Cauliflower mosaic virus reverse transcriptase. Activation by proteolytic processing and functional alteration by terminal deletion.

We have previously expressed the cauliflower mosaic virus (CaMV) reverse transcriptase (RTase) gene, the ORFV gene, in yeast in an active form (RTase-Y). An activity gel analysis revealed that the molecular size of RTase-Y as well as an RTase associated with the CaMV particles (RTase-V) is 60 kDa. This size is about 18 kDa smaller than that of the inactive form previously expressed in Escherichia coli (RTase-E) (78 kDa), which corresponds to the coding capacity estimated for the ORFV gene. To investigate the possible involvement of proteolytic processing in the de novo synthesis of CaMV RTase, we constructed a series of deletions from either terminus or both termini of the ORFV coding sequence and expressed them in E. coli. Among the various truncated RTases, those (denoted delta N) that lack N-terminal peptide fragments 143-185 amino acids long were active on the synthetic RNA template-primer, poly(rC)-oligo(dG). Those RTases (denoted delta C) lacking C-terminal peptide fragments 50-102 amino acids long and those lacking both termini (denoted delta NC) were also active on this template. However, only the delta N RTases showed enzyme properties indistinguishable from the RTase-Y in that they transcribed natural RNA into DNA and required either Mg2+ or Mn2+ for their activity. The length of the deletion corresponded approximately to the difference of the molecular weights between RTase-Y and RTase-E. These results suggest that CaMV RTase is translated in an inactive precursor form and then converted to an active form by proteolytic processing during de novo synthesis. We have also demonstrated that C-terminal deletions cause a loss of activity on a natural RNA template accompanied by an alteration in metal ion requirement. The inability to incorporate dTTP accounts for the loss of activity on the natural RNA template. However, the affinities for dTTP and the corresponding template, poly(rA)-oligo(dT), were found to be unaltered.


Elucidation of structural requirements on plasminogen activator inhibitor 1 for binding to heparin.

Plasminogen activator inhibitor 1 (PAI-1), a member of the serpin superfamily of proteins, has been demonstrated previously to interact functionally with the glycosaminoglycan heparin (Ehrlich, H.J., Keijer, J., Preissner, K. T., Klein Gebbink, R., and Pannekoek, H. (1991) Biochemistry 30, 1021-1028). Heparin specifically enhances the rate of association between PAI-1 and thrombin about 2 orders of magnitude, whereas no effect is detected with other serine proteases (e.g. factor Xa). For the heparin-dependent serpins antithrombin III and heparin cofactor II, basic amino acid residues in and around the helix D subdomain were proposed to be involved in the binding of glycosaminoglycans. Here we employed site-directed mutagenesis of full-length PAI-1 cDNA to identify the amino acid residues that mediate heparin binding. To that end, 15 single-point mutants of PAI-1, each having individual arginyl, lysyl, or histidyl residues replaced by a neutral (alanyl) residue ("ala-scan"), and one double mutant were constructed, expressed in Escherichia coli, and purified to apparent homogeneity. The purified biologically active proteins were subjected to the following analyses: (i) heparin-dependent inhibition of thrombin; (ii) heparin-dependent formation of sodium dodecyl sulfate-stable complexes with thrombin; and (iii) binding to and elution from heparin-Sepharose. Based on the data presented, we propose that the amino acid residues Lys65, Lys69, Arg76, Lys80, and Lys88 constitute major determinants for heparin binding of PAI-1. These residues are located in and around the helix D domain and are conserved in the other heparin-dependent thrombin inhibitors, antithrombin III and heparin cofactor II.


Reconstitution of interactions between the Src tyrosine kinases and Ras GTPase-activating protein using a baculovirus expression system.

Ras GTPase-activating protein (GAP) has been implicated in mitogenic signal transduction downstream of oncogenic and receptor tyrosine kinases. Previous studies have suggested that GAP is phosphorylated by oncogenic viral Src (v-Src) and that GAP is associated with a complex containing normal cellular Src (c-Src) in vertebrate fibroblasts. To investigate molecular interactions between the Src kinases and GAP, we developed an in vitro system for reconstituting Src-GAP complexes. For this purpose, we constructed recombinant baculovirus vectors that direct expression of Rous sarcoma virus v-Src, chicken c-Src, and bovine GAP in infected Sf9 insect cells. In vitro reconstitution experiments using baculovirus-expressed proteins demonstrate that both v-Src and c-Src associate in complexes with GAP. In addition, in vitro and in vivo phosphorylation analyses indicate that GAP serves as a substrate for both the v-Src and c-Src tyrosine kinases. To determine which structural features of GAP are involved in interactions with the Src kinases, we constructed recombinant baculoviruses that encode deletion mutants of bovine GAP. Deletion of the GAP amino-terminal portion containing Src homology 2 regions, which are highly conserved structural motifs postulated to mediate interactions among proteins, diminishes GAP phosphorylation and association with Src. This reconstitution system should facilitate further studies of molecular interactions between the Src kinases and GAP.


The association of insulin-elicited phosphotyrosine proteins with src homology 2 domains.

The interactions of the phosphotyrosine (Tyr(P))-containing proteins in basal and insulin-stimulated 3T3-L1 adipocytes with src homology 2 (SH2) domains from phosphatidylinositol 3-kinase (PI3K), ras GTPase-activating protein (GAP), and phospholipase C gamma have been examined. The Tyr(P) forms of the insulin receptor and its 160-kDa substrate protein (pp160) associated with fusion proteins containing either or both the SH2 domains of PI3K, but not with fusion proteins containing the two SH2 domains of GAP or phospholipase C gamma. These results demonstrate a specificity for the association of the Tyr(P) form of the insulin receptor and pp160 with SH2 domains that parallels the reported effects of insulin on PI3K, GAP, and phospholipase C gamma in vivo. Immunoprecipitates of pp160 from the cytosol of insulin-treated, but not basal, 3T3-L1 adipocytes contained PI3K activity. Moreover, the Tyr(P) form of pp160 with associated PI3K activity migrated at 10 S on a sucrose velocity gradient, whereas the Tyr(P) form without associated activity migrated at 6 S. These findings indicate that the Tyr(P) form of pp160 associates directly with PI3K in vivo.


Use of Romanowsky type (Diff-3) stain for detecting Helicobacter pylori in smears and tissue sections.

A Romanowsky type (Diff-3) stain was used for identifying Helicobacter pylori in gastric biopsy specimens from 50 patients with ulcer and non-ulcer dyspepsia. Air dried smears were prepared from fresh biopsy tissue and histological sections were prepared from paraffin wax processed tissue. The Diff-3 technique is accomplished in five steps and takes about 30 seconds. Results using the Diff-3 stain correlated 100% with those using the Giemsa stain. The Diff-3 stain is reliable, simple, rapid, easy and clean, and smears prepared from fresh biopsy tissue can be examined and an immediate report given. The method is recommended for the identification of H pylori in smears prepared from fresh tissue as well as in sections prepared from processed tissue.


HIV-1 sensitivity to zidovudine: a consensus culture technique validated by genotypic analysis of the reverse transcriptase.

In order to select and standardize a reliable assay for the analysis of sensitivity of HIV isolates to AZT, we have compared two culture methods. The first assay (Cell-Associated Isolate Sensitivity Assay: CAISA) quantified AZT-resistant HIV isolates by end-point dilution cultures of peripheral blood mononuclear cells (PBMCs) in the presence of various concentrations of AZT. In the second assay (Cell-Free Isolate Sensitivity Assay: CFISA), following a conventional isolation of HIV, dilutions of infected cell-free supernatants were cultivated with fresh normal donor PBMCs in the presence of increasing concentrations of AZT. Samples from 64 untreated and AZT-treated patients were studied by CAISA (41), CFISA (43) or both assays (20). The CFISA, which allows the determination of titration parameters with respect to various kinetics patterns of viral replication was selected, and some of the CFISA phenotypically characterized isolates were further studied by nucleotide sequence analysis of the reverse transcriptase gene. CFISA showed that isolates from untreated patients were susceptible to AZT while the frequency of resistance increased with the duration of therapy. Genotypic analysis of CFISA-resistant isolates exhibited mutations at crucial positions, particularly at residue 215. We consider CFISA as a consensus culture technique for longitudinal studies of isolates from patients receiving AZT or other analogs of nucleosides.


Fibrous mastocytoma in a patient with generalized cutaneous mastocytosis.

A 57-year-old woman with cutaneous mastocytosis of 23 years duration developed a hyperpigmented abdominal plaque composed of confluent indurated papules that enlarged for a period of 1 year to 12 x 8 cm. Biopsy showed dermal infiltration by closely packed spindle-shaped mast cells, fibroblasts, collagen, and scattered lymphocytes, predominantly T-suppressor cells. Electron microscopy showed close contact between mast cells, fibroblasts, and lymphocytes. Piecemeal mast cell degranulation and extrusion of mast cell granules was seen, with rare mast cell granules in fibroblasts, and collagen fibers in peripheral and perinuclear endoplasmic reticulum of mast cells. the term Fibrous mastocytoma is suggested for this tumor-like dermal fibrosis, possibly induced by lymphokines.


Fibroepithelioma-like changes associated with anogenital epidermotropic mucinous carcinoma. Fibroepitheliomatous Paget phenomenon.

We describe two patients with crusted perineal plaques that were biopsied and diagnosed as Paget's disease. Resection specimens of each contained a dermal mucinous carcinoma with extensive epidermotropism and coexistent epidermal basaloid proliferations closely resembling fibroepithelioma (Pinkus). The presence of the Paget phenomenon was supported by histochemical, immunohistochemical, and ultrastructural evidence. No other primary neoplasms were found in either patient. Followup at 2 1/2 and 3 1/2 years, respectively, has been negative. We conclude that either the fibroepitheliomatous changes may be induced by or may represent a collison (unlikely) with the epidermotropic mucinous carcinoma. It is proposed that the concept fibroepitheliomatous Paget phenomenon be used to stand for the histologic changes common to our cases as well as those previously reported.


Cutaneous lymphadenoma with focal mucinosis.

Cutaneous lymphadenoma of the eyebrow occurred in a 72-year-old man. The nodular tumor was well circumscribed but non encapsulated and invaded the reticular dermis and superficial hypodermis without any connection with the epidermis. Tumor cells had a basal-like appearance and were arranged in small lobules and islands with peripheral palisading and areas of central keratinization. They were often dissociated by an extracellular mucinous material. The stroma was fibroblastic and contained a marked lymphoid infiltrate that surrounded and penetrated the neoplastic lobules. Occasional germ-like hair follicle structures were noted. The immunohistochemical pattern was similar to that of hair follicles, and the leucocytic component was predominantly composed of T-lymphocytes and Langerhans cells, which were specially numerous within the neoplastic lobules. We believe that the tumor, in the present case, has morphologic features consistent with epithelial and stromal attributes observed in hair follicles.


Neurotropic malignant melanoma occurring in a stable burn scar.

Malignant melanoma occurring in burn scars is extremely rare. Nine cases are reported in the literature. We report a case of a small malignant melanoma arising in a stable thermal burn scar after a long latent period of 58 years. The case was unique in that it was of the de novo neurotropic variant. Fascicles of amelanotic, S100 protein-positive atypical spindle cells with neuroid appearance infiltrated deeply in the scarred dermis and perineural spaces. The pigmentation of the lesion was due to solar lentigo instead of atypical melanocytic proliferation in the overlying epidermis.


Tricholemmal carcinoma: a study of seven cases.

Seven cases of tricholemmal carcinoma (TLC), a rarely recognized cutaneous adnexal neoplasm of external hair sheath origin, are described. Most occurred on sun-exposed skin; five involved the head and neck, one the right leg, and one the right thigh. TLC had a generally short history and all were treated by local excision. The lesions had an exophytic (3 cases) or polypoid (4 cases) gross appearance. Histologically, TLC exhibited a sharply circumscribed, lobular epithelial proliferation in continuity with the epidermis. A cytologic hallmark of these tumors was the presence of large cells with PAS-reactive, diastase-sensitive, clear or pale eosinophilic cytoplasm. High mitotic rate was a constant feature. Four tumors were infiltrative, with pushing borders, whereas three were intraepithelial. One case showed acantholysis. Immunocytochemistry revealed positivity for prekeratin and negativity for CEA and EMA, supporting the trichogenic origin of these tumors. Ultrastructural examination gave clear indication of epithelial origin for the cells but did not verify hair follicular differentiation. Despite locally aggressive growth, the clinical course of TLC appeared indolent. Moreover, there are no cases with metastases reported in the literature.


Antiallergic effect of epinastine (WAL 801 CL) on immediate hypersensitivity reactions: (I). Elucidation of the mechanism for histamine release inhibition.

Epinastine caused an inhibition of histamine release from rat peritoneal mast cells induced by both antigen-antibody reaction and compound 48/80. Epinastine was similarly effective in inhibiting compound 48/80-induced histamine release not only from isolated rat peritoneal mast cells but also from rat mesenterial pieces. Also, histamine release from lung pieces obtained from actively sensitized guinea pigs after exposure to antigen challenge was markedly inhibited by epinastine. The drug was effective in inhibiting not only Ca2+ uptake into lung mast cells in actively sensitized guinea pigs but also Ca2+ release from the intracellular Ca store of rat peritoneal mast cells exposed to both compound 48/80 and substance P. No significant changes were observed in phosphodiesterase activity in rat peritoneal mast cells treated with epinastine, while adenylate cyclase activity was augmented by epinastine. Epinastine has no inhibitory effect on histamine release induced by Ca2+ or IP3 from permeabilized mast cells. However, the drug significantly and dose-dependently suppressed calmodulin activity suggesting that histamine release inhibition due to epinastine may be partly attributable to Ca(2+)-calmodulin dependent process(es). The drug caused no visible changes in thermodynamic behavior of lipids, either in order parameter or in differential scanning calorimetry, indicating that the drug has no influence on membrane fluidity.


Immunomodulatory activity of a novel nucleoside, 7-thia-8-oxoguanosine. II. Characterization of induced effector cells and the mechanism of induction.

In a preceding paper we characterized the in vivo and in vitro induction of cytotoxic effector cells elicited by a novel synthetic immuno-stimulator 7-thia-8-oxoguanosine (7T8OG)2. In the present study we further characterized the cells responsible for the induced cytotoxicity and the mechanisms together with the lymphokines mediating the immunological response to 7T8OG. Removal of macrophages from 7T8OG activated spleen cell suspensions by various methods resulted in a significant increase in cytotoxicity to YAC-1 targets. 7T8OG induced effectors did not exert cytotoxic effect on macrophage sensitive P815 target cells. In vivo activated effectors when incubated with anti-asialo-GM1 antibody plus complement lost completely their ability to lyse YAC-1 targets. Together, these findings indicate that the 7T8OG induced effector cells are not macrophage like. Spleen cells from nude mice were readily activated by 7T8OG. The induced effectors were resistant to complement mediated lysis using anti-L3T4, anti-Lyt1 or anti-Lyt2 antibodies. Pretreatment of spleen cells with macrophage depleting agents both, in vitro and in vivo and subsequent activation of cells by 7T8OG resulted in effectors with reduced cytotoxicity. When injected in vivo, 7T8OG induced strong IFN production which paralelled the kinetics of NK cell activation. Furthermore, antibodies to alpha & beta-IFN but not to gamma-IFN diminished the induction of the cytotoxic activity. Although these findings suggest that activation of NK cells by 7T8OG is most likely to be mediated by alpha & beta-IFN involvement of other cytokines can not be ruled out.


Immunomodulatory effects of gamma-HCH (Lindane) in mice.

Mice were fed for 24 weeks with three different subtoxic dosages of gamma-HCH (0.012, 0.12 and 1.2 mg/kg) mixed in powdered feed. The immunological profile was assessed at an interval of one month during the entire exposure period. Both the cell mediated and humoral components of immunity showed a biphasic response characterized initially by stimulation followed by suppression in a dose dependent manner. However, gamma-HCH did not affect the functional properties of peritoneal macrophages. Histological changes in lymphoid organs were in accordance with the biphasic immunomodulatory effects of gamma-HCH.


Isotype determination of anti-Trypanosoma cruzi antibody in murine Chagas' disease.

Isotypic analysis of anti-parasite humoral responses of C57B1/6 and C3H (He) mice surviving acute Trypanosoma cruzi infection showed that both mouse strains demonstrate IgG1, IgG2a, IgG2b, and IgM enzyme-linked immunosorbent assay titers from days 21 to 300 of infection. Using the western blot technique to determine the antigen specificity of the isotypic responses, 100-day infected C3H mice showed strong IgG1, IgG2a, and IgG2b responses to many antigens, whereas C57B1/6 mice showed weak responses to fewer antigens. Isotype western blots showed that reactivity to the T. cruzi antigen of 75-77 kDa is present in the humoral response of day 21-infected mice that will survive and missing in those that will not survive. In general, surviving immunized C3H mice respond with IgG1, IgG2a, and IgG2b reactions to the 75-77-kDa and other antigens, whereas resistant B6 mice concentrate their anti-T. cruzi response in the IgG2b isotype to the 75-77-kDa antigen. Perhaps induction of ineffective antibody responses to nonprotective antigens is beneficial to the parasite and detrimental to the host.


Role of GTP-binding proteins in the regulation of mammalian cardiac chloride conductance.

Beta-Adrenoceptor agonists activate a time- and voltage-independent Cl- conductance in mammalian cardiac myocytes. To characterize the cellular signaling pathways underlying its regulation, wide-tipped pipettes fitted with a pipette perfusion device were used to record whole-cell current and to introduce nucleotides to the interior of guinea pig ventricular myocytes. Replacement of pipette GTP with GDP beta S prevented activation of the Cl- conductance by Iso, suggesting a requirement for G protein turnover. With GTP in the pipette, the effect of Iso could be abolished by the beta-adrenoceptor antagonist propranolol, and mimicked by histamine or forskolin. These actions of Iso and forskolin are mediated exclusively via cAMP-dependent protein kinase (PKA), because (a) maximal activation of the Cl- conductance by forskolin or pipette cAMP occluded the effect of Iso, and (b) switching to pipette solution containing a synthetic peptide inhibitor (PKI) of PKA completely abolished the Cl- conductance activated by Iso and prevented the action of forskolin, but had no further effect. These results argue against basal activation of the Cl- conductance, and make it extremely unlikely that the stimulatory G protein, Gs, has any direct, phosphorylation-independent influence. The muscarinic receptor agonists acetylcholine (ACh) and carbachol diminished, in a reversible manner, Cl- conductance activated by Iso or forskolin, but not that elicited by cAMP. The muscarinic inhibition was abolished by replacing pipette GTP with GDP beta S, or by preincubating cells with pertussis toxin (PTX), and was therefore mediated by an inhibitory G protein, presumably Gi, influencing adenylyl cyclase activity. Nonhydrolyzable GTP analogues (GTP gamma S or GppNHp) applied via the pipette did not themselves activate Cl- conductance, but rendered Cl- current activation by brief exposures to Iso or histamine, but not to forskolin, irreversible. The Cl- conductance persistently activated by Iso was insensitive to propranolol or ACh, but could still be abolished by pipette application of PKI. The data indicate that stimulation of beta-adrenergic or histaminergic receptors in the presence of nonhydrolyzable GTP analogues causes persistent activation of Gs and uncouples it from the receptors. We conclude that autonomic regulation of cardiac Cl- conductance reflects accurately the underlying modulation of adenylyl cyclase activity and, hence, that this system is a suitable mammalian model for in situ studies of the interactions between adenylyl cyclase, Gs, Gi, and forskolin.


New thymidine triphosphate analogue inhibitors of human immunodeficiency virus-1 reverse transcriptase.

Several novel imidotriphosphate analogues of thymidine have been synthesized and have been shown to be effective inhibitors of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT). When the alpha,beta-bridging oxygens of thymidine triphosphate (TTP) and 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP) were replaced by a nitrogen, the resulting analogues were no longer substrates but instead became competitive inhibitors of HIV-1 RT. The most potent of the alpha,beta-imidotriphosphate derivatives tested was thymidine 5'-[alpha,beta-imido]triphosphate (TMPNPP, 1a). This analogue has a Ki value of 2.4 microM, inhibiting HIV-1 RT 400-fold more potently than it inhibits DNA polymerase I large fragment (Klenow). 3'-Azido-3'-deoxythymidine 5'-[alpha,beta-imido]triphosphate (AZTMPNPP, 1b) gave a Ki value about 10-fold greater than that for TMPNPP, indicating that a 3'-azido substituent decreases the affinity of AZTTP to HIV-1 RT relative to the normal 3'-OH substituent. Dideoxythymidine 5'-[alpha,beta-imido]triphosphate (ddTMPNPP, 1c) was intermediate in potency, giving a Ki value of 15 microM. In contrast, substitution at the beta,gamma-bridging oxygen by nitrogen did not block the enzymatic cleavage of the adjacent alpha,beta-phosphate linkage, and 3'-azidothymidine 5'-[beta,gamma-imido]triphosphate (AZTMPPNP, 1e), the 5'-[beta,gamma-imido]triphosphate analogue of AZTTP, is therefore both a substrate for and a potent inhibitor of HIV-1 RT with an observed Ki value of 87 nM. Further nitrogen substitution of the bridging oxygens in the phosphate chain decreases the inhibitory potency by approximately 10-fold, as in the case of thymidine 5'-[alpha,beta:beta,gamma-diimido]triphosphate (TMPNPNP, 1d).


Synthesis and biological evaluation of N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine.

A novel folic acid analogue, N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine, 3, was prepared via a multistep synthetic sequence. The key steps involved the conversion of 5-deazapteroic acid to its N10-formyl derivative followed by catalytic hydrogenation of the pyridine ring and subsequent heating in dilute sodium hydroxide to afford the new 5-deaza-5,6,7,8-tetrahydropteroic acid. After trifluoroacetylation, this compound was coupled to N delta-(tert-butyl-oxycarbonyl)-L-ornithine using conventional peptide bond forming conditions. Deprotection first in base and then in acid gave the title compound. Compound 3 was an effective inhibitor of hog liver folylpolyglutamate synthetase (Kis, estimated = 64 nM), and was shown to retard the formation of polyglutamates of a structurally related folic acid analogue in HCT-8 cells in vitro.


Development of potent and selective CCK-A receptor agonists from Boc-CCK-4: tetrapeptides containing Lys(N epsilon)-amide residues.

A series of Boc-CCK-4 derivatives represented by the general structure Boc-Trp-Lys(N epsilon-COR)-Asp-Phe-NH2, where R is an aromatic, heterocyclic, or aliphatic group, are potent and selective CCK-A receptor agonists. These amide-bearing compounds complement the previously described urea-based tetrapeptides (Shiosaki et al. J. Med. Chem. 1991, 34, 2837-2842); structure-activity studies revealed parallel as well as divergent trends between these two series. A significant correlation was observed between pancreatic binding affinity and the resonance constant R of the phenyl substituent in one particular series of derivatives. Sulfation of phenolic amides appended onto the epsilon-amino group of the lysine did not affect affinity for the CCK-A receptor in contrast to the 500-fold increase in binding potency observed upon sulfation of CCK-8, suggesting that the lysine appendage and the sulfated tyrosine in CCK-8, both key structural elements that impart high affinity for the CCK-A receptor, are interacting differently with the receptor. The amide-bearing tetrapeptides are full agonists relative to CCK-8 in stimulating pancreatic amylase release while being partial agonists in eliciting phosphoinositide (PI) hydrolysis. Both effects were blocked by selective CCK-A receptor antagonists.


Studies on neurokinin antagonists. 1. The design of novel tripeptides possessing the glutaminyl-D-tryptophylphenylalanine sequence as substance P antagonists.

To discover a novel and low molecular weight substance P (SP) antagonist we postulated that the essential binding domain of peptide ligands was only a small portion in the whole structure. On the basis of this assumption, we selected the known octapeptide SP antagonist D-Pro-Gln-Gln-D-Trp-Phe-D-Trp-D-Trp-Phe-NH2 (1) as a lead and synthesized its fragment tripeptides which were evaluated for their activity to block 3H-SP binding on guinea pig lung membranes. The protected tripeptide N alpha-[N alpha-[N alpha-(tert-butyloxycarbonyl)-L-glutaminyl]-N1-formyl-D-tryptophyl]- L-phenylalanine benzyl ester [Boc-Gln-D-Trp(CHO)-Phe-OBzl (4a)], corresponding to the Gln-D-Trp-Phe part of 1, exhibited 7-fold potent inhibitory activity in comparison with 1. Studies on structure-activity relationships revealed that the D-tryptophan, L-phenylalanine, and benzyl ester were quite important to maintain the high binding affinity. It was also indicated that 4a antagonized the SP-induced contraction of isolated guinea pig trachea strips (IC50 = 4.7 x 10(-6) M).


High-dose heparin suppresses platelet alpha granule secretion.

Platelet degranulation has been implicated in the pathophysiology of acute arterial thrombosis, intimal hyperplasia, and atherogenesis. Most previous studies that examined the effect of heparin on platelet function have used platelet aggregometry. These studies have resulted in contradictory data and, by the nature of the assay, reveal no information with regard to platelet degranulation. In contrast, flow cytometry allows accurate quantification of the extent of platelet degranulation by measurement of the platelet surface binding of a GMP-140 specific monoclonal antibody (S12). GMP-140 is only expressed on the platelet surface after platelet alpha granule release. In the present study increasing concentrations of heparin were added to whole blood anticoagulated with sodium citrate. Platelets were activated with a panel of agonists, and the extent of platelet degranulation was quantified by whole blood flow cytometry. Heparin concentrations as high as 100 units/ml were found to suppress platelet alpha granule release induced by either a thromboxane A2 analog (U46619) or a combination of adenosine diphosphate and epinephrine. Heparin suppressed alpha granule release induced by thrombin both in whole blood and in washed platelets. The addition of heparin after platelet activation had no effect on S12 binding. In summary, heparin in high concentrations is a potent inhibitor of platelet degranulation, an action that is unrelated to its effect on the coagulation cascade. Although the heparin concentrations used in this study exceed those used clinically by a factor of 10 or more, future studies of heparin fractions may allow the separation of the anticoagulant and antiplatelet properties of the molecule and allow the administration of an agent that selectively suppresses platelet degranulation without the humoral anticoagulant effect.


The immunosuppressive effect of FK 506 on canine lung transplantation.

The immunosuppressive potency of FK 506 was studied after left lung transplantation in adult mongrel dogs in comparison with cyclosporine. Fiberoptic bronchoscopy, bronchial mucosal blood flow measurement with laser Doppler velocimetry, and chest x-ray and pathologic examinations were performed. Group A had no immunosuppression (n = 5); group B received FK 506 (0.10 mg/kg/day intramuscularly (n = 5); group C received cyclosporine (20 mg/kg/day orally) (n = 5). In group A four dogs died of rejection on the seventh to the twenty-first postoperative days. Another one was killed on the fourteenth postoperative day because bronchial dehiscence occurred at the anastomosis. In group B one died of alveolar rejection on the seventh postoperative day. The remaining four survived 28 days and were put to death. In group C all five dogs survived 28 days and were put to death. In group A bronchial stenosis or dehiscence at the anastomosis was found in every one during the early postoperative period. In group B stenosis did not develop in any of the dogs, including the one that died on the seventh postoperative day. In group C slight stenosis was seen in one dog, severe narrowing in another, and good healing in the remaining three. The transplanted lungs were almost normal histologically in four animals of group B, and one showed alveolar phase rejection. In all animals of group A severe rejection was observed, and in group C two of five animals showed vascular phase rejection, two latent phase, and one fibrosis. Histologic examination of the bronchial anastomosis in group B showed almost normal bronchial epithelium and slight submucosal infiltration of mononuclear cells. In group A there was desquamation of epithelium and mild to moderate mononuclear cell infiltration. In group C hyperplasia of the epithelium was observed in two animals, an abscess at the site of anastomosis in one, and mild to moderate mononuclear cell infiltration in all five. With use of laser Doppler velocimetry, bronchial blood flow in group B was found to be the same as in group C. Laser Doppler velocimetry values reached preoperative levels by the twenty-eighth postoperative day in both groups. Although diarrhea developed in two dogs of group B, no other significant side effect of FK 506 was seen.


Role of nitric oxide in the oxidant stress during ischemia/reperfusion injury of the liver.

The potential role of nitric oxide (NO) and its reaction product with superoxide, peroxynitrite, was investigated in a model of hepatic ischemia-reperfusion injury in male Fischer rats in vivo. Pretreatment with the NO synthase inhibitor nitro-L-arginine (10 mg/kg) did neither affect the post-ischemic oxidant stress and liver injury during the initial reperfusion phase nor the subsequent infiltration of neutrophils into the liver and the later, neutrophil-induced injury phase. Furthermore, no evidence was found for a postischemic increase of the urinary excretion of nitrite, a stable oxidation metabolite of NO. In contrast, the administration of Salmonella enteritidis endotoxin (1 mg/kg) induced a significant diuresis in Fischer rats and an 800-fold enhancement of the urinary nitrite excretion. Nitro-L-arginine pretreatment inhibited the endotoxin-induced nitrite formation by 97%. Hepatic cGMP levels, as index of NO formation in the liver, were only increased significantly after endotoxin administration but not after ischemia and reperfusion. Our results provide no evidence for any enhanced generation of NO or peroxynitrite either systemically or locally during reperfusion and therefore it is unlikely that any of these metabolites are involved in the oxidant stress and liver injury during reperfusion after hepatic ischemia.


Antinociceptive effects of intrathecal taurine and calcium in the mouse.

Taurine (Tau), calcium (Ca+2) and opiates each produce antinociception when injected i.t. in mice. This study was initiated to determine whether there is a common mechanism underlying their antinociceptive effects. Using the abdominal stretch assay, the antinociceptive effects of both Tau (12 nmol) and Ca+2 (72 nmol) were antagonized by i.t. TAG (4.4 nmol), a Tau antagonist, but not by i.p. injection of the opiate antagonist naloxone (5 mg/kg). The antinociceptive effects of Tau and Ca+2 correlated with their ability to inhibit the intensity of caudally-directed biting and scratching behaviors produced by i.t. NMDA or kainic acid. The inhibitory effects of both Tau and Ca+2 on the biting and scratching behaviors behaviors induced by substance P or excitatory amino acids were reversed by TAG, suggesting a common mediation by Tau. These data indicate that the antinociceptive effects of both Tau and Ca+2 appear to be mediated, at least in part, by Tau but not by the release of endogenous opioid compounds. In addition, inhibition of chemical irritant-induced nociception may be produced by a simple blockade of excitatory amino acid activity.


[Use of lectins for the study of carbohydrate determinants of cholinoreceptors of various subtypes in mollusk neurons].

Chemical nature of carbohydrate components in identified neurons of mollusc Helix pomatia influencing acetylcholine responses was investigated using different lectins (Con A, RCA, WGA and LPA). Differences between Con A-evoked changes in acetylcholine-induced chloride and sodium-potassium currents as well as differences in the time and temperature dependences of these changes showed various mechanisms of action of carbohydrate component with mannose residues on the function of acetylcholine receptors. The analysis of the time and temperature dependences of depressing effects of WGA and RCA on acetylcholine-induced currents permits supposing endocytosis of lectin-receptor complexes with N-acetyl-D-glucosamine residues.


Comparison of late and early stage surgery for ruptured intracranial aneurysms.

Surgical indications and timing in serious cases (Hunt and Hess grades 4 and 5) of intracranial aneurysms were investigated. The outcomes in early surgery were compared to the clinical courses of cases intended for late surgery. The survival rate of early surgery patients was 64.4%, better than the total of improved (28.3%) and survived (6.5%) patients for late surgery. There were 28.8% good outcomes in the early surgery group, compared to 2.2% in those treated late. Both of these figures are statistically significant. We consider that early surgery is indicated for serious cases, but not for grade 5 patients with hematoma.


Microprolactinoma invading the cavernous sinus--report of three cases.

Three cases of microprolactinoma with cavernous sinus invasion on magnetic resonance imaging are reported. High-resolution computed tomographic scans did not demonstrate the cavernous sinus involvement. Magnetic resonance imaging is indispensable to detect cavernous sinus invasion before treatment (transsphenoidal surgery or bromocriptine treatment) of a microprolactinoma.


Repeated intracerebral hemorrhage associated with impaired platelet aggregation--report of two cases.

The authors report two cases of hypertensive intracerebral hemorrhage (ICH) repeated at the same site within 1 or 2 days causing death in a 53-year-old male and a 48-year-old female. In both cases, platelet aggregation was significantly impaired. Acquired platelet dysfunction may be important in the expansion of hemorrhage in patients with repeated hypertensive ICH. In such cases administration of normal platelets may be required to prevent devastating hemorrhage.


Ruptured anterior communicating artery aneurysm causing bilateral abducens nerve paralyses--case report.

A rare case of bilateral abducens nerve paralyses after rupture of an anterior communicating artery (AcoA) aneurysm occurred in a 56-year-old female after sudden onset of severe headache. Bilateral abducens nerve paralyses were present without additional neuro-ophthalmological signs. Computed tomography revealed subarachnoid hemorrhage (SAH). Angiography showed an AcoA aneurysm (15 mm in diameter, directed antero-inferiorly) that was successfully clipped. Postoperatively, the bilateral abducens nerve paralyses gradually recovered and disappeared 3 months after onset. Bilateral abducens nerve paralyses may occur after SAH due to ruptured AcoA aneurysm, and neurosurgeons should be alert to this possibility.


Surgical treatment of extracranial distal internal carotid artery dissecting aneurysm--case report.

A 53-year-old male presented with a dissecting aneurysm of the extracranial distal internal carotid artery (ICA) treated by aneurysm removal with interposition of a saphenous vein graft. The surgical approach involved sectioning of Riolan's nosegay.


Delayed postcontrast CT and MR imaging of chondroid chordoma--case report.

A rare case of intracranial primary chondroid chordoma is reported with special reference to neuroradiological findings. A precontrast computed tomographic (CT) scan revealed an isodense mass with multiple flecks in the right cerebellopontine region. A postcontrast CT scan showed slight ring enhancement. A delayed postcontrast CT scan demonstrated marked homogeneous enhancement extending from the middle cranial fossa to the cerebellopontine region on the right. Magnetic resonance (MR) imaging demonstrated a well-defined lesion as a high-intensity mass on T2-weighted image and a low-intensity mass on T1-weighted image. A MR image 5 minutes after gadolinium-diethylenetriaminepenta-acetic acid administration showed heterogeneous enhancement. A MR image 30 minutes after the contrast administration showed more marked, homogeneous enhancement. Such delayed postcontrast CT and MR imaging are useful in the differentiation of chondroid chordoma from classical chordoma.


Radiation-induced osteosarcoma of the calvaria--case report.

The authors report a case of radiation-induced calvarial osteosarcoma. A 58-year-old female received subtotal removal of the pituitary adenoma and 5000 rads postoperative irradiation. Seven years later, an osteoblastic osteosarcoma occurred in the frontotemporal region. She received total tumor removal and chemotherapy. However, computed tomography subsequently revealed multiple small lesions at the margin of the bone flap. A chest x-ray film demonstrated lung metastasis. Local recurrence and lung metastasis require careful attention in radiation-induced osteosarcoma patients.


Subfascial temporalis dissection preserving the facial nerve in pterional craniotomy--technical note.

The subfascial temporalis dissection and reconstruction of the temporalis muscle for pterional craniotomy are described. These procedures preserve the frontotemporal branch of the facial nerve and increase exposure along the sphenoid ridge. A good cosmetic appearance and good temporalis muscle function are achieved postoperatively.


Modified head fixation system for intraoperative CT scanning--technical note.

A head fixation system using four carbon fiber head pins and modified pin supporting devices is described. The fixation system was developed for intraoperative computed tomographic scanning and provides artifact-free images during open-field neurosurgery.


STA-MCA bypass surgery for internal carotid artery occlusion--comparative follow-up study.

Sixty-three patients with internal carotid artery occlusion manifesting as transient ischemic attack or minor stroke received superficial temporal artery-middle cerebral artery bypass surgery and medical treatment (n = 27) or medical treatment only (n = 36). Long-term follow-up showed that there was no significant difference in the outcomes. However, positron emission tomography studies suggested that patients with misery perfusion in the chronic stage benefited from extracranial-intracranial bypass surgery.


Ruthenium red affects the contractile apparatus but not sarcoplasmic reticulum Ca2+ release of skinned papillary muscle.

Ruthenium red has been shown to have a positive inotropic effect on isolated perfused hearts. The cellular mechanism of this action is not clear. Ruthenium red is able to block the Ca2+ release channel in isolated sarcoplasmic reticulum (SR) vesicle and reconstituted channel preparations. However, the effect of ruthenium red on SR Ca2+ release has not been studied in skinned cardiac muscle preparations. In the present study we investigated the actions of ruthenium red on both the characteristics of force generation by the contractile apparatus and Ca2+ release from the SR in chemically skinned rat papillary muscle. Ruthenium red (2 and 10 microM) significantly increased the Ca2+ sensitivity of the contractile apparatus (decreasing Ca2+ required for the half-maximal response from 1.56 +/- 0.04 microM to 1.46 +/- 0.05 microM) but had no effect on the maximal Ca(2+)-activated force in triton X-100 treated fibers. This result may suggest one explanation for the positive inotropic effect of ruthenium red on the heart. On the other hand, ruthenium red had no significant effect on either caffeine-induced Ca2+ release or Ca(2+)-induced Ca2+ release from the SR in saponin-skinned muscle fibers. Lack of a blocking effect on SR Ca2+ release by ruthenium red in skinned fibers suggests that the SR Ca2+ channels in intact preparations have characteristics that are different from those of either vesicular or reconstituted channel preparations.


Activation of nonselective cation channels in the basolateral membrane of rat distal colon crypt cells by prostaglandin E2.

Ion channels in the basolateral membrane of colonic crypts were investigated with the patch-clamp technique during stimulation of secretion. Intact crypts were isolated from rat distal colon and the cell potential was recorded by addition of nystatin to the pipette solution. The cell resting potential in the base of the crypt was -74 +/- 1 mV (n = 90). Addition of 100 microM carbachol to the bath resulted in a transient hyperpolarization by 9 mV, which was probably due to the opening of basolateral K+ channels. In contrast, application of prostaglandin E2 (PGE2, 1 nM-1 microM) caused a dose-dependent depolarization in the base of the crypt. With 1 microM PGE2 cells depolarized from -74 +/- 1 to -27 +/- 2 mV (n = 26). Cell potential recordings in the midcrypt showed only a slight and transient depolarization after application of PGE2, whereas cells close to the surface of the crypt had no response. In the base of the crypt the PGE2-induced depolarization could be completely inhibited by addition of 50 microM flufenamic acid, a known blocker of nonselective cation channels. After substitution of all monovalent cations by N-methyl-D-glucamine in the bath, PGE2 had no significant effect on the cell potential. Cell-attached experiments with no nystatin in the patch pipette revealed the activation of ion channels in the basolateral membrane after application of PGE2. After excision of the membrane patch, these channels could be identified as nonselective cation channels. Experiments involving substitution of the bath solution showed that the channel is impermeable for Cl- and scarcely permeable for Ca2+ ions. The permeability sequence for monovalent cations, as calculated from reversal potentials, is NH4+ greater than Na+ = K+ greater than Rb+ = Li+ much greater than TRIS+ = NMDG+. Single channels are completely inhibited by flufenamic acid (50 microM), mefenamic acid (200 microM), as well as by 3',5-dichlorodiphenylamine-2-carboxylate. In conclusion, PGE2 activates nonselective cation channels in the basolateral membrane of cells in the base of colonic crypts. It is suggested that this mechanism initiates the secretion of K+ ions. Na+ influx through the nonselective cation channel will stimulate the Na+/K+ pump and active uptake of K+ at the basolateral side. K+ can leave the cell at the luminal side through K(+)-selective channels.


Effects of activators and inhibitors of protein kinase A on increases in quantal size at the frog neuromuscular junction.

Adrenaline, permeable cyclic adenosine monophosphate (cAMP) derivatives and insulin are known to elicit an increase in quantal size at the frog neuromuscular junction, primarily by increasing the amount of acetylcholine (ACh) per quantum. The quantal size increases produced by adrenaline or cAMP were antagonized by the protein kinase inhibitor H8 N-[2-(methylamino)ethyl]-5-isoquinolonesulfonamide. The increase in quantal size produced by insulin was not prevented by H8. Quantal size is also increased by pretreatment in hypertonic solution; this increase was also antagonized by H8. The H8 did not alter the increase in miniature endplate potential (MEPP) frequency produced by the hypertonic solution. A permeable cGMP derivative had no effect on quantal size. The diastereomer (Sp)-cAMPS (cyclic 3',5'-phosphothoate) activates protein kinase A(PKA). It elicited an increase in quantal size. The (Rp)-cAMPS isomer is known to inhibit PKA; it had no effect on quantal size. The increase in quantal size produced by hypertonic solution was antagonized by (Rp)-cAMPS but not by (Sp)-cAMPS. Brief exposure to a hypertonic solution containing a phosphodiesterase inhibitor followed by incubation in the inhibitor leads to an increase in quantal size. We conclude that one pathway for signaling for an increase in quantal size involves activation of PKA and that hypertonic pretreatment acts via this pathway.


Mechanism of acetylcholine action on pacemaker current (i(f)) in canine Purkinje fibers.

We have recently reported in canine Purkinje fibers that acetylcholine (ACh) can reverse the positive voltage shift of the pacemaker current (i(f)) induced by beta-adrenergic stimulation while having no direct action of its own. We have now investigated this effect of ACh on the cyclic adenosine monophosphate (cAMP) cascade in more detail. We find that addition of a membrane permeable analogue of cAMP (8-chlorophenylthio cAMP), 0.5-1 mM, increased the amplitude of i(f). This action was not reversed by 1 microM ACh, implying that ACh acts at a step prior to cAMP action. We then looked at the steps controlling intracellular concentration of cAMP. Inhibiting the phosphodiesterase with 100 microM isobutyl-1-methylxanthine (IBMX) increased i(f). This action, however, was reversed by ACh. Finally we investigated whether the action of forskolin, a direct activator of adenylyl cyclase, could be reversed by ACh. Forskolin (10-20 microM) increased i(f), and ACh at 1 microM partially reversed this action of forskolin. These results suggest that, in canine Purkinje fibers, ACh reverses the positive action of beta-adrenergic agents on i(f) via a decrease in cAMP production.


DNA synthesis blocking lesions induced by singlet oxygen are targeted to deoxyguanosines.

In vitro DNA synthesis on single stranded templates damaged by singlet oxygen was investigated in the supF tRNA gene sequence, using several DNA polymerases. Singlet oxygen was generated by the thermal decomposition of the water soluble with the endoperoxide of disodium 3,3'-(1,4-naphthylidene) dipropionate (NDPO2). The data demonstrated that damage at deoxyguanosine residues interrupts DNA polymerization. Modified T7 phage and Thermus aquaticus DNA polymerases were found to synthesize DNA fragments which terminated opposite deoxyguanosine, while T4 phage DNA polymerase and avian myeloblast virus reverse transcriptase were blocked one nucleotide 3' to deoxyguanosine positions on the template. DNA polymerase I (Klenow fragment) from Escherichia coli was inhibited at both positions, before and at the putative damaged sites. The blocking lesions, induced by 5 mM NDPO2, were estimated to be approximately 1.5 per 260 nucleotides, corresponding to 2% of deoxyguanosines. The distribution of lesions in the supF gene did not reveal any specific sequence context which showed distinct susceptibility to the attack of singlet oxygen.


Human HeLa cell enzymes that remove phosphoglycolate 3'-end groups from DNA.

We have purified three chromatographically distinct human enzyme activities from HeLa cells, that are capable of converting bleomycin-treated DNA into a substrate for E. coli DNA polymerase I. The bleomycin-treated DNA substrate used in this study has been characterized via a 32P-postlabeling assay and shown to contain strand breaks with 3'-phosphoglycolate termini as greater than 95% of the detectable dose-dependent lesions. The purified HeLa cell enzymes were shown to be capable of removing 3'-phosphoglycolates from this substrate. Also 3'-phosphoglycolate removal and nucleotide incorporation were enzyme dependent. In addition, all three Hela cell enzymes have been determined to possess Class II AP endonuclease activity. The enzymes lack 3'----5' exonuclease activity and are, therefore, dissimilar to exonuclease III--an E. coli enzyme that can remove 3'-phosphoglycolate.


Symbolic association between individuals and objects by a chimpanzee as an initiation of ownership.

A chimpanzee, named Ai, received feeding training with two other apes. Ai was always fed with the green bowl; the other two apes were fed with different colored bowls. After this training, Ai could make symbolic associations between objects and individuals by using an artificial visual language, based only on her observation of bowls used in feeding without specific training.


Compilation of small RNA sequences.

This is an update containing small RNA sequences published during 1991. Approximately two hundred small RNA sequences are available in this and earlier compilations. The hard copy print out of this set will be available directly from us (inquiries should be addressed to R. Reddy). These files are also available on GenBank computer. Sequences from various sources covered in earlier compilations (see Reddy, R. Nucl. Acids Res. 16:r71; Reddy, R. and Gupta, S. Nucl Acids Res. 1990 Supplement, 18:2231 and 1991 Supplement, 19:2073) are not included in this update but are listed below.


The neuraminidases of Trypanosoma cruzi and Acanthamoeba castellanii are immunologically related.

We have recently reported the presence of neuraminidase (NA) activity in Acanthamoeba castellanii. We now show that the NAs of T. cruzi and A. castellanii share cross-reactive determinants using TCN-2, a monoclonal antibody (mAb) against the T. cruzi NA and a mouse polyclonal Ab (anti-TR) raised against a tandemly repeated dodecapeptide which contains the epitope recognized by TCN-2 (Prioli et al., submitted). This cross-reactivity was demonstrated by the reaction of TCN-2 and anti-TR with A. castellanii parasites using immunofluorescence, immunoblotting and ELISA. Inhibition and immunoprecipitation of enzyme activity confirmed that the A. castellanii antigen recognized by TCN-2 was the NA. Immunoprecipitation of [35S] labeled trophozoite lysates showed the A. castellanii NA to have a molecular weight of 115 kDa. In addition, immunoblot analysis of subcellular fractions obtained by ultracentrifugation showed the A. castellanii NA to be associated with the parasite membrane but not with the cytosol or cytoskeleton fractions. These results suggest that the TCN-2 epitope, contained within a dodecamer tandem repeat unit, is present in T. cruzi and A. castellanii NAs.


Association of a 59-kilodalton immunophilin with the glucocorticoid receptor complex.

Immunophilins, a family of proteins that exhibit rotamase (peptidyl-prolyl cis-trans isomerase) activity in vitro, are expressed in many organisms and most tissues. Although some immunophilins can mediate the immunosuppressive actions of FK506, rapamycin, and cyclosporin A, the physiological role of the unligated proteins is not known. A 59-kilodalton member of the FK506- and rapamycin-binding class was found to associate in the absence of these drugs with two heat shock proteins (hsp90 and hsp70) and the glucocorticoid receptor (GR). Together, these proteins make up the inactive GR, thus biochemically linking two families of proteins proposed to be involved in protein folding and assembly as well as two potent immunosuppressive modalities.


[Effects of substance P on the activities of oviductal isthmic smooth muscle in rabbits].

The effect of substance P (SP) on the activities of oviductal isthmic smooth muscle in rabbits was studied. Rabbits were divided into estrous, diestrous and ovariectomized groups. The results were as follows: (1) SP suppressed the activity of the oviductal isthmic smooth muscle in diestrous rabbit (P less than 0.05). (2) SP did not suppress the activities of the oviductal isthmic smooth muscles in estrous and ovariectomized rabbits (P greater than 0.05).


[Prognostic factors for postoperative mortality in cancer of the esophagus. Analysis of 46 cases].

Forty six patients with esophageal cancer underwent surgery between January 1986 and January 1990. In 14 patients (30.4%) distant metastases were recognized before surgery, whereas in 29 cases (63%) regional neoplastic lymph node infiltration was observed during surgery. Complications during and after surgery occurred in 32 (69.6%) patients and in 30 cases (65.2%) respectively. During the first 30 days after surgery 12 patients died. This represents a postoperative mortality of 26.1%. Among a total number of 51 variables analyzed in this study, 11 influenced the postoperative mortality: duration of intubation, previous history of toxic syndrome, presence of distant metastases before surgery, presence of neoplastic node involvement during surgery, tumor size greater than 4 cm, localization of the tumor at the middle third of the esophagus, respiratory insufficiency, cardiac failure, septic shock, and suture failure during the postoperative phase. However, multivariate analysis revealed that only three of these variables had an independent prognostic value on postoperative mortality: tumor size, presence of distant metastases, and development of respiratory insufficiency during the postoperative period.


[Guanethidine blockade for palliative treatment in reflex dystrophy].

Guanethidine blockade was employed in 22 patients with causalgia, hyperpathia and reflex dystrophy in an upper limb. Guanethidine was administered by means of regional intravenous technique in which 20 mg guanethidine was mixed with 400 IU of heparin and 70 mg lignocaine. Avascularity was present for 20 minutes. The number of blockades was determined in advance. In 68% of the patients, beneficial results occurred as they were relieved of pain or improved considerably. Employment of lignocaine in the injection solution made the blockade less painful.


[The effect of chronic gamma irradiation in an exponentially decreasing dose rate on the nucleic acid content in the hematopoietic organs and blood of rats].

The effect of continuous gamma irradiation at exponentially decreased dose rates (from 562 mGy/h to 13 mGy/h with a total cumulative dose of 14.355 Gy delivered over a period of 10 days) on the nucleic acid content of rat hemopoietic tissues and blood was followed up. The radiation model used simulated a decrease in the radioactivity of a fission mixture in the contaminated environment resulting from a nuclear device accident. We have found that the dynamics of the changes seems to be similar to that observed after acute exposure, and the hemopoiesis recovery starts just at the time of irradiation. In evaluating the damage and recovery extent after accidental irradiation, we consider it expedient to complement the biological dosimetry with the indices studied work including the determination of DNA and RNA concentrations in blood of irradiated human beings.


Carcinosarcoma of the prostate.

A very rare case of carcinosarcoma of the prostate is reported. The patient was a 77-year-old man in whom both primary and metastatic tumors presented the pathology of carcinosarcoma of the prostate. The carcinosarcoma was resistant to anti-androgen therapy, and the patient showed low level of serum prostatic acid phosphatase and was free from bony metastases despite multiple metastases to the lung, liver, pancreas, para-aortic lymph nodes, spleen and penis. The sarcomatous component consisted of chondrosarcoma and fibrosarcoma, both of which were positive for vimentin. The carcinomatous component was positive for both keratin and prostatic acid phosphatase.


Behavior of epithelial differentiation antigens (carcinoembryonic antigen, epithelial membrane antigen, keratin and cytokeratin) in transitional cell carcinomas of the bladder.

Results of an immunohistochemical study in normal urothelium and transitional cell carcinomas of the bladder are presented. Paraffin-embedded material was confronted with immunoantisera against carcinoembryonic antigen (CEA), keratin (K), cytokeratin (CK) and epithelial membrane antigen (EMA). Immunohistochemical findings confirm the changes in reactivity of dysplastic urothelium and carcinoma in situ for CEA, CK and EMA, in comparison with normal urothelium. Statistically significant differences were also found, depending upon tumor stage, in staining of transitional cell carcinomas for K and CK. Expression of CK correlated with the tumor differentiation grade: normal urothelium and well-differentiated carcinomas showed a specific pattern of immunostaining for the basal cells, this pattern being lost in poorly differentiated carcinomas.


Glycolipid expression in prostatic tissue and analysis of the antigen recognized by antiprostatic monoclonal antibody APG1.

The expression patterns of glycolipid from prostatic hyperplasia, prostatic cancer and normal prostate tissue were observed. A further analysis of antigen recognized by mouse monoclonal antibody APG1, which was gained by immunizing glycolipids extracted from human prostate cancer, was also performed. In cancer tissue, both of the lactosyl and globoside series glycolipids were found to be generally reduced, although in the ganglioside series, GM3 and GD3 were not reduced and only the glycolipids with longer chains than GD2 were found to be reduced. These results indicated that the inhibition of sugar chain elongation, but not sialylation, was the main synthetic change occurring with carcinogenesis of the human prostate. APG1 reacted with only two bands near GM2 and GD2 of the ganglioside fraction on a thin-layer chromatography plate, but it did not react with any of the known gangliosides of the ganglioside series including GM2 and GD2. Histochemically, APG1 showed intense reaction only in frozen tissue sections of human prostate, and the reactivity decreased with the increasing grade of cancer. Therefore, this antigen was considered to be a prostate-specific and differentiated antigen reacting with nonganglioseries gangliosides.


Calcification in human osteoblasts cultured in medium conditioned by the prostatic cancer cell line PC-3 and prostatic acid phosphatase.

A medium that had been conditioned by PC-3 cells stimulated the calcification of a human osteoblastic cell line, Tak-10, in a nonmitogenic culture. The calcification of the osteoblasts was stimulated maximally at a 25% concentration of the conditioned medium. Calcification activity was markedly enhanced by the addition of both prostatic acid phosphatase (PAP) and its substrate, alpha-glycerophosphate, to the medium; however, PAP added alone did not enhance this activity. These results suggest that human prostatic carcinoma cells produce a factor that stimulates the calcification of the human osteoblasts. Results have also suggested that PAP is a requisite for osteogenesis provided that its substrates are abundant in the medium.


Synthetic recombinant vaccine expressing influenza haemagglutinin epitope in Salmonella flagellin leads to partial protection in mice.

The influenza virus haemagglutinin epitope 91-108, which is a conserved amino acid sequence in all type A H3 strains, was expressed in Salmonella flagellin, to evaluate its potential as a vaccine. For that purpose, a synthetic oligonucleotide comprising 54 bases coding for the corresponding sequence was inserted into the plasmid pLS408 and transformed into Escherichia coli JM101. Colonies containing the recombinant plasmid were used to transform Salmonella typhimurium LB5000 and were then transduced to a flagellin negative 'live vaccine' aroA mutant of Salmonella dublin. Rabbits immunized either with the live recombinant S. dublin or with the flagellin isolated from it, showed significant levels of IgG response against the synthetic peptide 91-108 as well as against the intact A/Texas/77 influenza virus. Mice immunized with the same preparations developed influenza-specific IgG antibodies in the blood and secreted IgA antibodies in their lungs. Furthermore, these mice showed about 50% protection against challenge infection with the virus. The most successful results were achieved by intranasal immunization with the isolated recombinant flagellin, when employed without the aid of adjuvant.


Vascular remodeling after laser photocoagulation: concurrent regeneration and atrophy.

Laser photocoagulation is associated with paradoxical results: it causes obliteration of vessels, but leads also to the formation of new ones. In an attempt to better understand this dual vascular response we conducted an ultrastructure study of the choroidal vascular repair following krypton laser injury in rats. Three processes were observed: recanalization, neovascularization, and atrophy of both recanalized and newly formed capillaries. Post-lasering repair of the choroidal vasculature can therefore be described as a remodeling process, characterized by both regeneration and involution. The latter appears to be a secondary process of atrophy, contributing to permanent vascular obliteration. These mechanisms might explain why, in spite of initial vascular regeneration, laser photocoagulation treatment has a beneficial effect on choroidal subretinal neovascularization.


[Effects of trihexyphenidyl on slow action potentials in guinea pig papillary muscles].

Effects of trihexyphenidyl (THP) were examined on the slow action potentials (SAP) induced by isoproterenol (Iso), histamine (His), Bay k 8644 or tetrodotoxin (TTX), in guinea pig papillary muscles. THP 10, 50, and 100 mumol.L-1 depressed the maximal upstroke velocity (Vmax) and the action potential amplitude (APA) of SAP. These effects were reversed partially by the elevation of the concentration of calcium from 2.0 to 5.6 mmol.L-1. The results suggest that THP can inhibit calcium inflow in myocardium.


Prediction of N-acetylprocainamide disposition kinetics in rat by combination of gamma variate and physiological pharmacokinetic model.

Clearances and tissue/blood drug concentration ratios of N-acetylprocainamide (NAPA) in rats were determined. The clearances of NAPA in rat blood, liver, and kidney were 13.1, 4.88, and 8.24 ml.kg-1.min-1, respectively. Disposition kinetics of NAPA in rats was predicted with combination of gamma variate and physiological pharmacokinetic model. Equation for estimating the concentration of NAPA in rat blood following iv NAPA 40 mg.kg-1 was C = 55.06t(-0.220) exp(-0.00713t). Using r2 value as a criterion, we found a good agreement between predicted and observed concentrations in blood, lung, small intestine, heart, brain, and skin.


Genetic determination of exocrine pancreatic function in cystic fibrosis.

We showed elsewhere that the pancreatic function status of cystic fibrosis (CF) patients could be correlated to mutations in the CF transmembrane conductance regulator (CFTR) gene. Although the majority of CF mutations--including the most common, delta F508--strongly correlated with pancreatic insufficiency (PI), approximately 10% of the mutant alleles may confer pancreatic sufficiency (PS). To extend this observation, genomic DNA of 538 CF patients with well-documented pancreatic function status were analyzed for a series of known mutations in their CFTR genes. Only 20 of the 25 mutations tested were found in this population. They accounted for 84% of the CF chromosomes, with delta F508 being the most frequent (71%), and the other mutations accounted for less than 5% each. A total of 30 different, complete genotypes could be determined in 394 (73%) of the patients. The data showed that each genotype was associated only with PI or only with PS, but not with both. This result is thus consistent with the hypothesis that PI and PS in CF are predisposed by the genotype at the CFTR locus; the PS phenotype occurs in patients who have one or two mild CFTR mutations, such as R117H, R334W, R347P, A455E, and P574H, whereas the PI phenotype occurs in patients with two severe alleles, such as delta F508, delta I507, Q493X, G542X, R553X, W1282X, 621 + 1G----T, 1717-1G----A, 556delA, 3659delC, I148T, G480C, V520F, G551D, and R560T.


Analysis of four diverse population groups indicates that a subset of cystic fibrosis mutations occur in common among Caucasians.

To determine the nature and frequency of non-delta F508 cystic fibrosis (CF) mutations among diverse populations, we have sequenced exons 9-12 and 19-23 of the CF transmembrane conductance regulator (CFTR) gene from 128 CF chromosomes (39 U.S. Caucasian, 27 African-American, 42 Northern Irish, and 20 Israeli chromosomes). These regions were chosen because they encode the two putative ATP-binding folds of CFTR, domains which appear to have functional significance. In addition, CFTR exons 1 and 2 were analyzed in the American patients. Mutations were found on 49 of the 128 CF chromosomes. Nineteen different mutations were observed; six were novel, while the remaining 13 had been reported previously by our group or by other investigators. Six of nine different mutations found in African-American patients were unique to that population. However, the vast majority of the mutations found in U.S. Caucasians (eight of nine), Northern Irish (four of five), and Israelis (three of three) also occurred in other Caucasian groups. The preponderance of previously reported mutations in these three groups suggested that a subset of the non-delta F508 mutations occur in common among Caucasians. A survey of mutation frequencies in other Caucasian groups confirmed this observation. Unfortunately, this subset accounts for less than half of non-delta F508 CF mutations in most groups. These data suggest that screening for delta F508 and this select group of mutations will efficiently and economically maximize the number of CF mutations identified in Caucasian groups. However, it will be difficult to detect more than 90% of mutant CFTR alleles except in ethnically and geographically discrete populations where CF is the result of founder effect.


A molecular defect in human protoporphyria.

Protoporphyria is generally an autosomal dominant disease that is characterized clinically by photosensitivity and hepatobiliary disease and that is characterized biochemically by elevated protoporphyrin levels. The enzymatic activity of ferrochelatase, which catalyzes the last step in the heme biosynthetic pathway, is deficient in all tissues of patients with protoporphyria. In this study, sequencing of ferrochelatase cDNAs from a patient with protoporphyria revealed a single point mutation in the cDNAs resulting in the conversion of a Phe(TTC) to a Ser(TCC) in the carboxy-terminal end of the protein, F417S. Further, the human ferrochelatase gene was mapped to chromosome 18q21.3 by chromosomal in situ suppression hybridization. Finally, expression of recombinant ferrochelatase in Escherichia coli demonstrated a marked deficiency in activity of the mutant ferrochelatase protein and of mouse-human mutant ferrochelatase chimeric proteins. Therefore, a point mutation in the coding region of the ferrochelatase gene is the genetic defect in some patients with protoporphyria.


Subretinal fibrosis after laser photocoagulation for diabetic macular edema.

Seven eyes had subretinal fibrosis after grid laser photocoagulation for diabetic macular edema. The fibrosis caused persistent loss in visual acuity, and in six of the seven eyes, was not associated with detectable laser-induced Bruch's membrane rupture or subretinal hemorrhage. Choroidal neovascularization was detected in only one patient, who was notably younger (27 years) than the median age of 70 years in this series. The median preoperative visual acuity was 20/80 (range, 20/40 to 20/400); the median postoperative visual acuity was 20/400 (range, 20/80 to counting fingers). The subretinal fibrosis was detected at a median of three months (range, 14 days to 4 1/2 months) after laser therapy. In one of five bilaterally treated patients (20%), subretinal fibrosis developed in both eyes. Subretinal fibrosis may be caused by undetected choroidal neovascularization or by excessive proliferation after stimulation of an aged retinal pigment epithelium. Subretinal fibrosis may be a potential cause of loss in visual acuity after laser treatment for diabetic macular edema.


Macrophage markers in diagnostic neuropathology.

It can be difficult to differentiate the cells of infiltrating gliomas from macrophages associated with demyelinating lesions or cerebral infarcts, especially in tissue with freezing artifact or tissue that is technically suboptimal. We therefore sought to identify a reliable immunohistochemical marker for central nervous system macrophages that could be used on routinely processed tissue. The reactions of commercially available markers [Ham-56, Mac 387, CD68, CR3/43, DR alpha, lysozyme, CD16, and Ricinus communis agglutinin-1 (RCA-1)] were studied in normal cerebellum, subacute infarcts, viral encephalitis with microglial nodules, radiation necrosis, Alzheimer's disease, medulloblastoma, T-cell lymphoma, oligodendroglioma, gliomatosis cerebri, and microgliomatosis cerebri. We also performed a more extensive investigation of Ham-56 with largely surgical material. We concluded that (a) RCA-1 is a sensitive marker for microglia, either resting or responding to injury, but staining must be interpreted carefully because endothelial cells and reactive astrocytes frequently stain as well. This marker was useful in highlighting focal inflammation in HIV encephalitis. (b) CD68 is a specific marker for resting microglia. A case of microgliomatosis cerebri was intensely stained. (c) Ham-56 is a sensitive, specific (with the exception of endothelial cells), and reliable marker of central nervous system macrophages in routinely processed autopsy and surgical material. It is especially useful in distinguishing the macrophages in demyelinating disease and cerebral infarcts from neoplastic glial cells.


Multilocular renal cyst. Immunohistochemical and lectin-binding study.

Multilocular renal cyst is an uncommon lesion of uncertain pathogenesis seen in children and adults. We report the immunohistochemical and lectin-binding profiles of three MRC occurring in adults. All cases had strong and uniform cytoplasmic staining of lining epithelial cells for keratin and binding sites for arachis hypogaea lectin, similar to that seen for the distal convoluted tubules or collecting ducts in normal kidney. However, we found variable expression of other distal nephron markers, including epithelial membrane antigen and Ber-EP4. Furthermore, lining cells in some lesions coexpressed proximal nephron markers such as alpha-1-antitrypsin and lysozyme, as well as binding sites for lotus tetragonolobus lectin. Immunostaining for type IV (basement membrane) collagen demonstrated a continuous subepithelial basement membrane zone and basal laminae surrounding desmin-positive stromal cells. Areas of active collagen synthesis and stromal procollagen deposition were visualized within the interlocular septae using a monoclonal antibody to type I procollagen. Significant proliferative activity was not detected in the lining epithelium or stroma using the anti-proliferating cell nuclear antigen. In conclusion, MRC show aberrant tubular epithelial glycoprotein and glycoconjugate expression, low proliferative activity, and associated activation of interlocular stromal cells.


Argyrophilia and granin (chromogranin/secretogranin) expression in female breast carcinomas. Their relationship to survival and other disease parameters.

Ninety-one tumors (5.6%) containing argyrophilic cells were identified in a series of 1,628 consecutive primary breast carcinomas diagnosed between 1968 and 1972 at the Istituto Nazionale Tumori, Milan, Italy. Histological evaluation of these argyrophilic tumors showed the presence, either throughout the whole tumor mass (pure form) or in some areas (mixed form), of distinctive though not pathognomonic cellular features. Immunocytochemistry revealed the presence of chromogranin A or chromogranin B (secretogranin I) immunoreactivity in 86% of these argyrophilic carcinomas and of neuron-specific enolase (NSE) immunoreactivity in all of them. The three neuroendocrine markers were also immunolocalized at the ultrastructural level in the dense-core granules (granins) and the cytoplasmic matrix (NSE). Immunoblotting studies confirmed the chromogranin A and B and NSE immunoreactivities and documented the presence of secretogranin II. We also studied the relation of the histologic, histochemical, and immunocytochemical features to prognosis. There was no significant correlation between argyrophilia and such clinical parameters as age, menopausal status, tumor size, and overall survival; however, the pure form of argyrophilic tumors had a significant association with less frequent lymph node involvement and a low histologic grade.


Epithelioid hemangioendothelioma of the brain.

Epithelioid hemangioendothelioma is a recently described vascular neoplasm characterized by epithelioid tumor cells and borderline biologic behavior. Its four principal sites of occurrence are the soft tissue, liver, lung, and bone. We report a case of primary cerebral epithelioid hemangioendothelioma in a 4-month-old male infant. The tumor consisted of loose aggregates of epithelioid cells with a focal cordlike or bridging-branching pattern, supported in a fibromyxoid stroma. The tumor cells displayed frequent intracytoplasmic vacuoles. Immunohistochemically, the tumor cells showed positive staining for Ulex europaeus agglutinin, vimentin, and cytokeratin. The tumor pursued an indolent clinical course. The patient was alive 28 months after initial presentation, but he was left with a severe neurological deficit because of the location and growth of the tumor.


The fetal omentum in mice and humans. A site enriched for precursors of CD5 B cells early in development.

From these studies the fetal omentum appears to be an important site of B-cell generation in humans and a source of CD5 B cells in mice. We have analyzed the fetal omentum in other species and have found that B-cell development as determined by the presence of cytoplasmic IgM+ pre-B cells is also detected in the fetal rabbit omentum. We do not know if there is bias towards the production of CD5 B cells in this species; however, these preliminary results demonstrate that this site may be conserved throughout evolution in mammals as a site of B-cell generation. Because the fetal liver is also a source of precursors that can reconstitute this B-cell subset, what is the relationship between omentum and liver during the development of Ly-1 B cells? The most obvious relationship between these two sites is that cells simply migrate from one location to the other; that is, precursor cells may migrate from the fetal liver into the fetal omentum and in this milieu give rise to exclusively Ly-1+ B cells or the sister population. Alternatively, precursors of Ly-1 B cells may arise in the omentum and migrate to the liver. This is demonstrated graphically in the diagram (Fig. 6a) of a transverse section through an 8-week human fetus. In this paper, however, we suggest a model for the development of Ly-1+ B cells from the omentum and liver in which Ly-1 B cells arise from distinct precursors located in situ in the mesodermally derived omentum and mesothelial-derived liver capsule. The omentum primordia forms as the back to back fusion of the mesodermally derived lining of the peritoneal cavity, and this lining surrounds the developing gut when the liver begins to develop as an outgrowth of the intestinal primordia at approximately 3.5 weeks gestation; the outer covering or capsule of the liver is derived from the same tissue of origin as the omentum. Figure 6B is a diagram of a section through the same plane as Figure 6A but the body wall has been omitted. We propose that the Ly-1+ B cells arise in situ in the omentum and lining of the liver as indicated in Figure 6B. That Ly-1+ B cells arise from distinct precursors has been suggested by others, but ours is the first evidence for a developmental site that apparently contains B-cell progenitors for this B-cell subset.


Loss of CD23 is a consequence of B-cell activation. Implications for the analysis of B-cell lineages.

When splenic CD5- B cells are stimulated with antiimmunoglobulin they become CD5+ and have a prolonged in vitro life. Further treatment with IL-6 induces a loss of surface CD23 and IgD; that is, they resemble freshly isolated peritoneal CD5+ cells. These data suggest that the CD5 phenotype is induced after sIg-mediated B-cell activation. Additional support for this view arises from the observation that the loss of CD23 and IgD can be induced by another activation inducer, LPS, although in this case CD5 is not expressed. Thus, activation by anti-Ig plus IL-6 or by LPS induces CD23 loss. Consistent with the hypothesis that the loss of CD23 is a consequence of activation, we now report that the surface expression of CD23 varies inversely with the amount of total cellular RNA. We also find both CD23 positive and negative B cells among freshly isolated splenic CD5- B cells. In young mice a proportion of small splenic CD5+ B cells are CD23+, providing additional evidence that CD23 is present on all B cells prior to activation. A comparison of the features of CD5+ B cells and the antibody responses to thymus-dependent and thymus-independent antigens leads us to hypothesize that the CD5 phenotype arises as a consequence of thymus-independent type 2 (TI-2) stimulation. The relationship of CD5 expression to B-cell lineage (fetal vs. adult bone marrow) is discussed.


Lymphokine production by B cells from normal and HIV-infected individuals.

Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are both secreted by in vivo-activated normal B cells and by in vivo-activated B cells from patients with polyclonal B-cell activation, including individuals infected with the human immunodeficiency virus (HIV). Furthermore, IL-6 and TNF-alpha are involved in autocrine and paracrine regulation of human B-cell differentiation. Following in vitro stimulation of normal B cells with Staphylococcus aureus Cowan strain I and IL-2, there is a rapid but brief increase in supernatant levels of TNF-alpha. There is also an initial increase followed by a subsequent and more sustained increase in IL-6 production. The secondary rise in IL-6 production is dependent upon the prior production of TNF-alpha. There is no significant difference in IL-6 and TNF-alpha secretion by CD5 positive versus CD5 negative tonsillar B cells. Ig production by normal in vitro-activated B cells and freshly isolated B cells from patients with hypergammaglobulinemia is largely dependent upon TNF-alpha and IL-6 production. As another measure of B-cell TNF-alpha and IL-6 production, freshly isolated B cells from HIV-infected individuals induce virus production by chronically HIV-infected cells in which HIV production is known to be triggered by a variety of cytokines. By contrast, freshly isolated B cells from normal controls fail to increase HIV production unless they are stimulated in vitro. Thus, the spontaneous production of IL-6 and TNF-alpha by B cells from individuals infected with HIV may contribute to viral expression as well as to the hypergammaglobulinemia often associated with HIV infection.


Signaling to a CD5+ B-cell clone through surface Ig and MHC class II molecules.

Cells of the CD5+ mouse B-cell clone CH12.LX are induced to become antibody-secreting cells by costimulatory signals delivered by binding of their surface Ig and MHC class II molecules. Class II-mediated signals can be delivered by the binding of either T cells or class II-specific monoclonal antibodies. Divalent, but not monovalent antigen-binding fragments of mAbs are effective in signalling, and cross-linking intact anti-class II mAbs with isotype-specific Ab enhance class II-mediated signaling. Class II-mediated signaling is accompanied by a rise in cAMP and is blocked by an adenyl cyclase inhibitor. The cAMP analogue dibutyryl cAMP, can partially but not completely substitute for the class II-mediated signal. The costimulatory Ig-mediated signal can be delivered by binding of either antigen or antiidiotype Ab, but anti-IgM Abs are much less effective. Antibodies specific for Ig constant regions are much more effective, however, if they engage both the mIgM and mIgD molecules; anti-kappa Abs or a combination of anti-IgM and anti-IgD Abs were more effective in signaling.


VH12 rearrangements in adult peritoneal B cells.

About half of the phosphatidylcholine (PtC)-binding peritoneal B cells of B10.H-2aH-4bp/Wts mice express the VH12 gene. In these cells the D-region gene segments are restricted in length and sequence and are always rearranged to JH1, suggesting that PtC-specific B cells are clonally selected. To assess the extent to which this VH gene is used by peritoneal B cells to encode antibodies of other specificities, we have analyzed the length and sequence of D-region gene segments of VH12-D-JH1 rearrangements in peritoneal B cells independent of antigen specificity by PCR. We find that all 34 randomly chosen VH12-D-JH1 rearrangements analyzed are productive and have D regions that are restricted identically to those of PtC-specific B cells. These data suggest that essentially the entire repertoire of VH12-D-JH1 rearrangements are used by B cells that bind PtC, further illustrating the degree to which this repertoire is shaped by antigen selection.


T cell-dependent antibody production by Ly-1 B cells.

Through the use of a SCID transfer system, we have demonstrated that under certain conditions, the production of Ig by Ly-1 B cells can be modulated by T cells. This modulation can take the form of enhanced isotype production or isotype-switch induction and to some extent appears to be dependent on the activation state of the T cells. Furthermore we have shown that Ly-1 B cells can mount an idiotypically restricted T cell-dependent immune response to the antigen PC-KLH. This result suggests that the previous failure to observe T cell-dependent responses by Ly-1 B cells has been due to these B cells being "blind" to the antigens used and is not due to some inherent property of these B cells. When one considers the previous reports of the substantial contribution of Ly-1 B cells to the natural serum immunoglobulin levels and the ability of T cells to affect Ig production by Ly-1 B cells documented in this report, it is clear that the interaction of T cells with the Ly-1 B-cell population is important in determining the "natural" serum Ig repertoire of the mouse.


Characteristics and development of the murine B-1b (Ly-1 B sister) cell population.

In this paper we have outlined the evidence for two distinct branches of the B-1 cell lineage. The data show that phenotypically B-1a and B-1b cells are essentially identical, distinguished only by the presence or absence of the CD5 antigen. Functionally no differences between the two populations have yet been identified. Both produce anti-PtC antibodies, a specificity not observed in conventional B cells. Both produced high levels of IgM as measured in adoptive transfer experiments. Developmentally, B-1a and B-1b cells are indistinguishable with respect to generation from progenitors present in fetal liver and omentum, feedback regulation of new B-1a and B-1b cells from bone marrow, self-replenishment from Ig+ cells following adoptive transfer, and the generation of clonal populations. The major difference in the two populations is seen in the development of B-1a and B-1b cells from B220- progenitors in the adult bone marrow. Although B220- B-1a progenitors are rare in adult (greater than 6 weeks) bone marrow, the progenitors for B-1b cells persist well into adulthood. Our understanding of B-1b cell ontogeny is at a stage similar to that of B-1a cells five years ago. We have evidence from transfer experiments that strongly suggests the existence of two distinct progenitors for B-1a and B-1b, but we have yet to physically separate these progenitors as Solvansen et al. have done for B-1 and conventional B cells. Furthermore we must determine whether the B-1b cells that develop from fetal liver and bone marrow are functionally and developmentally equivalent to those that develop from adult bone marrow. As with B-1a cells, the role of B-1b cells in the immune system is unclear. Although we have not yet discerned functional differences between B-1a and B-1b, given the recent identification of CD72 (Lyb-2) as the ligand for CD5, it is tempting to speculate that B-1a cells are more involved in B-B cell interactions such as idiotype-anti-idiotype regulation of the early B-cell repertoire and that B-1b cells are more involved in B-T cell interactions. Whatever their function, it is clear that in trying to understand the role of the B-1 lineage it is important to consider both the B-1a and B-1b lineages.


Immunoglobulin V gene expression in CD5 B-cell malignancies.

Chronic lymphocytic leukemia (CLL) and small lymphocytic lymphomas (SLL) generally are malignancies of CD5 B cells. Immunophenotypic and clinicopathologic data, however, are required to distinguish subtypes that apparently have a different cytogenesis than that of conventional CLL or SLL. In addition to expressing CD5, neoplastic cells of the latter are also distinctive in that they frequently coexpress surface immunoglobulin (Ig), bearing one or more cross-reactive idiotypes (CRIs) (e.g. 17.109, G6,) that commonly are found on monoclonal IgM autoantibodies. The frequent occurrence of such CRIs reflects both the biased rearrangement and subsequent selected expression of Ig V genes with little or no somatic mutation. IgM/L CLL, for example, frequently (8/33) harbor abortive Ig rearrangements involving Humkv325, the VK gene encoding the 17.109-CRI. Also, the VH1 gene(s) encoding the G6 CRI accounts for over 10% of all VH genes and over 60% of all the VH1 genes used in randomly selected common CLL/SLL. Furthermore, comparison with the Ig expressed by nonmalignant G6 CRI+ B cells reveals an apparent restriction in the CDR3 of IgH expressed by G6 CRI+ CLL. Coupled with the observed potential bias in antibody light chain and heavy chain pairing in B-CLL, these data suggest that the autoantibodies expressed in this disease are selected based on antigen-binding activity. Collectively, our studies indicate that nonstochastic Ig V gene rearrangement and subsequent selection may influence the Ig repertoire expressed in this common B-cell malignancy.


Large-cell "mixed-phenotype" lymphoma in AIDS. Identification of a CD5-expressing subset of B-cell non-Hodgkin's lymphoma.

We have characterized cells from an AIDS-associated non-Hodgkin's lymphoma (NHL). We found a malignant CD5+ B cell (approximately three-fourths of cells), with the balance of cells comprised of T cells having an activated phenotype. The lymphoma was unusual, in that all cells bear "T-lineage" markers, CD3 and CD5, and "B-lineage" markers, CD19 and CD20. Thus, conventional immunohistologic techniques were insufficient to type this mixture of cells; single-cell analysis was required. These "mixed-phenotype" lymphomas may occur frequently in AIDS-associated NHL. Our results show striking similarity to those obtained in the analysis of certain murine CD5+ B-cell lymphomas, suggesting that the study of lymphomagenesis in the mouse may aid in the understanding of AIDS-associated NHL.


CD20 and CD5 expression in B-chronic lymphocytic leukemia.

In order to quantitate a previously noted decrease in CD20 fluorescence intensity (FI) on B-CLL lymphocytes, binding capacities [BC x 10(3) +/- 1SD = number of antibodies bound per cell] were calculated. The mean (N = 5) BC x 10(3) +/- 1SD of CD20 reagents for normal B-PBL and B-CLL lymphocytes confirmed this observation. B-PBL and B-CLL were 56 +/- 11 and 61 +/- 14, and 19 +/- 15 and 18 +/- 16, respectively, for Leu 16 and B1. Although adequate compensation standards for the determination of CD5 and CD20 coexpression are not available, qualitatively, the density of CD5 on both normal B-PBL and B-CLL is less compared to the expression of CD5 by normal T cells. CD5 expression on B-CLL seems to be linked to the lower levels of CD20, whereas CD5 expression may appear to be absent on CLL lymphocytes expressing normal levels of CD20. Levels of CD20 in B-CLL suggest involvement of one or two genes (alleles) whose decreased expression may be linked to CD5 expression.


Immunoregulatory capability of murine CLL-like CD5+ B cells.

NZB mice spontaneously develop clones of hyperdiploid CD5+B cells, which are much like the malignant cells of CLL. We have found that these clonal CD5+B cells are immunoregulatory. The development of these cells is accompanied by the loss of detectable normal percentages of IgM+ B220(6B2)+ B cells secondary to hyperplasia of splenic non-B cells, which are positively regulated by the hyperdiploid B-cell clone.


Genetic regulation of CD5+ B cells in autoimmune disease and in chronic lymphocytic leukemia.

CD5+ B cells have attracted much attention, because of their involvement in both autoimmunity and B cell-type chronic lymphocytic leukemia (B-CLL). B-CLL is a type of leukemia most often occurring among close relatives and is partly associated with the major histocompatibility complex (MHC), a finding relevant to autoimmune disease. We established MHC (H-2)-congenic NZB x NZW (NZB/W) F1 mice (H-2d/z, H-2z/z, and H-2d/d), in that only H-2d/z heterozygotes developed severe SLE, associated with IgG anti-DNA antibodies, as the animals aged. Such age-associated changes occurred in parallel with the decrease in the splenic, but not peritoneal, CD5+ B cells. By contrast, H-2z/z homozygotes did not develop SLE but, in turn, a marked clonal proliferation of CD5+ B cells resembling B-CLL did occur. H-2d/d homozygotes also did not develop the typical SLE, and a moderate CD5+ B frequency persisted. Despite the finding that all the three H-2-congenic NZB/W F1 strains produced IgM anti-DNA antibodies, only the H-2d/z heterozygotes produced IgG antibodies. Whereas the surface phenotype of major IgM producers was CD5+ sIgM+, that of IgG producers was CD5-sIgM-. Genetic and cellular analyses supported our thesis that in the heterozygotes IgM to IgG isotype switching probably emerges in CD5+ B cells and that this event is associated with the loss of CD5 molecules. Because of the lack of genetic elements required for differentiation, only signals for proliferation would be functioning in CD5+ B cells in the H-2z/z homozygotes. These observations infer that certain different, but related, MHC haplotypes may predispose either to B-CLL or to autoimmune disease in close relatives.


Specificity and idiotope expression of IgM produced by CD5+ and CD5- cord blood B-cell clones.

Epstein-Barr virus (EBV)-immortalized monoclonal B-cell lines were established from CD5+ and CD5- cord-blood B cells. IgM from many of both CD5+ and CD5- clones reacted with IgG-Fc, ssDNA, and a variety of other autoantigens. More CD5+ B cells that used light chains of the kappa isotype reacted with IgG-Fc and ssDNA than kappa-bearing CD5- B cells. Because many of the clones reacted with IgG-Fc, they were analyzed for the expression of cross-reactive idiotypes (CRI) associated with rheumatoid factor and cold agglutinin paraproteins using murine antibodies (mAb) recognizing V kappa and VH subgroup-associated determinants. Expression of the V kappa IIIb sub-subgroup-associated idiotope recognized by 17.109 mAb was expressed at significantly higher frequency (32%; p less than 0.05) and IgM antibodies derived from the CD5+ compared with the CD5- clones (5%). Both CD5+ and CD5- clones expressed the RF paraprotein-associated idiotope recognized by G8 mAb to the same extent. Similar results were obtained using binding to SpA as a marker of VH III family usage. Furthermore, no differences in frequency of expression of RF paraprotein-associated idiotopes recognized by B6 and/or D12, and characteristic of some antibodies using VH III family genes, were found between the CD5+ and CD5- populations. Although a higher than expected frequency of VH IV-gene expression was demonstrated (around 30%) in both CD5+ and CD5- cells, there were differences in expression of CRI recognized by mAb Lc1 and R2.1A2 with specificities for two VH IV subfamilies. While some CD5+ and CD5- clones were identified in which their IgM reacted with mAb Lc1, only CD5+ clones were recognized by another mAb R2.1A2. Analysis of the relationships between antigen specificities and V kappa- and VH-family gene usage indicated that auto- or polyreactivity was not associated with V kappa III nor any particular VH family. The higher frequency of the V kappa IIIb sub-subgroup-associated idiotope recognized by 17-109 in the CD5+ clones and the association of CD5+ B cells with the VH IV subfamily recognized by mAb R2.1A2 and 9G4 may suggest that CD5+ B cells in cord blood are expanded as a result of recruitment within the fetal environment.


CD5+ B lymphocytes and T-cell subsets in a case of juvenile rheumatoid arthritis.

Recent research has demonstrated that CD5+ B lymphocytes have an important role in autoimmune and rheumatic diseases. In a case of juvenile rheumatoid arthritis (JRA), we observed the behavior of these cells and other subpopulations of T lymphocytes of peripheral blood before and after therapy with thymopentin (TP5). All the lymphocyte subpopulations returned to normal range, except the CD5+ B cells, where the percentage remained abnormally high (60%). These data suggest that CD5+ B cells can be a useful monitoring index and confirm their important role in the pathogenesis of juvenile rheumatoid arthritis.


Comparative evaluation of CD5 B cells in patients with rheumatoid arthritis and essential mixed cryoimmunoglobulinemia using FACS analyzer and FACSCAN flow cytometer.

In this study, using both a FACSCAN flow cytometer and the FACS analyzer, and two-color fluorescence, CD5 B cells have been enumerated in the peripheral blood (PB) of normal controls (NC) and patients with rheumatoid arthritis (RA) or essential mixed cryoimmunoglobulinemia (EMC). Using a FACSCAN flow cytometer, no significant difference was observed between the percentage of CD5 B cells in the NC (21.24% +/- 3.71) and RA (24.83% +/- 3.85), or EMC (21.03% +/- 6.38). Using the FACS analyzer, however, there was significant difference between the NC (9.14% +/- 3.82) and EMC (2.84% +/- 2.58) (p less than 0.05), but no significant difference between the NC and RA (7.17% +/- 2.55). Further analysis of 10,000 B cells selected by live gating showed that there were three populations of B cells, with the majority of B cells unstained, a small number of cells brightly stained for CD5 in the NC (3.78% +/- 1.09) or RA (2.80% +/- 0.96), and about 6.45% to 9.43% with intermediate staining in patients with RA and the NC, respectively. In addition, serum low molecular weight IgM (LMW IgM) from patients with RA was detected by an enhanced chemiluminescence detection system combined with a modified immunoblot technique. A significant linear correlation was observed between the percentage of CD5 B cells and the height of LMW IgM peak (r = 0.69, p less than 0.05).


B-cell subsets defined by the Fc epsilon R.

The data summarized herein demonstrate the utility of the low-affinity Fc epsilon R in delineating murine B-cell subsets. In the peritoneal cavity, the Fc epsilon R appears to be a reliable marker in distinguishing between conventional (Fc epsilon R+) and Ly-1/sister (Fc epsilon R-) B cells. In the spleens of normal animals, flow cytometric and histologic studies established that a distinct population of Fc epsilon R- B cells is also present and comprises the marginal zones. Thus in the spleen, the Fc epsilon R may be the first murine marker to allow for selective purification and analysis of marginal zone B cells. Although it is unlikely that splenic Fc epsilon R- B cells are directly related to peritoneal Fc epsilon R- Ly-1/sister B cells, further studies will be required to address this question. Analysis of autoimmune mice revealed that the splenic Fc epsilon R- subset is greatly expanded in these animals and indicates that the Fc epsilon R may be a sensitive indicator of abnormalities within the B-cell compartment. Additional studies compared the functional capacity of Fc epsilon R+ and Fc epsilon R- B cells and tested the ability of these populations to isotype-switch and respond to polyclonal stimuli. The results showed that Fc epsilon R+ and Fc epsilon R- B cells from both the peritoneum and spleen can switch to produce IgG, and all but the peritoneal Fc epsilon R- B cells can switch to the IgE class. This latter result is certainly interesting and demonstrates an important functional difference between peritoneal and splenic Fc epsilon R- B cells. Finally, experiments with B-cell mitogens showed further differences between the Fc epsilon R+ and Fc epsilon R- subsets. Whereas Fc epsilon R- B cells appeared to be more sensitive to LPS stimulation, Fc epsilon R+ B cells were clearly more responsive to an anti-IgM signal. Taken together, the results show that the Fc epsilon R is likely to be useful in separating B cells with different phenotypic, histologic, and functional characteristics.