Effect of the lymphocytosis-stimulating factor from Bordetella pertussis on murine lymphoid and hemopoietic cells

Effect of B. pertussis lymphocytosis-promoting factor (LPF) on the lympho-hematopoietic system of mice was studied. The injection of LPF was shown to sharply enhance endogenous colony formation and to induce a severe depletion of thymus cells, reaching its maximum of day 4. Thymocytes obtained on day 2 or 3 after the injection of LPF produced a suppressive effect on endogenous colony formation. The proliferative activity of hematopoietic stem cells sharply increased under the influence of LPF, though it had no radioprotective action. On the following day after the injection of LPF a steep rise in the number of hematopoietic stem cells was observed in the blood of mice: their content increased 20-fold in comparison with the control level. These data may be important for the evaluation of the side effects of pertussis vaccine on the lympho-hematopoietic system.     

The effect of Bordetella pertussis lymphocytosis-promoting factor (LPF) on antibody response in mice: its enhancing and suppressive effects

The effect of lymphocytosis-promoting factor (LPF) on antibody response in mice was estimated under different sets of experimental conditions. Four- and 6-week-old mice were intravenously inoculated with LPF. Three days later these mice were inoculated either intraperitoneally or intravenously with sheep red blood cell (SRBC) or human serum albumin (HSA) as an antigen. The adjuvant effect of LPF was demonstrated on antibody response in 6-week old mice to intraperitoneally inoculated SRBC but not to intravenously-inoculated one. When 4-week-old mice were immunized, hemagglutinin production in response to intraperitoneally inoculated SRBC was not enhanced by LPF. In addition, a rather suppressive effect of LPF at a comparatively high dose was demonstrated on hemagglutinin production in response to intravenously inoculated SRBC. Anti-HSA production was enhanced by inoculation of LPF in any combination of the mouse age and the route of antigen administration. These findings indicate that the adjuvant effect of LPF on antibody response in mice depends upon experimental conditions: the age of mice, the quality of antigen and the route of antigen administration used for immunization.     

The quantitative assay of the clustering activity of the lymphocytosis-promoting factor (pertussis toxin) of Bordetella pertussis on Chinese hamster ovary (CHO) cells

An experimental design and a statistical method for the estimation of the clustering-response activity of lymphocytosis-promoting factor (LPF) in Chinese hamster ovary cells growing in wells on a microplate were investigated. The scoring method introduced by Ipsen was adopted to express the grade of the clustering response rather than the end-point method generally used. The scoring method was validated by statistical analyses. The grade of response varied with the location of the wells on a microplate, and thus the expression of the clustering activity of a test sample in terms of the end-point may be inadequate in terms of accuracy and reproducibility. It was shown that the allocation of test samples to individual wells according to a Latin square design minimized the effect of the location of wells on the clustering response. Under such experimental conditions, a fairly precise and reproducible method for the quantification of the clustering activity was developed.     

Modulation of the biologic activities of IgE-binding factors. III. Switching of a T cell hybrid clone from the formation of IgE-suppressive factor to the formation of IgE-potentiating factor

Incubation of rat-mouse T cell hybridoma cells, 23B6, with rat immunoglobulin E (IgE) results in the formation of the 15,000-dalton IgE-suppressive factor and the 30,000-dalton IgE-binding factor, which has neither potentiating activity nor suppressive activity on the IgE response. Another T cell hybridoma, 23A4 cells, produces the 30,000-dalton "inactive" IgE-binding factor upon incubation with IgE. Both the 15,000-dalton IgE-suppressive factor and the 30,000-dalton IgE-binding factor lacked affinity for lentil lectin but bound to peanut agglutinin. When the 23B6 cells were incubated with IgE in the presence of lysolecithin, the majority of the 15,000-dalton IgE-binding factor formed by the cells gained affinity for lentil lectin, and this factor selectively potentiated the IgE response. The glycosylation-enhancing factor, which was formed by stimulation of normal spleen cells with lymphocytosis-promoting factor (LPF or pertussigen), also switched 23B6 cells from the formation of IgE-suppressive factor to the formation of IgE-potentiating factor. It was also found that the 30,000-dalton "inactive" IgE-binding factor, formed by both 23B6 and 23A4 cells, gained the ability to potentiate the IgE response, when the cells were cultured with IgE in the presence of glycosylation-enhancing factor. The results indicate that IgE-potentiating factor and IgE-suppressive factor share common precursors, and that biologic activities of IgE-binding factors are decided by their carbohydrate moieties. Incubation of the two hybridoma cells with lysolecithin or glycosylation-enhancing factor results in an increase in the proportion of FC epsilon R+ cells, suggesting that the assembly of N-linked oligosaccharide to precursor molecules is intrinsic for the expression of FC epsilon R.     

Modulation of the biologic activities of IgE-binding factor. V. The role of glycosylation-enhancing factor and glycosylation-inhibiting factor in determining the nature of IgE-binding factors

Glycosylation-enhancing factor (GEF) and IgE-potentiating factor were detected in culture supernatants of rat mesenteric lymph nodes (MLN) cells obtained 14 days after infection with Nippostrongylus brasiliensis (Nb), but not in supernatants of MLN cells of 8-day Nb-infected rats. Both factors were also released from T cells upon antigenic stimulation of KLH + alum-primed spleen cells. The GEF from the Nb-infected rats and KLH + alum-primed spleen cells had affinity for p-aminobenzamidine agarose and were inactivated by phenylmethylsulfonylfluoride, an inhibitor of serine proteases. These results indicate that the GEF obtained in the two systems is a serine protease and is identical to that obtained by stimulation of normal T cells with lymphocytosis-promoting factor (LPF) from Bordetella pertussis. The concomitant formation of IgE-potentiating factor and GEF by Nb infection, by antigenic simulation of KLH + alum-primed spleen cells, and by treatment of rats with Bordetella pertussis vaccine suggests that the serine protease is involved in a common pathway leading to the selective formation of IgE-potentiating factor. In contrast, glycosylation-inhibiting factor (GIF) is always found during the selective formation of IgE-suppressive factor. IgE-suppressive factor and GIF were formed by MLN cells of 8-day Nb-infected rats and KLH-CFA-primed spleen cells. GIF was detected in culture supernatants of T cell hybridomas 23A4 and 23B6, which form unglycosylated IgE-binding factors upon incubation with IgE. GIF obtained from all of these sources bound to monoclonal anti-lipomodulin. These findings indicate that GIF or lipomodulin is involved in all systems, which leads to the selective formation of IgE-suppressive factor. However, the formation of GIF was not restricted to those conditions in which IgE-suppressive factor was selectively formed. The culture supernatants of MLN cells of 14-day Nb-infected rats and antigen-stimulated KLH + alum-primed spleen cells contained a small amount of GIF, which could be detected after inactivation of GEF. It appears that T cells from these sources formed GEF and GIF, but that GEF overcame the effect of GIF on glycosylation of IgE-binding factors. The results indicate that the nature and biologic activities of IgE-binding factors are decided by the balance between GEF and GIF formed by T cells.     

Effect of Bordetella pertussis leukocytosis (lymphocytosis)-promoting factor (LPF) on the physical lymphoepithelial-cell association studied with the use of an in vitro model of mouse thymus

The effect of highly purified leukocytosis (lymphocytosis)-promoting factor (LPF) of Bordetella pertussis on physical lymphocyte and reticuloepithelial (RE) cell association was studied in an in vitro thymus model. First, a simplified in vitro system to assess the lympho-RE-cell association was developed. A completely confluent layer of thymic RE cells was formed by cultivating trypsinized thymus cell suspensions from 2- to 7-day-old mice. When thymic lymphoid cells were seeded on this cell layer and cultivated overnight, a significant proportion of them were found underneath the RE cell layer. This physical lympho-RE-cell association was quantitated by counting the lymphoid cells underneath the RE cell layers. Second, the effect of LPF on this physical lympho-RE-cell association phenomenon was investigated. Addition of LPF to the culture markedly inhibited the formation of the lympho-RE-cell complex; that is, it inhibited the infiltration of lymphoid cells under the RE cell layer. LPF rendered a nearly maximal level of inhibitory effect at a dose of 0.1 ng/ml. Furthermore, LPF enhanced the liberation of lymphoid cells from preformed lympho-RE-cell complexes. On the other hand, LPF had no direct cytotoxic effect on lymphoid cells at doses below 1 microgram/ml. In order to investigate whether LPF produced the effect by acting on lymphoid cells, RE cells, or both, the following experiments were performed. When lymphoid cells were pretreated with LPF and added to normal RE cell layers, the lympho-RE-cell association was maximally inhibited above the dose of 1 ng/ml. Treatment of these LPF-treated lymphoid cells with anti-LPF antibodies failed to abrogate the effect of LPF. When RE cell layers were similarly pretreated with LPF and were cultivated with normal lymphoid cells, however, much higher doses of LPF, above 100 ng/ml, were required for maximal inhibition. Furthermore, treatment of these LPF-treated RE cells with anti-LPF antibodies abrogated the effect of LPF. Therefore, the apparent effect of LPF on RE cells was considered to be due to the carry-over by RE cells of LPF, which should directly act on lymphoid cells at extremely low doses. On the basis of these results, it was concluded that LPF acted directly on lymphoid cells without mediation of RE cells. These in vitro results appear to parallel the effects of LPF in vivo, where it induces a depletion of cells in the thymus. The model may be useful to study this phenomenon and the concomitant accumulation of blood lymphocytes.     

A biphasic serum glucose response in mice to inoculation with pertussis vaccine

The effect of B. pertussis vaccine on the serum glucose level of mice was investigated. The results show that at least two components in the vaccine interfere with glucose metabolism. A heat-stable component which is assumed to be LPS induced hypoglycemia 3-5.5 h after inoculation, especially in LPS-sensitized mice. A heat-labile component which is possibly equivalent with the LPF/HSF/IAP complex, is responsible for persistence of the hypoglycaemia for at least 6 days. If hypoglycaemia contributes to the neurological side effects after pertussis vaccination both components have to be considered as being responsible for these effects.     

吸附精制百日咳菌苗质量控制的研究

本文就吸附精制百日咳菌苗制检过程中使用的菌株、有效组分ＬＰＦ和ＦＨＡ提纯工艺以及毒性,效力等检定方法阳相应的质量控制指标等进行了研究。结果表明,中国Ｃ、Ｓ菌株产毒优于日本Ｔｏｈａｍａ株。ＬＰＦ和ＦＨＡ─ＥＬＩＳＡ用作菌苗工艺各步骤中质控有效组分的产量,是一种快速、敏感和特异的方法。根据５年中菌苗批量生产每ｍｌ培养基ＬＰＦ和ＦＨＡ产量逐年上升趋势,说明生产工艺不断改进完善和操作技术的熟练是提高菌苗产量和质量的关键。制检规程中用作毒性和效力试验的方法,是菌苗安全有效质控的有效实用方法。     

A new rabies vaccine in public health practice in the USSR

A new antirabies vaccine prepared on the basis of virus grown in the ovine brain, purified from 85-90% of brain-tissue ballast substances and inactivated with beta-propilactone has been developed at the Moscow Research Institute of Viral preparations (USSR Acad. Med. Sci.). The preparation produces no neuro-allergenic effect in tests on guinea pigs. When injected to humans, the vaccine shows much lower reactogenicity than Fermi vaccine. High antigenic and immunogenic activity of the new vaccine has made it possible to work out a less intensive immunization schedule in comparison with that used for immunization with Fermi vaccine and nonconcentrated tissue-culture vaccine, viz. doses of 3 ml for 12 days or doses of 3 ml for 20 days with two booster immunizations. The preparation has been introduced into medical practice.     

Modulation of the biologic activities of IgE-binding factor. IV. Identification of glycosylation-enhancing factor as a kallikrein-like enzyme

Stimulation of normal rat splenic T cells with pertussigen (lymphocytosis-promoting factor, LPF, from Bordetella pertussis) resulted in the release of a soluble factor that enhanced the glycosylation of IgE-binding factors during their biosynthesis. The soluble factor was detected by the ability of a culture filtrate of LPF-stimulated spleen cells to switch a T cell hybridoma, 23A4, from the formation of unglycosylated IgE-binding factor to the formation of glycosylated IgE-binding factor. The glycosylation-enhancing factor (GEF) had affinity for D-galactose, and the binding of the factor to hybridoma cells via a cell surface galactose was essential for modulation of IgE-binding factors. The GEF was inactivated by irreversible inhibitors of serine proteases such as phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, and p-nitrophenyl ethylpentylphosphonate but was not affected by nonphosphorylating analogues of the organophosphorus compounds. Benzamidine, a competitive and reversible inhibitor of trypsin, also inhibited the glycosylation of IgE-binding factors by GEF. The factor could be purified by absorption to p-aminobenzamidine agarose followed by elution with benzamidine. The capacity of GEF to enhance the glycosylation of IgE-binding factors was inhibited by synthetic substrates of trypsin but not by substrates of chymotrypsin, indicating that GEF is a trypsin-like enzyme. Indeed, trypsin, plasmin, and kallikrein enhanced the glycosylation of IgE-binding factors during their biosynthesis. An inhibitor of trypsin-like enzyme(s), N-alpha-p-tosyl-L-lysine chloromethylketone (TLCK), inhibited trypsin and plasmin but not kallikrein, and TLCK failed to inhibit the GEF-mediated enhancement of glycosylation. It was also found that bradykinin, the biologically active product of cleavage of kininogen by kallikrein, enhanced the glycosylation of IgE-binding factors. The results indicate that GEF is a kallikrein-like enzyme.     

Separation and characterization of two distinct hemagglutinins contained in purified leukocytosis-promoting factor from Bordetella pertussis.

1. The leukocytosis-promoting factor of Bordetella pertussis was found to contain two hemagglutinins with different susceptibilities to papain and separable from each other by agarose gel filtration with Tris - HCl buffer containing 1 M NaCl. 2. One hemagglutinin, referred to as hemagglutinin HA, had a high hemagglutinating activity, but neither leukocytosis-promoting nor histamine-sensitizing activity. The other hemagglutinin, referred to as hemagglutinin LPF appeared to be identical with the leukocytosis-promoting factor and possessed a low hemagglutinating and high leukocytosis-promoting and histamine-sensitizing activities. 3. The hemagglutinating activity of hemagglutinin HA was highly sensitive to papain. The hemagglutinating, leukocytosis-promoting, and histamine-sensitizing activities of hemagglutinin LPF were fairly resistant to the enzyme. 4. The two hemagglutinins were distinct from each other in immunological and chemical properties. 5. Morphologically, hemagglutinin HA showed itself to be filamentous molecules of approx. 2 X 40 nm, while hemagglutinin LPF comprised of spherical molecules of approx. 6 nm diameter. 6. The molecular weight values of hemagglutinin HA estimated by sodium dodecylsulfate polyacrylamide gel electrophoresis and sucrose density gradient centrifugation were approx. 126 000 and 133 000, respectively. Those of hemagglutinin LPF estimated by polyacrylamide gel electrophoreis at pH 4.5, sucrose density gradient centrifugation and gel filtration on a 10% agarose column were 107 000, 103 000 and 30 000, respectively. A possible reason for obtaining such a low molecular weight value by gel filtration is discussed.     

Genetic Control of Immune Responses to HIV-1 env DNA Vaccine

We investigated the genetic control of immunoglobulin production and the delayed-type hypersensitivity (DTH) response produced by an HIV-specific DNA vaccine using several strains of mice. Murine antigen-specific immunoglobulin production was determined by ELISA. The DTH response was assessed in terms of the footpad swelling reaction. All strains of mice, except for B10.RIII and B10.T(6R), exhibited strong immunoglobulin production and footpad swelling in response to the DNA vaccine. In vitro treatment of lymphoid cells with monoclonal antibodies showed that the footpad swelling response was mediated by CD4⁺8^? and Ia— T cells. However, CD8⁺ T cells did not suppress footpad swelling. There was no difference in the induction of HIV-specific immunoglobulin production or DTH response induced by the DNA vaccine among the strains, suggesting that HIV-specific DNA vaccine is useful for immunizing various populations against HIV-1.     

Purified Rabies Vaccine (Suckling Rat Brain Origin)

A 10% suckling rat brain rabies vaccine free from encephalitogenic activity was prepared and inactivated with 1:8,000 beta-propiolactone (BPL), or ultraviolet light, or a combination of ultraviolet light and BPL, or 1% phenol. Potency was excellent in all samples, with the exception of the phenolized product which was marginal. A purified suckling rat brain (SRB) vaccine prepared by zonal centrifugation and inactivated with 1:8,000 BPL contained about 0.01 the amount of protein nitrogen of the unpurified 10% SRB vaccine. This purified product passed the National Institutes of Health potency test for rabies vaccine after administration of a quantity equivalent to a standard 10% brain suspension.     

弹性髓针在青少年锁骨中段骨折治疗中的应用研究

目的:回顾性研究弹性髓针(EMN)在青少年锁骨中段骨折(MCF)治疗中的应用效果。方法:纳入2016年1月-2019年6月于我院接受治疗的MCF患者66例,依据手术方法分为EMN组(n=35)与锁定钢板内固定(LPF)组(n=31)。EMN组行EMN治疗,LPF组行LPF治疗。观察两组术中出血量、手术时间、切口长度、住院时间、骨折愈合时间等手术相关指标。比较两组术前及术后3 d纤维蛋白原(Fg)、凝血酶原时间(PT)、活化部分凝血酶原时间(APTT)等凝血功能指标。比较两组术前及术后7 d视觉模拟量表(VAS)评分及血清去甲肾上腺素(NE)、五羟色胺(5-HT)、P物质(SP)水平。比较两组术前及术后30 d肩关节活动度及肩关节功能评分,记录两组术后并发症。结果:EMN组术中出血量少于LPF组,手术时间、切口长度、住院时间短于LPF组(P0.05);两组骨折愈合时间无差异(P0.05)。术后3 d,EMN组PT水平高于LPF组,Fg、APTT水平均低于LPF组(P0.05)。术后7 d,EMN组VAS评分、5-HT、SP、NE水平均低于LPF组(P0.05)。术后30d,两组肩外展、肩前屈活动度及肩关节功能评分均无差异(P0.05)。EMN组术后并发症发生率低于LPF组。结论:EMN治疗青少年MCF创伤小,对机体凝血功能影响小,可有效缓解患者术后疼痛,促进患者肩关节功能恢复,降低术后并发症发生风险。     

Differential Response of Neural Cells to Trauma-Induced Swelling In Vitro

Brain edema and the associated increase in intracranial pressure are major consequences of traumatic brain injury (TBI) that accounts for most early deaths after TBI. We recently showed that acute severe trauma to cultured astrocytes results in cell swelling. We further examined whether trauma induces cell swelling in neurons and microglia. We found that severe trauma also caused cell swelling in cultured neurons, whereas no swelling was observed in microglia. While severe trauma caused cell swelling in both astrocytes and neurons, mild trauma to astrocytes, neurons, and microglia failed to cell swelling. Since extracellular levels of glutamate are increased in brain post-TBI and microglia are known to release cytokine, and direct exposure of astrocytes to these molecules are known to stimulate cell swelling, we examined whether glutamate or cytokines have any additive effect on trauma-induced cell swelling. Exposure of cultured astrocytes to trauma caused cell swelling, and such swelling was potentiated by the exposure of traumatized astrocytes to glutamate and cytokines. Conditioned medium (CM) from traumatized astrocytes had no effect on neuronal swelling post-trauma, while CM from traumatized neurons and microglia potentiated the effect of trauma on astrocyte swelling. Further, trauma significantly increased the Na–K–Cl co-transporter (NKCC) activity in neurons, and that inhibition of NKCC activity diminished the trauma-induced neuronal swelling. Our results indicate that a differential sensitivity to trauma-induced cell swelling exists in neural cells and that neurons and microglia are likely to be involved in the potentiation of the astrocyte swelling post-trauma.     

Extended Preclinical Safety,Efficacy and Stability Testing of a Live-attenuated Chikungunya Vaccine Candidate

We recently described a new, live-attenuated vaccine candidate for chikungunya (CHIK) fever, CHIKV/IRES. This vaccine was shown to be well attenuated, immunogenic and efficacious in protecting against CHIK virus (CHIKV) challenge of mice and nonhuman primates. To further evaluate its preclinical safety, we compared CHIKV/IRES distribution and viral loads in interferon-α/β receptor-incompetent A129 mice to another CHIK vaccine candidate, 181/clone25, which proved highly immunogenic but mildly reactive in human Phase I/II clinical trials. Compared to wild-type CHIK virus, (wt-CHIKV), both vaccines generated lower viral loads in a wide variety of tissues and organs, including the brain and leg muscle, but CHIKV/IRES exhibited marked restrictions in dissemination and viral loads compared to 181/clone25, and was never found outside the blood, spleen and muscle. Unlike wt-CHIKV, which caused disrupted splenic architecture and hepatic lesions, histopathological lesions were not observed in animals infected with either vaccine strain. To examine the stability of attenuation, both vaccines were passaged 5 times intracranially in infant A129 mice, then assessed for changes in virulence by comparing parental and passaged viruses for footpad swelling, weight stability and survival after subcutaneous infection. Whereas strain 181/clone25 p5 underwent a significant increase in virulence as measured by weight loss (from <10% to >30%) and mortality (from 0 to 100%), CHIKV/IRES underwent no detectible change in any measure of virulence (no significant weight loss and no mortality). These data indicate greater nonclinical safety of the CHIKV/IRES vaccine candidate compared to 181/clone25, further supporting its eligibility for human testing.     

Na-K-Cl Cotransporter-1 in the mechanism of ammonia-induced astrocyte swelling

Brain edema and the consequent increase in intracranial pressure and brain herniation are major complications of acute liver failure (fulminant hepatic failure) and a major cause of death in this condition. Ammonia has been strongly implicated as an important factor, and astrocyte swelling appears to be primarily responsible for the edema. Ammonia is known to cause cell swelling in cultured astrocytes, although the means by which this occurs has not been fully elucidated. A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. In particular, activation of the Na-K-Cl cotransporter (NKCC1) has been shown to be involved in cell swelling in several neurological disorders. We therefore examined the effect of ammonia on NKCC activity and its potential role in the swelling of astrocytes. Cultured astrocytes were exposed to ammonia (NH(4)Cl; 5 mm), and NKCC activity was measured. Ammonia increased NKCC activity at 24 h. Inhibition of this activity by bumetanide diminished ammonia-induced astrocyte swelling. Ammonia also increased total as well as phosphorylated NKCC1. Treatment with cyclohexamide, a potent inhibitor of protein synthesis, diminished NKCC1 protein expression and NKCC activity. Since ammonia is known to induce oxidative/nitrosative stress, and antioxidants and nitric-oxide synthase inhibition diminish astrocyte swelling, we also examined whether ammonia caused oxidation and/or nitration of NKCC1. Cultures exposed to ammonia increased the state of oxidation and nitration of NKCC1, whereas the antioxidants N-nitro-l-arginine methyl ester and uric acid all significantly diminished NKCC activity. These agents also reduced phosphorylated NKCC1 expression. These results suggest that activation of NKCC1 is an important factor in the mediation of astrocyte swelling by ammonia and that such activation appears to be mediated by NKCC1 abundance as well as by its oxidation/nitration and phosphorylation.     

The detoxification of Bordetella pertussis with glutaraldehyde

To improve the whole-cell pertussis vaccine we have studied the inactivation of the biological properties characteristic of Bordetella pertussis phase I bacteria, i.e. histamine-sensitizing, lymphocytosis-promoting and mouse protective activities, by treating a concentrated bacterial suspension with various concentrations of glutaraldehyde. Under the experimental conditions, treatment with 10 mM glutaraldehyde at 37 degrees C for 30 min resulted in a marked reduction of the toxic activities without grossly diminishing the protective potency. Further tests were performed on the stability of the protective potency, on the agglutinin production in mice, and on the freedom from abnormal toxicity in guinea-pigs.     

Physicochemical and immunobiological properties of the dialysate antigen of Bordetella pertussis

The study of the physicochemical and immunobiological properties of B. pertussis dialysate antigen indicates that the antigen has a complex composition and possesses hemagglutinating and lymphocytosis-promoting activity, which permits further studies with a view to developing diagnostic and prophylactic preparations on the basis of this antigen.