Relevance of level IIb neck dissection in patients with papillary thyroid carcinoma.

BACKGROUND: Cervical nodal metastasis is a key prognostic factor in patients with papillary thyroid carcinoma. The role of lymph nodes in papillary thyroid carcinoma management and prognosis remains controversial. METHODS: Level IIb lymph nodes obtained from 44 patients with papillary thyroid carcinoma were histopathologically examined retrospectively. Specimens were classified as ipsilateral or contralateral. The number of dissected nodes and prevalence of level IIb metastasis were compared according to pre-operative clinical nodal stage. RESULTS: In the node-negative neck, the prevalence of contralateral and ipsilateral IIb nodes was 0 out of 20 and 0 out of 3, respectively. In the node-positive neck, the prevalence of contralateral and ipsilateral IIb nodes was 1 out of 13 (7.70 per cent) and 3 out of 41 (7.32 per cent), respectively. Clinically determined and pathologically confirmed level IIb node negativity were significantly associated. Thirty-four patients (77.3 per cent) developed accessory nerve complications from level IIb dissection. CONCLUSION: Level IIb neck dissection for papillary thyroid carcinoma may be required if pre-operative examination reveals multilevel, level IIa or suspicious level IIb metastasis.

The interaction of Herpes Simplex Virus with murine lymphocytes. I. Mitogenic properties of herpes simplex virus.

Herpes simplex virus (HSV) stimulates DNA synthesis in mouse spleen cultures prepared from normal, macrophage-depleted, and T-cell-depleted spleen cells, but not from thymocytes. In addition, a polyclonal antibody response is observed in HSV-infected spleen cultures. These findings indicate that the cells stimulated to undergo DNA synthesis after HSV infection appear to be the bone marrow-derived lymphocytes. The newly synthesized DNA is host cell and not of viral origin. Heat treatment and ultraviolet irradiation of HSV before addition to spleen cultures prevents the induction of DNA synthesis. We consider the use of this system as assay for the study of cell transformation by HSV and also for the study of host cell control of the expression of the viral genome.

Biochemical and biological characterization of lymphocyte regulatory molecules. I. Purification of a class of murine lymphokines.

Murine spleen cells activated by concanavalin A (Con A) in culture produce a class of lymphokine molecules which possess biological activity in a number of lymphocyte response assays. Lymphokines with a mol wt of 30,000, as estimated from gel filtration studies, can be resolved into two components which differ by charge, with isoelectric point (pI) values of 4.3 and 4.9, respectively. Both components stimulate (a) the growth of established T-cell lines in culture, (b) the proliferation of thymocytes in the presence of Con A under culture conditions where Con A alone is nonmitogenic, (c) the induction of antibody responses to heterologous erythrocyte antigens in athymic (nude) spleen cultures, (d) the generation of cytotoxic T lymphocytes (CTL) in thymocyte cultures, and (e) the generation of CTL in nude spleen cultures. In each of these culture systems we suggest that the assays are detecting a single class of lymphokine which acts directly on activated T cells. Nonactivated T cells must be stimulated by either antigen or mitogen before becoming responsive to lymphokine, but do not require antigen or mitogen for continued growth with lymphokine. The two molecular species, separable by isoelectric focusing are referred to as the T-cell growth factor (TCGF). A lymphokine, similar in size (30,000 daltons) to TCGF but heterogeneous in charge (pI 3.0--4.0), stimulates immune responses to erythrocyte antigens in T-cell-depleted spleen cultures but has no stimulatory activity in the other lymphocyte assay systems described. The data have been interpreted as showing the two molecular forms of murine TCGF (pI 4.3 and 4.9) are responsible for many of the lymphokine activities described elsewhere as thymocyte mitogenic factor, nonspecific T-cell-replacing factor and killer helper factor or costimulator. The other lymphokine, separable from TCGF by charge, appears to have true T-cell-replacing activity.

B cell precursor growth-promoting activity. Purification and characterization of a growth factor active on lymphocyte precursors.

We have used a biological assay system we developed to biochemically purify a previously uncharacterized murine lymphopoietic growth factor designated lymphopoietin 1 (LP-1). This factor is capable of stimulating the proliferation and extended maintenance of precursor cells of the B lineage. A stromal cell line producing LP-1 was established after transfection of primary stromal cultures with a plasmid encoding the transforming genes of SV40. This factor was purified to a single 25-kD species from the culture supernatant of an adherent stromal cell line. This material acts on immature lymphocytes, it binds to specific receptors on cells, and is distinct from previously described hematopoietic factors. LP-1 has been purified some 10(7)-fold with an overall recovery of 35%. The purified protein exhibits a specific activity of approximately 4 X 10(6) U/micrograms of protein and is active at a half-maximal concentration of 10(-13) M.

Purification to homogeneity of B cell stimulating factor. A molecule that stimulates proliferation of multiple lymphokine-dependent cell lines.

Murine B cell stimulating factor 1 (BSF-1) was purified to homogeneity from supernatants of a stimulated thymoma cell line. A protein of 18.4 kD with a unique N-terminal amino acid sequence was identified. BSF-1 had a sp act of at least 3.28 X 10(8) U/mg. In addition to its B cell-stimulatory activity, BSF-1 also stimulated the proliferation of several IL-2- and IL-3-dependent cell lines. We conclude that BSF-1 is both a growth factor and a differentiation factor. Finally, these results also suggest additional biologic properties of BSF-1 on lineages besides B lymphocytes.

Human interleukin 1. Purification to homogeneity.

We have purified human interleukin 1 (IL-1) to homogeneity by a simplified procedure that results in excellent yields of pure material that retains a high level of biological activity. IL-1, secreted by human peripheral blood macrophages that have been stimulated with Staphylococcus aureus, was purified by ion exchange chromatography and affinity chromatography on Procion Red agarose. The pure protein has a specific activity of 3.2 X 10(8) U/mg in the thymocyte mitogenesis assay, and is pyrogenic. No molecular weight heterogeneity was observed, in contrast to findings for mouse IL-1 and earlier reports of human IL-1. Purified IL-1, as analyzed by two-dimensional electrophoresis/electrofocusing gels, exhibited a series of charged species with isoelectric points ranging from 6.0 to 4.9, all with a molecular weight of approximately 17,500. Amino acid analysis indicated an abundance of acidic residues, in agreement with the low isoelectric points. There is little or no cysteine in the molecule. No evidence was found for the presence of carbohydrate moieties. The overall yield for this procedure was approximately 31% of the activity contained in the initial culture supernatant.

Induction of macrophage tumoricidal activity by granulocyte-macrophage colony-stimulating factor.

Monocytes are a subpopulation of peripheral blood leukocytes, which when appropriately activated by the regulatory hormones of the immune system, are capable of becoming macrophages--potent effector cells for immune response to tumors and parasites. A complementary DNA for the T lymphocyte-derived lymphokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), has been cloned, and recombinant GM-CSF protein has been expressed in yeast and purified to homogeneity. This purified human recombinant GM-CSF stimulated peripheral blood monocytes in vitro to become cytotoxic for the malignant melanoma cell line A375. Another T cell-derived lymphokine, gamma-interferon (IFN-gamma), also stimulated peripheral blood monocytes to become tumoricidal against this malignant cell line. When IFN-gamma activates monocytes to become tumoricidal, additional stimulation by exogenously added lipopolysaccharide is required. No such exogenous signals were required for the activation of monocytes by GM-CSF.

Homologous recombination repair is regulated by domains at the N- and C-terminus of NBS1 and is dissociated with ATM functions.

The proteins responsible for radiation sensitive disorders, NBS1, kinase ataxia-telangiectasia-(A-T)-mutated (ATM) and MRE11, interact through the C-terminus of NBS1 in response to the generation of DNA double-strand breaks (DSBs) and are all implicated in checkpoint regulation and DSB repair, such as homologous recombination (HR). We measured the ability of several NBS1 mutant clones and A-T cells to regulate HR repair using the DR-GFP or SCneo systems. ATM deficiency did not reduce the HR repair frequency of an induced DSB, and it was confirmed by findings that HR frequencies are only slightly affected by deletion of ATM-binding site at the extreme C-terminus of NBS1. In contrast, The HR-regulating ability is dramatically reduced by deletion of the MRE11-binding domain at the C-terminus of NBS1 and markedly inhibited by mutations in the FHA/BRCT domains at the N-terminus. This impaired capability in HR is consistent with a failure to observe MRE11 foci formation. Furthermore, normal HR using sister chromatid was completely inhibited by the absence of FHA/BRCT domains. These results suggested that the N- and C-terminal domains of NBS1 are the major regulatory domains for HR pathways, very likely through the recruitment and retention of the MRE11 nuclease to DSB sites in an ATM-independent fashion.

Emergent thyroidectomy with sternotomy due to acute respiratory failure with severe thyroid storm.

Huge cervical and mediastinal masses may lead to acute respiratory failure caused by laryngotracheal compression and airway obstruction. Thyroid storm is also a life-threatening endocrine emergency originating almost exclusively from uncontrolled Graves' disease. We report a case of a 42-year-old man with acute upper airway obstruction and tachycardia from progressive swelling of a giant thyroid, in conjunction with thyroid storm resulting from uncontrolled Graves' disease. Fibreoptic-assisted nasal intubation was performed while the patient was awake, immediately followed by emergency total thyroidectomy via a cervical and sternal approach. The patient had an uneventful postoperative course and recovered well. Respiratory failure due to swelling of a giant thyroid is a life-threatening condition and should be treated immediately with endotracheal intubation while the patient is awake following emergent total thyroidectomy, even with a sternotomy.

Role for cyclic AMP in the postreceptor control of cytokine-stimulated stromal cell growth factor production.

We examined the role of augmented formation of intracellular cyclic AMP (cAMP) in the mediation of stromal cell growth factor production that occurs constitutively or upon cytokine stimulation. Clonal murine marrow adherent cell lines were stimulated under serum-free conditions by interleukin-1 (IL-1) or lipopolysaccharide (LPS) and one (+/+ -1.LDA11) was found to produce low quantities of granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF identity was confirmed by the ability of supernatants from stromal cells to promote proliferation of the factor-dependent cell line FDC-P1, neutralization of this activity by antiserum to GM-CSF, and by Northern blot analysis. However, optimal concentrations of IL-1 and tumor necrosis factor-alpha (TNF-alpha), in combination, led to synergistic (greater than 5-fold higher quantity) GM-CSF production compared with either stimulus alone in the +/+ -1. LDA11 cell line, capable of GM-CSF production after only single stimulation with IL-1 or LPS. In addition, synergistic stimulation by IL-1 and TNF-alpha led to equivalent high amounts of GM-CSF in another cell line incapable of GM-CSF production after induction with only IL-1 or LPS. Any of several means to raise intracellular cAMP levels, including addition of 8-bromo-cyclic AMP (8Br cAMP) (0.25-1mM), pertussis toxin (20-100 ng/ml), or addition of prostaglandin E1 (PGE1) (1 microM), failed to stimulate GM-CSF production alone and strongly inhibited GM-CSF production in stromal cells stimulated by IL-1, LPS, or the synergistic combination of IL-1 and TNF-alpha. In addition, PGE1 and pertussis intoxication were agonists of adenylate cyclase in membranes of marrow adherent cells, whereas IL-1 and LPS were not. The role for regulators of intracellular cAMP was specific because any of the cAMP agonists alone, or in the presence of cytokine stimulators of stromal cells, strongly enhanced IL-6 production, an event known to be cAMP-responsive. Thus, acute formation of intracellular cAMP is a negative regulator of stromal cell GM-CSF production mediated by cytokines, but positively regulates IL-6 production and may be an important determinant of cytokine-directed marrow microenvironmental function. These findings on the requirement for augmentation versus inhibition of cytokine-mediated production of hemopoietic growth factors might be applied to an analysis of marrow stromal cell heterogeneity.

Interleukin 4 alone or in combination with interleukin 1 stimulates 3T3 fibroblasts to produce colony-stimulating factors.

Colony-stimulating activity (CSA) can be produced by fibroblasts when stimulated by interleukin 1 (IL-1). We show that like IL-1, interleukin 4 (IL-4) can stimulate 3T3 fibroblasts to produce CSA. Biological and molecular analyses show that a significant portion of the CSA is colony-stimulating factor 1 (CSF-1) and granulocyte colony-stimulating factor (G-CSF). CSF-1 production in cells stimulated with a combination of both IL-1 and IL-4 was greater than that observed when cells were stimulated with either cytokine alone. However, the data show a synergistic induction of the expression of high levels of G-CSF mRNA and protein in cells incubated in the presence of both IL-1 and IL-4. The concentration of G-CSF in supernatants from cells stimulated with both IL-1 and IL-4 was at least tenfold higher than that measured in supernatants harvested from cells stimulated with either IL-1 or IL-4 alone. Previous investigations have shown that IL-4 had direct effects on hematopoietic progenitor cell growth. The studies described herein indicate that IL-4 is also involved in the regulation of hematopoiesis in an indirect manner, that is, by playing a role in the regulation of hematopoietic growth factor production.

A novel mast cell growth factor (MCGF-3) produced by marrow-adherent cells that synergizes with interleukin 3 and interleukin 4.

A clonal marrow-adherent stromal cell line, +/+-1 LDA11, was derived and found to produce hemopoietic stimulatory activity for an interleukin 3 (IL-3)-dependent mast cell line, NFS/N1. This factor-dependent mast cell line displayed restricted growth factor responsiveness to only IL-3, interleukin 4 (IL-4), and the stromal cell-produced factor. The factor produced by stromal cells was distinguished from IL-3 and IL-4 and was characterized biochemically. This factor appears to be a novel mast cell growth factor (MCGF-3) capable of synergizing with IL-3 and IL-4. It may have broader reactivity in hemopoiesis than simply IL-3-dependent mast cells, and it may prove relevant to stromal cell-mediated hemopoiesis.

Interleukin 1 plus interleukin 3 plus colony-stimulating factor 1 are essential for clonal proliferation of primitive myeloid bone marrow cells.

The clonal growth in nutrient agar at low cell densities of high-proliferative potential colony-forming cells (HPP-CFC) of bone marrow obtained from mice treated 2 days earlier with 5-fluorouracil (FU) (FU2dBM) has been shown to require a combination of three growth factors, interleukin 1 (IL-1), interleukin 3 (IL-3), and macrophage colony-stimulating factor (CSF-1). These HPP-CFC have been enriched 140-fold from FU2dBM by fluorescence-activated cell sorting of 7/4-, B220-, and L3T4-negative cells. The mean of the plating efficiencies of these enriched populations was 4.4% and no growth was observed when the factors were used singly. Similarly, enrichments of 16-fold were obtained from FU2dBM using immunomagnetic Dynabeads with anti-7/4 plus anti-B220 (meaning plating efficiency 0.5%). The further additions of human granulocyte CSF or mouse granulocyte-macrophage CSF or both to IL-1 plus IL-3 plus CSF-1 did not increase HPP-CFC colony formation, but both augmented the small colony formation with IL-1 plus IL-3, IL-3 plus CSF-1, or IL-1 plus CSF-1.

Expression, purification and characterization of recombinant murine granulocyte-macrophage colony-stimulating factor and bovine interleukin-2 from yeast.

Expression and secretion of two lymphokines, murine granulocyte-macrophage colony-stimulating factor (MuGM-CSF) and bovine interleukin-2 (BoIL-2), to levels of 50-60 mg per liter were achieved by placing these cDNAs in a Saccharomyces cerevisiae expression vector that utilized the yeast alcohol dehydrogenase-2 promoter and alpha-factor leader peptide. These lymphokines were purified to homogeneity by direct application of the crude yeast medium to reversed-phase high-performance liquid chromatography. Despite the fact that both lymphokines contain at least one N-glycosylation site and have identical N-terminal residues (Ala-Pro-Thr), recombinant (R) GM-CSF was found to be heterogeneously glycosylated by yeast while RBoIL-2 was secreted without glycosylation. Additionally, approximately 40% of the RGM-CSF was found to be proteolytically cleaved after the second amino acid residue, while RBoIL-2 was found to be intact.

Biochemical separation of interleukin 2.

Interleukin 2 (IL-2) is a class of T cell growth factors produced by T cells of a number of species. The unique growth-promoting properties of these molecules allow the development of antigen-specific effector T cell lines which can be used to analyze the molecular basis of lymphocyte interactions. A murine T cell tumor line has been identified as a source of IL-2. The purification and biochemical properties of murine IL-2 are described, and compared with rat and human Il-2.

(+/-)-(Z)-2-(aminomethyl)-1-phenylcyclopropanecarboxamide derivatives as a new prototype of NMDA receptor antagonists.

(+/-)-(Z)-2-(Aminomethyl)-1-phenylcyclopropane-N,N-diethylcarbo xamide (milnacipran, 1), a clinically useful antidepressant, and its derivatives were prepared by an improved method and were evaluated as NMDA receptor antagonists. Of these, milnacipran (1), its N-methyl and N,N-dimethyl derivatives, 7 and 8, respectively, and its homologue 12 at the aminomethyl moiety had binding affinity for the receptor in vitro (IC50: 1, 6.3 +/- 0.3 microM; 7, 13 +/- 2.1 microM; 8, 88 +/- 1.4 microM; 12, 10 +/- 1.2 microM). These also protected mice from NMDA-induced lethality. These compounds would be important as anovel prototype for designing potent NMDA-receptor antagonists because of their characteristic structure, which clearly differentiated them from known competitive and noncompetitive antagonists to the receptor.

(R)-1,2,3,4-tetrahydro[1]benzothieno[2,3-c]pyridines: novel optically active compounds with strong 5-HT1A receptor binding ability exhibiting anticonflict activity and lessening of memory impairment.

(R)-1,2,3,4-Tetrahydro[1]benzothieno[2,3-c]pyridine derivatives (60-114) were synthesized. The (R)-isomers have affinity for the 5-HT1A receptor while the (S)-isomers have no such ability. The affinity of the (R)-isomers was discussed on the basis of structure-activity relationships between the affinity and hydrophobicity of the (R)-isomers. Compounds 71 and 107, which are representative derivative compounds, have anticonflict activity and lessening of memory impairment. In particular, compound 107 cannot bind to receptors other than the 5-HT1A receptor, demonstrating that it is a unique compound with a different mechanism of action from that of conventional anxiolytics.

The treatment of induced immune deficiency with interleukin-2.

In vivo generation of alloreactive cytotoxic T lymphocytes (CTL) was found to be inhibited by treatment of mice with either cyclophosphamide or the glucocorticoid, hydrocortisone acetate. The effects of these immunosuppressive agents could be overcome, however, by the in vivo administration of IL-2 from both murine and human sources. Both human IL-2 derived by recombinant DNA techniques as well as the natural protein from mouse and man all reverse the unresponsive state. A single injection of IL-2 was sufficient to reverse the effect of cyclophosphamide treatment, while additional injections with as little as 8 micrograms of protein ablated the steroid-induced suppression. Furthermore, the responder cells generated in vivo following IL-2 therapy were shown to be antigen specific in terms of their lytic capacity. Thus, IL-2 therapy appears to restore the in vivo responsiveness of immunosuppressed recipients to allogeneic tumor cell challenge. These data demonstrate the importance of IL-2 as an immunoregulatory molecule in vivo and suggest its future use as a potent therapeutic.

Effect of the 'antidementia drug' pantoyl-GABA on high affinity transport of choline and on the contents of choline and acetylcholine in rat brain.

1. Effect of pantoyl-gamma-aminobutyric acid (pantoyl-GABA) on high affinity transport of choline into synaptosomes and on the choline (Ch) and acetylcholine (ACh) concentrations of rat brain were studied. 2. Pantoyl-GABA was injected intraperitoneally four times at a dose of 500 mg kg-1 at intervals of 30 min. One hour after the last injection, rats were killed by decapitation for measurement of high affinity transport of Ch into synaptosomes or by microwave irradiation for the measurement of Ch and ACh concentrations. 3. Transport of Ch was increased into synaptosomes prepared from the cerebral cortex and hippocampus, but not into those from the striatum. 4. In the cerebral cortex and hippocampus, Ch concentration was increased and ACh concentration decreased. 5. Since treatments that enhance the activity of cholinergic neurones in vivo are reported to increase high affinity transport of Ch measured in vitro, the present results suggest that pantoyl-GABA may increase cholinergic activity in vivo. This action of the drug may be related to changes in the Ch and ACh concentrations.

Blockade of intracellular actions of calcium may protect against ischaemic damage to the gerbil brain.

1. The brain cytoprotective effects of a putative calcium-associated protein kinase inhibitor, HA1077, as well as a calcium entry blocker nicardipine were evaluated in models of cerebral ischaemia in Mongolian gerbils. Morphological changes characterizing delayed neuronal death of selectively vulnerable CA1 pyramidal neurones in the hippocampus of the Mongolian gerbil brain occurred 7 days after transient bilateral occlusion of the common carotid arteries. 2. A single injection of HA1077 (1 and 3 mg kg-1, i.p.) 5 min after the occlusion led to a dose-dependent protection of the CA1 neurones. Repeated administrations of HA1077 (1 and 3 mg kg-1, i.p., twice daily for 7 days post-ischaemia) revealed an increase in the number of normal cells, compared to findings with a single administration. 3. In contrast to HA1077, nicardipine (0.3 and 1 mg kg-1, i.p.) did not reduce neuronal degeneration. 4. HA1077 did not interact with the ion channel within which MK-801 binds, as determined by receptor binding. 5. The calcium ionophore, A23187, caused a tonic contraction in canine cerebral arterial strips. HA1077, but not nicardipine, relaxed the A23187-induced contraction, concentration-dependently. 6. These results suggest that blockade of the intracellular actions of calcium may provide protection against ischaemic damage in the brain.