Integrin-associated protein (CD47) is a comitogenic molecule on CD3-activated human T cells

IAP is a glycoprotein functionally and physically associated with some integrins, i.e., the leukocyte response integrin and the beta3 integrin chain on placenta, platelets, and polymorphonuclear cells. IAP may act as a transducer element in activation mediated via these integrins. Since IAP is present at high density on peripheral T lymphocytes we have investigated its involvement in T cell activation. We tested three mAbs against IAP, namely B6H12, BRIC126, and 2D3, which recognize two distinct epitopes. IAP cross-linking with B6H12 or BRIC126, but not 2D3, transduces costimulatory signals within highly purified CD3-activated T lymphocytes, i.e., enhancement of proliferation, CD25 expression, and IL-2 secretion, while no effect was observed upon CD2 stimulation. However, we could not observe any functional association between IAP and integrins on peripheral T cells. In an attempt to explore further the activation signal delivered by IAP, we show here that IAP cross-linking with the comitogenic B6H12 mAb induces the phosphorylation on tyrosine of several proteins, one of which is identified as p56(lck) protein tyrosine kinase. Moreover, we observed that IAP is associated with p56(lck) on PMA-activated, but not on resting, T cells. These data suggest that on T cells, IAP may be involved directly via a specific ligand in cell-matrix or cell-cell interactions. Such interactions could trigger protein tyrosine phosphorylation pathways, which play an important role in both maturation and activation of T cells.     

Adoptive transfer of anti-CD3-activated CD4+ T cells plus cyclophosphamide and liposome-encapsulated interleukin-2 cure murine MC-38 and 3LL tumors and establish tumor-specific immunity

The infusion of anti-CD3-activated murine T cells plus interleukin-2 (IL-2) exerts antitumor effects against several tumors in murine immunotherapy models. This study compares the therapeutic efficacy of anti-CD3-activated CD4+ or CD8+ T-cell subsets, when given with cyclophosphamide (Cy) and liposome-encapsulated IL-2 (L-IL2) in a murine model. C57BL/6 mice bearing subcutaneous (S.C.) MC-38 colon adenocarcinoma, 3LL Lewis lung carcinoma, or 38C13 lymphoma for 7 to 14 days were pretreated with low-dose intraperitoneal (I.P.) Cy before intravenous (I.V.) injection of anti-CD3-activated T cells or T-cell subsets. Cell administration was followed by I.P. administration of L-IL2 for 5 days. Mice receiving activated CD4+ T cells showed significantly reduced tumor growth or complete remissions with prolonged disease-free survival in MC-38, 3LL, and 38C13. The timing of Cy doses in relation to adoptive transfer was critical in obtaining the optimal antitumor effect by CD4+ cells. Injecting Cy 4 days before the infusion of CD4+ cells greatly enhanced the antitumor effect of the CD4+ cells and improved survival of the mice compared with other Cy regimens. C57BL/6 mice cured of MC-38 after treatment with CD4+ T cells developed tumor-type immunologic memory as demonstrated by their ability to reject rechallenges with MC-38, but not 3LL. Similarly, mice cured of 3LL tumors rejected rechallenges of 3LL, but not MC-38. The immunologic memory could be transferred with an I.V. injection of splenocytes from mice cured of MC-38 or 3LL. No cytotoxic T-lymphocyte activity was detected in T cells or T-cell subsets from mice cured of MC-38 or 3LL. Increased IL-2 and interferon-gamma (IFN-gamma) production was observed from CD4+ subsets in cured animals when stimulated in vitro with the original tumor, but not with an unrelated syngeneic tumor. These results suggest that tumor-specific immunity can be achieved in vivo with anti-CD3-stimulated CD4+ T cells in this cellular therapy model.     

Paxillin phosphorylation and association with Lck and Pyk2 in anti-CD3- or anti-CD45-stimulated T cells

Antibodies to either CD3 or CD45 have been shown to induce dramatic changes in cell morphology, increased tyrosine phosphorylation of cellular proteins, and the association of a subset of these proteins with the tyrosine kinase Lck. The current study was initiated to determine the identity of the tyrosine-phosphorylated 70-80 kDa protein that becomes Lck-associated after stimulation with anti-CD45 or anti-CD3. We demonstrate that the cytoskeletal protein paxillin becomes tyrosine-phosphorylated when cells are plated on immobilized antibodies specific for CD45 or CD3. Only tyrosine-phosphorylated paxillin is associated with Lck, suggesting that the association is through the SH2 domain of Lck. Consistent with this we demonstrate that the SH2 domain of Lck binds tyrosine-phosphorylated paxillin. In contrast, the association of paxillin with the FAK-related kinase Pyk2 was found to be constitutive and not altered by the phosphorylation of either protein. Finally, we establish that the phosphorylation of paxillin is dependent on the expression of Lck. Taken together, these results demonstrate that paxillin is physically associated with kinases from two different families in T cells and suggest that paxillin may function as an adaptor protein linking cellular signals with cytoskeletal changes during T cell activation.     

Peripheral blood stem cells in acute myeloid leukemia: biology and clinical applications

Fifteen persons with profound mental retardation were divided into two groups. One group was identified with chronic training needs by habilitative staff and the other group served as a control. In an attempt to identify a reinforcer, each participant received a preference assessment and a simple, low-effort treatment procedure. In Experiment 1, only individuals who approached at least one stimulus on 80% or more of the preference assessment trials ("high preference") showed reinforcement effects in treatment. However, three individuals showing high preference failed to show treatment effects. All persons identified with chronic training needs failed to show reinforcement effects. Experiment 2 analyzed characteristics of the two groups and found significant differences in overall movement and response latency. Limitations of the current reinforcement technology were apparent for identifying reinforcers in the group with chronic training problems. Research is suggested for evaluating training alternatives for people with profound multiple disabilities who move very little or who respond with very long latencies.     

Monocyte-mediated lysis of acute myeloid leukemia cells in the presence of the bispecific antibody 251 x 22 (anti-CD33 x anti-CD64)

Immunotherapy using bispecific antibodies (BsAb) to direct immune effector cells toward target tumor cells has been shown to be effective in a number of studies. Several immune trigger molecules have been characterized. Among them, FcgammaRI appears to play an important role in antibody-dependent cellular cytotoxicity. It is expressed mainly on monocytes, macrophages, and neutrophils under certain clinical situations. The expression of FcgammaRI can be regulated by a variety of cytokines, primarily by IFN-gamma. Recent studies have shown that granulocyte-colony-stimulating factor (G-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF) can increase the number of the FcgammaRI-positive monocytes, increase the expression of FcgammaRI on circulating neutrophils after in vivo infusion, and greatly enhance the cytotoxic activity of circulating neutrophils. CD33 is a glycoprotein expressed on the cell surface of mature monocytes, myeloid progenitor cells, and myeloid leukemic blasts, but not on the earliest hematopoietic progenitor cells and other normal tissues. Herein, we report the construction of a BsAb, 251 x 22, by conjugating an anti-CD33 mAb (mAb 251) to an anti-FcgammaRI mAb (mAb 22). The BsAb 251 x 22 is capable of enhancing the cytotoxicity of several leukemia cell lines by cytokine-activated monocytes. Our data also show that G-CSF- and GM-CSF-stimulated monocytes can mediate cytotoxicity of target leukemia cells comparable to that of IFN-gamma-stimulated monocytes. The expression of FcgammaRI on monocytes after 24-h in vitro incubation with G-CSF and GM-CSF was increased, although not significantly. Prolonged incubation of monocytes with G-CSF for 48 h significantly increased the FcgammaRI expression. Because humanized anti-CD33 and anti-FcgammaRI mAb are available, and because GM-CSF and G-CSF have been used widely for patients after chemotherapy to stimulate the recovery of myeloid hematopoiesis, additional clinical development of this project is feasible. A BsAb comprised of humanized anti-CD33 and anti-FcgammaRI could have clinical application in the treatment of myeloid leukemia, especially in the management of minimal residual disease.     

Polymerase chain reaction (PCR) and its possible applications in diagnosis of infection in ophthalmology

Fecal incontinence resulting from pudendal canal syndrome has been treated by pudendal canal decompression (PCD) with satisfactory results. Considering the possible difficulty in exposing the pudendal canal and nerve by the open method, laparoscopic PCD was practiced in 9 women aged between 37 and 52 years. They were complaining of fecal incontinence; urinary stress incontinence was an additional complaint in 4/9 women. Neurologic, manometric, and EMG studies confirmed the diagnosis of pudendal canal syndrome. For laparoscopic PCD a 1-cm incision lateral to the anal orifice was performed. A balloon dilator was introduced in the ischiorectal fossa (IRF) to create a working space, and CO2 was insufflated. Under the guidance of a laparoscope, the IRF was entered and the inferior rectal nerve identified and followed to the pudendal canal. The latter was split open, releasing the pudendal nerve into the IRF. Fecal control was achieved in 7/9 patients and urinary control in 2/4. Fecal and urinary control were associated with improvement in perianal sensation, rectal neck pressure, EMG of external anal sphincter and levator ani muscle as well as in pudendal nerve terminal motor latency. Two women showed no improvement. Failure is suggested to be due to an advanced pudendal neuropathy. In conclusion, laparoscopic PCD is a simple, easy, and safe procedure. It allows for better exposure of the contents of the IRF than the open procedure, thus avoiding injury of the pudendal nerve and its branches during the performance of the PCD.     

Synthesis and photoluminescent properties of Sm~(3+)-activated La_3Si_6N_(11) as an orange-red emitting phosphor

A series of Sm³⁺-doped La₃Si₆N₁₁phosphor materials we re synthesized by a high temperature solid-state reaction method.The crystal structure,micro structure,photoluminescence properties,decay curves as well as thermal quenching properties of the as-prepared phosphors were investigated systematically.The excitation spectra contain a wide asymmetric band below 350 nm originating from the host absorption,several sharp excitation peaks in the range of 300-550 nm corresponding to f-f transition of Sm³⁺.Under the excitation of 369 and 414 nm light,the phosphors exhibit strong narrow-band orangered emission peaked at 605 nm.The average decay time of La_2.99Si₆N₁₁:0.01 Sm³⁺sample is fitted to be0.38 ms and the CIE coordinates were calculated to be(0.6105,0.3833).For water resistance,La₃Si₆N₁₁:Sm³⁺is better than K₂SiF₆:Mn⁴⁺phosphor.After soaking in deionized water for 300 min,the La₃Si₆N₁₁:Sm³⁺sample retains approximately 80%of its initial relative emission intensity.When the temperature rises to 423 K(150℃),the emission intensity of La_2.99Si₆N₁₁:0.01 Sm³⁺sample remains 85%in co mparison to that of room tempe rature.The activation energy was calculated to be 0.63253 eV,which is higher than those of Sm³⁺-activated oxide phosphors,indicating that the phosphor has relatively good thermal stability.     

Targeting of saporin to Hodgkin''s lymphoma cells by anti-CD30 and anti-CD25 bispecific antibodies

Many studies have demonstrated that allograft tolerance can be achieved in inbred rats and mice following intrathymic injection of donor cells or antigen and treatment with antilymphocyte serum (ALS). In outbred dogs, xenografts, and inbred rat strains with major MHC antigen difference, tolerance has not similarly been induced. The focus of this study was to determine whether allogeneic thyroid graft tolerance could be achieved in outbred rabbits. In the experimental group (n = 5), recipients received an intrathymic injection of donor lymphocytes and a single treatment of ALS. Controls (n = 5) received intrathymic cell culture medium and ALS treatment. Donor-recipient allogenicity was monitored with mixed lymphocyte culture (MLC) over 18 weeks. Donor thyroid tissue was placed into recipient gluteal muscle fibres one week following the last MLC measurement. A third group of rabbits (n = 4) received thyroid autografts without any other treatment. There were no differences in MLC stimulation indices (SI) between the control and experimental group nor did MLC (SI) change within groups. All thyroid autografts survived the two week monitoring period and demonstrated normal appearing thyroid follicles on histologic examination. All thyroid allografts showed severe acute rejection reactions on biopsy within one week. Further studies using outbred animals to examine the role of thymic inoculation are required to determine whether similar techniques might be successful in the human.     

Tumor-reactive immunoglobulins in ovarian cancer: diagnostic and therapeutic significance? (review)

Inhibition of the immune system has been observed in association with most stages of ovarian cancer; however, the mechanisms involved in the induction and maintenance of this chronic immune unresponsiveness associated with cancer progression are poorly understood. This immunosuppressed state is primarily defined as the failure to eradicate the tumor. This immunosuppressed state is generally associated with decreased numbers and reactivity of lymphoid cells in women with ovarian cancer. The degree of immune dysfunction in ovarian cancer patients has been demonstrated to correlate with patient survival. While ovarian cancer patients generally fail to exhibit effective immunosurveillance, as manifested by continued tumor growth and progression, the presence of tumor-reactive immunoglobulins can be demonstrated in these women, indicating the continued presence of immune recognition. We have not only demonstrated the presence of tumor-reactive antibodies in ovarian cancer patients, but have also shown that the levels of these antibodies increase as the disease progresses. The antigens recognized by the patients' humoral response have been identified as either membrane-associated or intra-cellular. In general, the localization of these antigens tend to be linked to the patient's prognosis. The presence of a humoral response against intracellular proteins are correlated with poor prognosis, while autoantibodies reactive with surface components appear to have a better prognosis. In addition to general antigen recognition, these reactive antibodies have been utilized to define specific epitopes on tumor-associated proteins. Certain specific antigenic epitopes exhibit common recognition among patients with the same tumor type. The specific recognition of certain epitopes can provide early evidence of aberrant protein expression and this aberrant expression of certain proteins, such as procathepsin D, appear to be linked to the tumor's acquisition of specific malignant characteristics, including metastasis formation and chemoresistance. Despite the existence of circulating tumor-reactive immunoglobulins, their presence correlates, in general, with poor prognosis and poor host survival. Since tumor-reactive immunoglobulins are elicited and can be detected early in the development of tumors and their enhanced synthesis is induced prior to the clinical manifestation of recurrence, the assessment of the tumor-reactive immune response against specific antigenic epitopes should represent an early significant diagnostic and prognostic marker in ovarian cancer.     

黄金选矿年评(续完)

评述了非氰化法提金工艺及难浸金矿石预处理技术的国内外最新进展。     

IP3-activated calcium-permeable channels in the inside-out patches of cultured cerebellar Purkinje cells

Inositol-1,4,5-trisphosphate (IP3)-activated calcium-permeable channels were recorded from inside-out patches of cultured cerebellar Purkinje cells. When 2-5 microM of IP3 was applied to the internal surface of the inside-out patches, inward Ba2+ currents were activated within 10 sec following the application in 11 out of 24 patches. In the presence of heparin (100 micrograms/ml), activation of Ba2+ currents by IP3 was inhibited. Unitary currents with different amplitudes and kinetics were observed; small and large unitary currents, and rapid fluctuations with various amplitudes. The small unitary currents (single channel conductance; 5.6 pS) were most frequent. Addition of inositol 1,3,4-trisphosphate (2-5 microM) slightly activated Ba2+ currents in 2 out of 10 patches, but the amount of the increment was much smaller than that produced by IP3. These results suggest a possibility that IP3 directly activates Ca(2+)-permeable channels in the plasma membrane of cerebellar Purkinje cells.     

Adoptive immunotherapy with vaccine-primed lymph node cells secondarily activated with anti-CD3 and interleukin-2

PURPOSE: In preclinical studies, we have reported the ability to induce immune T cells in lymph nodes (LN) primed by in vivo vaccination with tumor cells admixed with a bacterial adjuvant. These LN cells can be activated and expanded ex vivo for the successful immunotherapy of established tumors. We have applied these methods to generate vaccine-primed LN in patients with advanced melanoma and renal cell cancer (RCC) for therapy. MATERIALS AND METHODS: Irradiated autologous tumor cells admixed with bacille Calmette-Guérin (BCG) were used to vaccinate patients. Seven days later, draining LN were removed for activation with anti-CD3 monoclonal antibody (mAb) followed by expansion in interleukin-2 (IL-2). Activated LN cells were administered intravenously (IV) with the concomitant administration of IL-2. RESULTS: A total of 23 patients were evaluated (11 melanoma and 12 RCC). Vaccine-primed LN were expanded ex vivo with a mean of 8.4 x 10(10) cells administered per patient. Among 20 patients assessed, 15 demonstrated minimal cytotoxicity of autologous tumor cells by the activated LN cells, with the remaining mediating nonspecific cytotoxicity. By contrast, a majority of the activated LN cells showed highly specific release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon gamma (IFN-gamma) to autologous but not allogeneic tumor stimulation. This tumor-specific cytokine release was found to be major histocompatibility complex (MHC) class I-restricted, which indicates the involvement of CD8+ cells. Among 11 melanoma patients, one had a partial tumor response. Among 12 RCC patients, two had complete and two partial responses. A trend (P = .066) between the enhancement of delayed-type hypersensitivity (DTH) reactivity to autologous tumor after therapy and tumor regression was observed. CONCLUSION: Tumor vaccines can be used to induce immunologically specific T-cell responses against melanoma and RCC in draining LN. Anti-CD3/IL-2 activation of primed LN cells can be reliably performed for clinical therapy and appears to have activity in patients with metastatic RCC.     

Inelastic scattering of neutrons and possible biological applications

The field of neutron inelastic scattering has probably been developed to the stage where it can begin to help the biologist. Because essentially no experimental data have been obtained, it is difficult either to draw conclusions or to make forecasts except on the basis of general hypotheses. It seems likely, however, that the next stage is up to biologists. After reviewing those biological problems in which molecular dynamics might play an important role, they should suggest specimens of interest which can give inelastic peaks with existing spectrometers operating with 5 to 10-A neutrons at angles greater than 5degrees and with resolutions of approximately 50 mueV. These specimens may involve molecules slightly smaller and more mobile than some biologists would like, but a successful outcome might lead to the development of spectrometers capable of working in a more satisfactory range. In this event the return may well prove rewarding to the biologists.     

Adult alveolar type II cells lack cAMP and Ca(2+)-activated Cl-channels

The recently developed procedure of chromosomal DNA loop excision by topoisomerase II-mediated DNA cleavage at matrix attachment sites (S. V. Razin, R. Hancock, O. Iarovaia, O. Westergaard, I. Gromova, and G. P. Georgiev, Cold Spring Harbor Symp. Quant. Biol. 58:25-35, 1993; I. I. Gromova, B. Thompsen, and S. V. Razin, Proc. Natl. Acad. Sci. USA 92:102-106, 1995) has been employed for mapping the DNA loop anchorage sites in a 500-kb region of the Drosophila melanogaster X chromosome. Eleven anchorage sites delimiting 10 DNA loops ranging in size from 20 to 90 kb were found within this region. Ten of these 11 anchorage sites colocalize with previously mapped scaffold attachment regions. However, a number of other scaffold attachment regions are found to be located in loop DNA.     

Photoluminescence properties and thermal stability of Eu~(3+)-activated La_7Ta_3W_4O_(30) red-emitting phosphors for near-UV-excited w-LEDs

Novel trivalent europium(Eu~(3+))-activated La_7 Ta_3 W_4 O_(30):xEu~(3+)(x=0.5 mol%-40 mol%) red-emitting phosphors were synthesized by means of a high-temperature solid-state reaction.The structure,morphology,photoluminescence,thermal-stability properties,lifetime,and color-rendering of the prepared phosphors were investigated in detail.The La_7 Ta_3 W_4 O_(30):Eu~(3+) phosphors show five emission peaks under near-ultraviolet(n-UV) at 397 nm,and these peaks are ascribed to the transitions of ~5 D_0-~7 F_j(j=0,1,2,3 and 4) by Eu~(3+) ions.The optimal doping concentration of Eu~(3+) is 20 mol%,and the critical distance of the energy transfer between the Eu3+ions was calculated to be 1.768 nm.The quenching temperature(T_(0.5)) of La_7 Ta_3 W_4 O_(30):20 mol%Eu~(3+) is about 440 K.The quantum yield(QY) was measured to be 85.85%.The fabricated white-light-emitting diodes(w-LEDs) possess high color-rendering index(R_a) of 90,and high correlated color temperature(CCT) of 5810 K,respectively.The Commission Internationale de L'Eclairage(CIE) coordinates are(0.311,0.322).Therefore,the prepared phosphor has a promising application for w-LEDs.     

Glucagon-like peptide 1 (GLP-1): a potent gut hormone with a possible therapeutic perspective

This paper narrates Dr Héctor R Croxatto and collaborators' efforts over the past 50 years in search for peptidic hormones obtained by pepsin hydrolysis of blood plasma substrates. In the forties, Croxatto described three peptidic fractions characterized by their hypertensive, oxytocic and antidiuretic properties, designated as pepsitensin, pepsitocin and pepsanurin, respectively. While pepsitensin and pepsitocin were later identified as angiotensin I and metlys-bradykinin, pepsanurin was not identified and its research was halted for 35 years. During that time, Prof Croxatto and his group worked mostly on the renal kallikrein-kinin system, studying its physiological anti-hypertensive role, making significant contributions in the field of renovascular hypertension. After the discovery of atrial natriuretic peptide, Croxatto resumed his work with pepsanurin. In a series of papers from 1988 to 1998, it was shown that: 1) when injected intraperitoneally or in the intestinal lumen of anesthetized rats, or in the isolated perfused rat kidneys, pepsanurin is a potent inhibitor of the natriuretic effect of ANP; 2) plasma kininogens are identified as the substrates for pepsanurin formation; 3) bradykinin and prokinins exert the anti-ANP effect when injected either intravenously, intraperitoneally or intraduodenally, at small non-vasodilator doses; endogenous kinins also block ANP renal excretory effects; 4) a 20-amino acid peptide released by pepsin from domain 1 of purified LMW kininogen was isolated by Croxatto and collaborators, designed as PU-D1, and shown to exert similar anti-ANP effects as pepsanurin or kinins, but being more potent and longer lasting; 5) the anti-ANP effect of pepsanurin, kinins and PU-D1 is mediated by B2 kinin receptors, since it is blocked by a bradykinin receptor antagonist. Currently, Dr Croxatto is working on the hypothesis that intestinal-borne kinins and/or PU-D1 may reduce renal excretion during the prandial cycle.     

Gastroduodenal inflammation associated with Helicobacter pylori infection. Diagnostic and therapeutic implications (a review)

Site-directed mutagenesis has proved an effective experimental technique to investigate catalytic mechanisms and to determine relations between enzyme structure and function. This article invokes an analytical model based on evolution by mutation and natural selection-Nature's analogue of site-directed mutagenesis-to derive a set of general rules relating enzyme structure and activity. The catalysts are described in terms of the structural parameters, rigidity and flexibility, and the functional variables, reaction rate and substrate specificity. The evolutionary model predicts the following structure-activity relations: (a) rigid enzyme-flexible substrate: large variation in reaction rates, broad substrate specificity; (b) rigid enzyme-rigid substrate: diffusion controlled rates, absolute specificity; (c) flexible enzyme-rigid substrate: intermediate reaction rates, group specificity; (d) flexible enzyme-flexible substrate: slow rates, absolute specificity. Spectroscopic methods and X-ray crystallography now yield important characteristics of enzyme-substrate complexes such as molecular flexibility. The evolutionary analysis we have exploited provides general principles for inferring catalytic activity from structural studies of enzyme-substrate complexes.     

Synthesis,structure and NIR luminescence properties of Yb~(3+) and Bi~(3+)-activated vanadate GdVO_4

Near-infrared emission of Yb~(3+) and Bi~(3+)-co-doped GdVO_4 vanadate phosphors obtained via the modified Pechini's method was reported. The phase purity and structure of samples were characterized by X-ray powder diffraction(XRD), X-ray energy dispersion spectroscopy(EDS), scanning electron microscopy(SEM), Raman and infrared(IR) spectroscopy. Photoluminescence emission(PL) and excitation(PLE) spectra were recorded and investigated in details. Results indicated that upon near-UV excitation(340 nm) in the(Bi~(3+)-V~(5+)) charge transfer state this phosphor had a strong broad band yellow-green emission(~3P_1→~1S_0) of Bi~(3+) ions and also NIR emission(~2F_(5/2)→~2F_(7/2)) of Yb~(3+) ions in the range of 950–1050 nm, which matched well with the spectral response of the silicon-based solar cells. The decreasing Bi~(3+) emission with increasing Yb~(3+) concentration proved the occurrence of energy transfer from Bi~(3+) to Yb~(3+) ions. Results demonstrated that Yb~(3+) and Bi~(3+)-co-doped GdV O4 vanadate phosphors might act as a NIR down-conversion(DC) solar spectral converter to enhance the efficiency of the silicon-based solar cells.