Radiolocalization of 99mTc-labeled fragments in a melanoma xenografted model

A xenograft model of human malignant melanoma was used to compare, in terms of tumor localization, the specific antimelanoma monoclonal antibody (MoAb) 225.28S with an irrelevant antibody (4C4). Both specific and non-specific 99mTc-labeled fragments were injected in 12 nude mice bearing subcutaneous tumor. The animals were then sacrificed at 6 and 24 hours post-injection and immediately dissected. Radioactivity of the tumor and normal tissues was measured in a well scintillation counter and autoradiography of tumor, liver and kidneys was also obtained. Tumor localization of 99mTc-labeled MoAb 225.28S fragments was highly specific compared with 99mTc-labeled irrelevant antibody 4C4. With the exception of the kidneys, already at six hours there was a satisfactory tumor-to-normal tissue ratio, which improved at 24 hours. However, the percentage of injected dose per gram of tumor decreased with time, probably due to the weaker bond of radiolabeled Fab' fragments to tumor cells. These results would indicate 99mTc-fragments of the antimelanoma MoAb 225.28S as a suitable radiotracer in clinical nuclear medicine.     

Peroxynitrite-modified 99mTc-beta-VLDL: tissue distribution and plasma clearance rate

Free radicals superoxide (O(2)(-)) and nitric oxide (*NO) are generated by blood vessels and can rapidly react to produce a peroxynitrite anion (ONOO(-)), a powerful oxidant that modifies lipoproteins making them more atherogenic. The aim of this study was to investigate the effect of peroxynitrite-induced modifications on beta-very-low-density lipoprotein (beta-VLDL) as to its biodistribution and plasma clearance rate, as well as the uptake of these particles by THP-1 cells. After being injected into New Zealand White rabbits, the peroxynitrite-modified beta-VLDL (99mTc-per-beta-VLDL) was cleared from circulation faster than the native beta-VLDL (99mTc-nat-beta-VLDL) in both normocholesterolemic rabbits (NC) and in hypercholesterolemic rabbits (HC). In HC rabbits, the fractional clearance of 99mTc-labeled beta-VLDL was significantly lower than in NC rabbits. The in vivo studies showed that accumulation of 99mTc-labeled beta-VLDL, expressed per gram of tissue, followed the decreasing order: kidney > liver > spleen > adrenal gland >or= lung > aortic arch > heart >or= abdominal aorta > thoracic aorta > psoas muscle. The high accumulation in the kidneys suggests the processing of 99mTc-labeled apolipoproteins by receptors present in kidney cells. The accumulation of 99mTc-nat-beta-VLDL in the whole organ was the following: liver > kidney > heart > spleen > adrenal gland > aorta in HC and NC rabbits. The uptake of 99mTc-per-beta-VLDL by the spleen was greater than the uptake by the heart in both groups. The in vitro studies showed that the uptake of 99mTc-per-beta-VLDL by THP-1 cells was higher than that of 99mTc-nat-beta-VLDL. These results show that peroxynitrite-modified beta-VLDL is rapidly removed from plasma and accumulates in several tissues, mainly in the liver and kidney. This may be particularly important in hypercholesterolemic situations that could favor the accumulation of native and peroxynitrite-modified beta-VLDL in several tissues.     

The Streptococcus sanguinis competence regulon is not required for infective endocarditis virulence in a rabbit model

Streptococcus sanguinis is an important component of dental plaque and a leading cause of infective endocarditis. Genetic competence in S. sanguinis requires a quorum sensing system encoded by the early comCDE genes, as well as late genes controlled by the alternative sigma factor, ComX. Previous studies of Streptococcus pneumoniae and Streptococcus mutans have identified functions for the >100-gene com regulon in addition to DNA uptake, including virulence. We investigated this possibility in S. sanguinis. Strains deleted for the comCDE or comX master regulatory genes were created. Using a rabbit endocarditis model in conjunction with a variety of virulence assays, we determined that both mutants possessed infectivity equivalent to that of a virulent control strain, and that measures of disease were similar in rabbits infected with each strain. These results suggest that the com regulon is not required for S. sanguinis infective endocarditis virulence in this model. We propose that the different roles of the S. sanguinis, S. pneumoniae, and S. mutans com regulons in virulence can be understood in relation to the pathogenic mechanisms employed by each species.     

Ecto-5'-nucleotidase: a candidate virulence factor in Streptococcus sanguinis experimental endocarditis

Streptococcus sanguinis is the most common cause of infective endocarditis (IE). Since the molecular basis of virulence of this oral commensal bacterium remains unclear, we searched the genome of S. sanguinis for previously unidentified virulence factors. We identified a cell surface ecto-5'-nucleotidase (Nt5e), as a candidate virulence factor. By colorimetric phosphate assay, we showed that S. sanguinis Nt5e can hydrolyze extracellular adenosine triphosphate to generate adenosine. Moreover, a nt5e deletion mutant showed significantly shorter lag time (P<0.05) to onset of platelet aggregation than the wild-type strain, without affecting platelet-bacterial adhesion in vitro (P?=?0.98). In the absence of nt5e, S. sanguinis caused IE (4 d) in a rabbit model with significantly decreased mass of vegetations (P<0.01) and recovered bacterial loads (log(10)CFU, P?=?0.01), suggesting that Nt5e contributes to the virulence of S. sanguinis in vivo. As a virulence factor, Nt5e may function by (i) hydrolyzing ATP, a pro-inflammatory molecule, and generating adenosine, an immunosuppressive molecule to inhibit phagocytic monocytes/macrophages associated with valvular vegetations. (ii) Nt5e-mediated inhibition of platelet aggregation could also delay presentation of platelet microbicidal proteins to infecting bacteria on heart valves. Both plausible Nt5e-dependent mechanisms would promote survival of infecting S. sanguinis. In conclusion, we now show for the first time that streptococcal Nt5e modulates S. sanguinis-induced platelet aggregation and may contribute to the virulence of streptococci in experimental IE.     

Structural basis for the species selectivity of a fibrin-specific monoclonal antibody

The structural determinant underlying the species specificity of a monoclonal anti-fibrin antibody (59D8) is the leucyl residue at position 5 in beta-chains of human fibrin. Anti-fibrin antibody 59D8 which had been elicited by immunization with human beta(1-7) peptide, Gly-His-Arg-Pro-Leu-Asp-Lys, binds to human and canine fibrins but not to bovine, ovine, or porcine fibrins. A comparison of the available amino acid sequence data suggested that the ability of anti-fibrin antibody 59D8 to discriminate among various fibrin beta-chains might be due to the amino acid at position 5. This was confirmed by competitive inhibition studies using synthetic fibrin-like peptides and determination of the amino acid sequences of the N-termini of ovine and porcine fibrin beta-chains. Edman degradation employing o-phthalaldehyde blocking permitted use of fibrin monomer rather than its separated constituent polypeptide chains. The same sequencing strategy was used to obtain partial sequence data for the alpha-chains of bovine, ovine, and porcine fibrin.     

Injection of cells and monoclonal antibodies into mice: comparison of tail vein and retroorbital routes

Organ distribution and blood concentration profiles were compared following injection of mice with radiolabeled test agents via the lateral tail vein or retroorbital venous sinus. Monoclonal antibodies directed against B16 melanoma of C57BL/6 origin were labeled with iodine-125. Thymocytes from BALB/c mice and B16 melanoma cells were labeled with technetium-99m sodium pertechnetate (Na 99mTcO4). Animals were injected with 5 microCI of iodinated antibody, 5 X 10(5) syngeneic thymocytes, 2.5 X 10(5) melanoma cells, or 10 microCi Na 99mTcO4 in 0.2 ml saline via either route. In non-tumor-bearing C57BL/6 mice radiolabeled monoclonal antibody was found primarily in the gastrointestinal tract, liver, and blood. Na 99mTcO4 localized in the gastrointestinal tract, 99mTc-labeled thymocytes in the spleen and liver, and 99mTc-labeled B16 melanoma cells in the liver and lungs. Pharmacokinetic analysis of blood samples taken 4, 8, and 12 min following injection of the labeled agents suggested that the iodinated antibody had less vascular permeability than Na 99mTcO4 and that thymocytes and B16 melanoma cells were trapped in the pulmonary vasculature as they passed through the lungs. It is noteworthy that no biologically significant differences in organ distribution patterns or blood decay profiles were found between lateral tail vein and retroorbital routes. The data clearly indicate that these routes can be used interchangeably with one another for intravenous injections.     

Disseminated staphylococcal disease in laboratory rabbits (Oryctolagus cuniculus).

The pathologic findings in 13 cases of staphylococcal disease in New Zealand white rabbits were described. Subcutaneous abscesses and embolic pyemic abscesses in kidney, heart, brain, and lung were found. Conjunctivitis, rhinitis, otitis media, and fibrinous pneumonia also occurred. One rabbit had a valvular endocarditis. Staphylococcus aureus, coagulase-positive, and fermenting mannitol were isolated from the lesions described. Staphylococcal disease was diagnosed in 13 of the 171 (7.6%) rabbits necropsied during a 3-yr period. Disseminated staphylococcal lesions were observed only in rabbits during this time.     

Microbial adhesion to fibronectin in vitro correlates with production of endocarditis in rabbits

Microbial adhesion to the constituents of nonbacterial thrombotic endocarditis (NBTE) is an important early event in the pathogenesis of infective endocarditis. Fibronectin is a ubiquitous mammalian glycoprotein with diverse functions which binds to certain bacteria but not to others. In this study, we determined that fibronectin is present on the surface of NBTE (after catheter-induced aortic valve trauma) but not on normal rabbit cardiac valvular endothelium. The adhesion of various bacteria and yeasts to human fibronectin in tissue culture wells was then measured. Microorganisms with a high isolation frequency from endocarditis cases (Staphylococcus aureus, Candida tropicalis, C. albicans, Streptococcus faecalis, S. sanguis) bound significantly better (P less than 0.01) to fibronectin in vitro than other organisms (Escherichia coli, C. krusei, Pseudomonas aeruginosa) rarely implicated in this disease. Microbial adhesion to fibronectin correlated closely with the propensity of each organism to produce endocarditis in rabbits (e.g., ID50) with preexistent NBTE. A similar distribution was noted after binding of soluble radiolabeled fibronectin to bacteria in suspension. The results suggest that fibronectin, expressed on the surface of NBTE, may mediate microbial adhesion of circulating organisms to initiate colonization during the early pathogenesis of infective endocarditis.     

Role of D and E domains in the migration of vascular smooth muscle cells into fibrin gels

The structure of fibrin plays an important role in the organization of thrombi, the development of atherosclerosis, and restenosis after PTCA. In this study, we examined the mechanisms of the migration of vascular smooth muscle cells (SMCs) into fibrin gels, using an in vitro assay system. Cultured SMCs from bovine fetal aortic media migrated into fibrin gels prepared with thrombin, which cleaves both fibrinopeptides A and B from fibrinogen, without other chemotactic stimuli. Both desA fibrin gels prepared with batroxobin, which cleaves only fibrinopeptide A, and desB fibrin gels prepared with Agkistrodon contortrix thrombin-like enzyme (ACTE), which cleaves only fibrinopeptide B, similarly induced the migration of SMCs compared to fibrin gels prepared with thrombin. These results suggest that the cleavage of fibrinopeptides is not necessary, but rather that the three-dimensional structure of the gel may be important for the migration of SMCs. Furthermore, gels prepared with protamine sulfate, which forms fibrin-like gels non-enzymatically, similarly induced the migration of SMCs compared to the gels prepared with thrombin. Both anti-fibrin(ogen) fragment D and anti-fibrin(ogen) E antibodies inhibited the migration of SMCs into fibrin gels, suggesting that both the D and E domains of fibrin(ogen) are involved in the migration of SMCs into fibrin gels. The addition of GRGDS, a synthetic RGD-containing peptide, but not that of GRGES, a control peptide, partially inhibited the migration of SMCs into fibrin gels, suggesting that the migration of SMCs into fibrin gels is at least in part dependent on the RGD-containing region of the alpha chain. The migration of SMCs into fibrin gels was also inhibited by a monoclonal antibody for integrin alpha v beta 3 and alpha 5 beta 1, indicating that migration is dependent on these integrins. Furthermore, both fibrin(ogen) fragments D and E inhibited the migration of SMCs into fibrin gels, suggesting that these fragments, generated during fibrino(geno)lysis, may be relevant in the regulation of SMC migration into fibrin gels.     

纤维蛋白单体D区与E区聚合后E区结构的变化

应用纤维蛋白单克隆抗体IF 5 3,观察当纤维蛋白的“A”位点与另一纤维蛋白D区域的“a”位点结合后纤维蛋白E区的变化 .纤维蛋白原Aα链经赖氨酰肽链内切酶消化后 ,应用反相HPLC分离纯化 ;通过ELISA法检测单克隆抗体IF 5 3与纤维蛋白原及其衍生物的反应情况 ;应用放射免疫法检测RGD合成肽抑制纤维蛋白单体与IF 5 3反应的情况 .发现IF 5 3能与纤维蛋白原Aα链的一个片段反应 ,该片段经氨基酸序列分析显示为纤维蛋白原Aα链氨基末端 (1～ 2 9) .该抗体能与酸溶解的纤维蛋白单体和可溶性纤维蛋白及XDP反应 ,但不能与酸化纤维蛋白原或GPRP反应 ,因此IF 5 3的抗原决定簇在Aα 2 0～ 2 9,与凝血酶作用于纤维蛋白肽A ,暴露出的聚合位点“A”(Aα17～19)紧邻 .当GPRP存在于纤维蛋白原溶液时 ,经凝血酶作用产生这种纤维蛋白单体不能与IF 5 3反应 .Aα(93～ 99) (ILRGDFS)合成肽部分抑制纤维蛋白单体与IF 5 3的反应 .实验结果提示 ,当纤维蛋白单体相互聚合 ,或纤维蛋白单体与纤维蛋白原聚合时 ,纤维蛋白单体结构会发生变化 ,其中Aα2 0～ 2 9片段成为新抗原暴露于E区表面 ,并且Aα2 0～ 2 9与纤维蛋白原细胞粘附区域RGD1片段邻近     

A new high affinity technetium-99m-bombesin analogue with low abdominal accumulation

99mTc-labeled bombesin analogues have shown promise for noninvasive detection of many tumors that express bombesin (BN)/gastrin-releasing peptide (GRP) receptors. 99mTc-labeled peptides, however, have a tendency to accumulate in the liver and intestines due to hepatobiliary clearance as a result of the lipophilicity of the 99mTc chelates. This makes the imaging of lesions in the abdominal area difficult. In this study, we have synthesized a new high affinity 99mTc-labeled BN analogue, [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN, having a built-in pharmacokinetic modifier, DTPA, and labeled with 99mTc using a hydrophilic diaminedithiol chelator (Pm-DADT) to effect low hepatobiliary clearance. In vitro binding studies using human prostate cancer PC-3 cell membranes showed that the inhibition constant (Ki) for [DTPA1, Lys3(99Tc-Pm-DADT), Tyr4]BN was 4.1 +/- 1.4 nM. Biodistribution studies of [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN in normal mice showed very low accumulation of radioactivity in the liver and intestines (1.32 +/- 0.13 and 4.58 +/- 0.50% ID, 4 h postinjection, respectively). There was significant uptake (7.71 +/- 1.37% ID/g, 1 h postinjection) in the pancreas which expresses BN/GRP receptors. The uptake in the pancreas could be blocked by BN, partially blocked by neuromedin B, but not affected by somatostatin, indicating that the in vivo binding was BN/GRP receptor specific. Scintigraphic images showed specific, high contrast delineation of prostate cancer PC-3 xenografts in SCID mice. Thus, the new peptide has a great potential for imaging BN/GRP receptor-positive cancers located even in the abdomen.     

胶原/纤维蛋白止血效果观察

目的研究胶原/纤维蛋白对新西兰兔的止血作用,并与胶原蛋白海绵止血效果作比较。方法选用胶原/纤维蛋白止血贴,对新西兰兔耳部动、静脉出血、耳表创面、股动脉割伤、肝损伤、体表创面进行止血试验,同时与胶原蛋白海绵止血效果作比较,观察其止血时间、失血量、敷料与创面的粘合等情况,并定期观察创面愈合、体内吸收和抗炎情况。结果胶原/纤维蛋白复合止血贴组、胶原蛋白海绵组新西兰兔耳动、静脉、耳表创面、股动脉割伤、标准肝创伤的止血时间与对照组比较,差异显著（P〈0.05）。胶原/纤维蛋白复合止血贴组新西兰兔耳动、静脉、耳表创面的止血时间与胶原蛋白海绵组,差异显著（P〈0.05）。胶原/纤维蛋白复合止血贴组、胶原蛋白海绵组动物的耳表创面、标准肝创伤失血量与对照组比较,差异显著（P〈0.05）。体内标准肝创伤、体表创伤后期观察,胶原/纤维蛋白复合止血贴与胶原蛋白海绵均能在21d内完全吸收,未见炎症反应。结论胶原/纤维蛋白复合止血贴对新西兰兔耳动脉割伤、耳静脉割伤、耳标创伤、股动脉割伤和标准肝损伤模型都具有明显的止血作用,体表创面伤口恢复良好,体内吸收速度快,具有一定的抗炎作用,而且在新西兰兔耳动脉、耳静脉割伤和耳表创伤的止血效果明显优于胶原蛋白海绵。胶原/纤维蛋白复合止血贴是一种较安全有效的局部止血生物材料。     

Comparative binding of 125I-and 99mTc-d-labeled native and glycated low-density lipoprotein to human microvascular endothelial cells-potential for atherosclerosis imaging?

Native (n), glycated (g), and glycoxidated (go) low-density lipoproteins (LDL) were labeled with 125I or 99mTc, and the labeling efficiency and binding were assessed for potential use of these LDL compounds in imaging analysis of atherosclerotic lesions (PPAR-gamma receptors) by determining the number of specific receptors for nLDL, gLDL or goLDL on human microvascular endothelial cells as well as the KDs using either 125I-or 99mTc-labeled LDLs. The specific activity of labeled gLDL and goLDL was much higher (for goLDL 20 times higher) than that of nLDL. Gel filtration of labeled LDLs revealed, however, that 99mTc-g/goLDL is significantly degraded by the labeling reaction. No fragmentation was observed for 99mTc-nLDL and all the 125I-labeled LDL forms. Binding studies using both 125I-and 99mTc-nLDL indicated a weak binding affinity (KD 10- 7mol/L) to human microvascular endothelial cells. The binding affinity of 125I-g/goLDL to these cells was significantly higher (KD 10- 9mol/L) and could be increased further by preactivation of the endothelial cells using TNFalpha. Incubation with 99mTc-goLDL, however, did not result in specific binding of the ligand, possibly as a consequence of the fragmentation of the lipoprotein during the labeling. Scatchard transformation of the binding data with 99mTc-gLDL revealed the presence of only a few binding sites. This was in contrast to the results obtained with 125I-labeled gLDL, which revealed a much higher membrane density of scavenger receptors for this ligand. We conclude that for in vitro binding studies as well as for potential in vivo imaging, only 125I-labeled goLDL should be used, whereas nLDL may be applied as 125I-or 99mTc-labeled ligand.     

人接头蛋白NRBP的表达、纯化及多克隆抗体制备

目的:制备接头蛋白NRBP的兔多克隆抗体,并检测该抗体的效价及特异性。方法:PCR方法以重组质粒PEF-NRBP为模板,获得NRBP全长及NRBP(1-99Aa)cDNA,构建到原核表达质粒pET-21a及pGEX-6P-1中。分别转入大肠杆菌BL21菌株,IPTG诱导表达后,纯化并鉴定表达产物将其免疫家兔,间接ELISA法及免疫印迹等方法鉴定抗体。结果:成功获得人NRBP的cDNA,构建得到相关的原核表达质粒,在大肠杆菌中可诱导性高表达,纯化后蛋白免疫家兔制备得到的抗血清经ELISA检测为阳性结果,4只免疫家兔的抗体效价约为1:5 200～1:40 000。Western印迹结果显示,该抗体可特异性的识别真核细胞外源性及内源性约60kDa的NRBP蛋白,并且具有较强免疫沉淀能力。结论:NRBP多克隆抗体具有很好的特异性和敏感性,该抗体的成功制备为进一步研究NRBP的功能提供了重要工具。     

兔抗麻雀IgY酶标抗体的制备

目的制备辣根过氧化物酶（HRP）标记的兔抗麻雀IgY抗体,为禽类血清学检测体系的建立提供技术储备。方法硫酸铵盐析法粗提麻雀血清IgY,进一步在SDS-PAGE上分离后,切下带有目的条带的凝胶作为免疫原,免疫实验兔制备抗血清,Protein-A柱亲和纯化兔抗IgY血清IgG,,使用改良过碘酸钠法制备酶结合物。ELISA检测酶标抗体的工作浓度,western blotting检测酶标抗体的特异性。结果硫酸铵盐析法粗提IgY,可去除部分杂蛋白,SDS-PAGE上分离后切下带有目的条带的凝胶,可以得到足够纯度的抗原,将带有IgY的凝胶作为抗原免疫后获得的抗血清经Protein-A纯化后,二抗在SDS-PAGE上鉴定,纯度达到99%以上。改良的过碘酸钠法标记获得的抗体浓度为1.008 mg/mL,ELISA检测酶标抗体效价为1∶1000。Western blotting鉴定抗体具有特异性。结论获得了优质可靠的兔抗麻雀IgY酶标抗体。     

The effect of fibrin on endothelial cell migration in vitro

Endothelial cells are known to migrate and come into contact with fibrin during numerous physiological processes, such as in wound healing and in tumor growth. The present study was initiated to investigate the effect of fibrin on endothelial cell migration in vitro. Endothelial cell migration was assayed by wounding confluent monolayers of bovine aortic endothelial cells with a razor blade and counting the number of cells crossing the wound per unit time. Wound-induced proliferation of endothelial cells was inhibited by mitomycin C-treatment without affecting endothelial cell migration, indicating that in this assay migration could be measured independent of proliferation. Migration of endothelial cells in vitro was inhibited by fibrin in a concentration dependent manner. Endothelial cell migration under fibrin was further reduced by plasminogen depletion of the serum, and fibrin still inhibited the migration of mitomycin C-treated endothelial cells. Kadish et al. (Tissue and Cell, 11, 99, 1979) previously reported that fibrin did not affect EC migration in vitro. The inability to inhibit EC migration with fibrin appears to be due to their assay system which employed agarose, since pre-treating the wounded monolayer with agarose eliminated the inhibition of EC migration by fibrin. The present results indicate that EC migration in vitro can be used as a model system for studying the interaction of fibrin with EC.     

Targeting arterial wall sulfated glycosaminoglycans in rabbit atherosclerosis with a mouse/human chimeric antibody

The progression of atherosclerosis is favored by increasing amounts of chondroitin sulfate proteoglycans in the artery wall. We previously reported the reactivity of chP3R99 monoclonal antibody (mAb) with sulfated glycosaminoglycans and its association with the anti-atherogenic properties displayed. Now, we evaluated the accumulation of this mAb in atherosclerotic lesions and its potential use as a probe for specific in vivo detection of the disease. Atherosclerosis was induced in NZW rabbits (n = 14) by the administration of Lipofundin 20% using PBS-receiving animals as control (n = 8). Accumulation of chP3R99 mAb in atherosclerotic lesions was assessed either by immunofluorescence detection of human IgG in fresh-frozen sections of aorta, or by immunoscintigraphy followed by biodistribution of the radiotracer upon administration of ^99mTc-chP3R99 mAb. Immunofluorescence studies revealed the presence of chP3R99 mAb in atherosclerotic lesions 24 h after intravenous administration, whereas planar images showed an evident accumulation of ^99mTc-chP3R99 mAb in atherosclerotic rabbit carotids. Accordingly, ^99mTc-chP3R99 mAb uptake by lesioned aortic arch and thoracic segment was increased 5.6-fold over controls and it was 3.9-folds higher in carotids, in agreement with immunoscintigrams. Moreover, the deposition of ^99mTc-chP3R99 mAb in the artery wall was associated both with the presence and size of the lesions in the different portions of evaluated arteries and was greater than in non-targeted organs. In conclusion, chP3R99 mAb preferentially accumulates in arterial atherosclerotic lesions supporting the potential use of this anti-glycosaminoglycans antibody for diagnosis and treatment of atherosclerosis.     

Induction of immunological tolerance in neonatal and adult rabbits. Differences in the cellular events

A transient production of antibody does not occur in the induction of specific immunological tolerance to soluble BSA in neonatal rabbits. The newborn rabbit, however, has the immunologic potential to respond to BSA when it is given in adjuvant. These results suggest that a normal complement of specific B cells is present in the newborn rabbit and that the immunoincompetence to the soluble protein, that permits the induction of tolerance without a phase of antibody formation, may be a function of either T cells or macrophages. In contrast to the newborn, the induction of tolerance in adult rabbits following daily injections with large doses of BSA is preceded by a transient production of antibody-producing cells. In this situation, at least, one of the events involved in the induction of unresponsiveness appears to be interpretable as an exhaustive differentiation of competent cells capable of being stimulated. Therefore, two differing cellular patterns can be observed in the completion of seemingly identical end states of immunological tolerance.     

Diffusion of ofloxacin in the endocarditis vegetation assessed with synchrotron radiation UV fluorescence microspectroscopy

The diffusion of antibiotics in endocarditis vegetation bacterial masses has not been described, although it may influence the efficacy of antibiotic therapy in endocarditis. The objective of this work was to assess the diffusion of ofloxacin in experimental endocarditis vegetation bacterial masses using synchrotron-radiation UV fluorescence microspectroscopy. Streptococcal endocarditis was induced in 5 rabbits. Three animals received an unique i.v. injection of 150 mg/kg ofloxacin, and 2 control rabbits were left untreated. Two fluorescence microscopes were coupled to a synchrotron beam for excitation at 275 nm. A spectral microscope collected fluorescence spectra between 285 and 550 nm. A second, full field microscope was used with bandpass filters at 510-560 nm. Spectra of ofloxacin-treated vegetations presented higher fluorescence between 390 and 540 nm than control. Full field imaging showed that ofloxacin increased fluorescence between 510 and 560 nm. Ofloxacin diffused into vegetation bacterial masses, although it accumulated in their immediate neighborhood. Fluorescence images additionally suggested an ofloxacin concentration gradient between the vegetation peripheral and central areas. In conclusion, ofloxacin diffuses into vegetation bacterial masses, but it accumulates in their immediate neighborhood. Synchrotron radiation UV fluorescence microscopy is a new tool for assessment of antibiotic diffusion in the endocarditis vegetation bacterial masses.     

Diffusion of Ofloxacin in the Endocarditis Vegetation Assessed with Synchrotron Radiation UV Fluorescence Microspectrocopy

The diffusion of antibiotics in endocarditis vegetation bacterial masses has not been described, although it may influence the efficacy of antibiotic therapy in endocarditis. The objective of this work was to assess the diffusion of ofloxacin in experimental endocarditis vegetation bacterial masses using synchrotron-radiation UV fluorescence microspectroscopy. Streptococcal endocarditis was induced in 5 rabbits. Three animals received an unique IV injection of 150 mg/kg ofloxacin, and 2 control rabbits were left untreated. Two fluorescence microscopes were coupled to a synchrotron beam for excitation at 275 nm. A spectral microscope collected fluorescence spectra between 285 and 550 nm. A second, full field microscope was used with bandpass filters at 510–560 nm. Spectra of ofloxacin-treated vegetations presented higher fluorescence between 390 and 540 nm than control. Full field imaging showed that ofloxacin increased fluorescence between 510 and 560 nm. Ofloxacin diffused into vegetation bacterial masses, although it accumulated in their immediate neighborhood. Fluorescence images additionally suggested an ofloxacin concentration gradient between the vegetation peripheral and central areas. In conclusion, ofloxacin diffuses into vegetation bacterial masses, but it accumulates in their immediate neighborhood. Synchrotron radiation UV fluorescence microscopy is a new tool for assessment of antibiotic diffusion in the endocarditis vegetation bacterial masses.