus. The type strain, PB1, has been deposited in the National Collection of Industrial and Marine Bacteria as NCIMB 13113.


Characterization of three thermophilic strains of Methanothrix ("Methanosaeta") thermophila sp. nov. and rejection of Methanothrix ("Methanosaeta") thermoacetophila.

Three thermophilic Methanothrix ("Methanosaeta") strains, strains PTT (= DSM 6194T) (T = type strain), CALS-1 (= DSM 3870), and Z-517 (= DSM 4774), were characterized chemotaxonomically and compared with five mesophilic strains, Methanothrix soehngenii ("Methanosaeta concilii") GP6 (= DSM 3671), Opfikon (= DSM 2139), FE (= DSM 3013), UA, and PM. These methanogens were exclusively acetotrophic and had a characteristic sheathed structure. The DNA base compositions of the strains which we studied ranged from 50.3 to 54.3 mol% guanine plus cytosine. The thermophilic strains often had phase-refractive gas vesicles inside their cells. Denaturing electrophoresis of proteins showed that the mesophilic and thermophilic Methanothrix strains formed two distinct groups and that there were differences in protein patterns between the groups. The difference between the thermophiles and mesophiles was also verified by comparing partial 16S rRNA sequences (ca. 30 base differences in ca. 540 bases). On the basis of our results, we propose the name Methanothrix thermophila for the three thermophilic strains. The type strain of M. thermophila is strain PT (= DSM 6194). We also propose that the name Methanothrix thermoacetophila ("Methanosaeta thermoacetophila"), which was given to strain Z-517 (type strain), should be rejected because of its description, which was based on an enrichment culture, was inadequate.


Phylogenetic position of Cowdria ruminantium (Rickettsiales) determined by analysis of amplified 16S ribosomal DNA sequences.

The 16S ribosomal DNA sequence of Cowdria ruminantium, the causative agent of heartwater disease in ruminants, was determined. An analysis of this sequence showed that C. ruminantium forms a tight phylogenetic cluster with the canine pathogen Ehrlichia canis and the human pathogen Ehrlichia chaffeensis. Although a close relationship between the genus Cowdria and several members of the tribe Ehrlichieae has been suspected previously, the tight phylogenetic cluster with E. canis and E. chaffeensis is surprising in view of known differences in host preference and target cells.


Comparison of the solophenyl-red polarization method and the immunohistochemical analysis for collagen type III.

In the present study, we have compared the staining pattern of the Solophenyl-Red 3 BL-method for the visualization of collagen type III with the immunohistochemical staining in serial sections from 7 skin wounds (wound age 3 days up to 4 weeks) to elucidate the specificity of the histochemical staining method. Large amounts of collagen type III were clearly detectable in the investigated wounds using the immunohistochemical technique. In the sections stained with Solophenyl-Red, however, only 3 out of 7 skin lesions showed a significant positive red staining at the wound margin or in the granulation tissue, while the adjacent normal connective tissue revealed a typical intensive staining. Using polarization microscopy no characteristic bright green fibrils, as reported for collagen type III, could be seen in the wound areas without positive Solophenyl-Red staining. Since the localization of collagen type III detected by immunohistochemistry and the presumed distribution of this collagen type by the Solophenyl-Red method was not identical, the histochemical polarization method has to be regarded as non-specific for visualization of this collagen type.