Blood Microbiota and Cancer: Cell Wall-Deficient L-Forms of Bacteria and Fungi as Cancer-Promoting Environment

In recent years, valuable experience and insights have been gained into L-forms (cell-wall-deficient variants) of bacteria and fungi and their disease-trigger potential in cases with chronic infections, autism spectrum disorders, autoimmune and neurodegenerative diseases. Based on the concept of “internal” blood microbiota, consisting of L-forms and its relevance to health and disease, the current study aims to outline the profile of dysbiotic disorders in three cancer patients (with endometrial cancer, breast cancer and acute myeloid leukemia), all in a phase before chemotherapy. Venous blood samples from the patients and from one control healthy person, were microbiologically studied. The used novel methodology of blood microbiota assessment was based on the following phases: isolation of L-forms, development and propagation, cultivation and conversion of L-forms into classical bacteria and fungi, as well as their identification with MALDI-TOF method. From the patients were isolated L-forms of opportunistic bacteria (Enterococcus faecalis, Esherichia coli, Enterobacter cloacae and Pseudomonas oryzihabitans) and fungi such as Rhodotorula mucilaginosa, Aspergillus fumigatus and Mucorales. In conclusion, the common feature found for the three cancer patients was the isolation from the blood of highly associated communities consisting of morphologically indistinguishable L-bodies, which through reversion in broth, were identified as distinct bacterial and fungal species. Unlike classic bacteria or fungi causing sepsis and bacteremia/fungemia, the presence of L-forms in blood is hidden, it does not demonstrate clinical signs nor it can be detected by conventional methods. It should be noted, however, that the dysbiotic blood microbiota shows unique and individual characteristics for the concrete cancer patient, correlates to the common state of the organism and tumor localization in the body, as well as it outlines the cancer promoting role of L-forms in processes of malignization, cancer genesis and progression.

Novel Approach to Microbiological Study of Chronic Inflammations at Upper Respiratory Tract: Research of Blood L-Form Microbiota

Background: The recognition of human blood microbiota, consisting of cell wall-deficient microbes (L-forms), is a major challenge today in the field of microbiology. There are accumulating data confirming the concept of “internal” blood L-form microbiota and its significance for health and diseases. Finding out whether the blood microbiota can be of diagnostic and prognostic importance for detection and evaluation of chronic infections anywhere in the body is a major objective. In the context of chronically infected upper respiratory tract (URT), the aim of the current study was to understand whether a local infection can be a source for entry of bacteria and fungi in the blood. Methods: Blood samples from six persons with chronic inflammations in URT diagnosed with hypertrophied adenoids, chronic sinusitis, nasal polyps, chronic naso-pharyngitis and one control healthy person were studied. Blood microbiota assessment methodology that be used, included three phases: 1) isolation of L-form cultures from blood-development and propagation;2) cultivation directed to conversion of L-forms into bacterial and fungal cultures;3) isolation of pure classical bacterial and fungal cultures and their identification by MALDI-TOF method. Results: From the patients were isolated L-forms of opportunistic bacteria (Streptococcus mitis, Roseomonas mucosa, Dermacoccus nishinomiyaensis, Enterococcus faecalis, Acinetobacter johnsonii, Pseudomonas putida, Staphylococcus aureus, Pseudomonas luteola, Enterobacter cloacae) and fungi such as Rhodotorula mucilaginosa, Aspergillus niger, Aspergillus fumigatus and Mucorales. Conclusion: The novel innovative methodology for assessment of blood L-form microbiota was successfully applied for detection of microbes responsible for chronic infections at URT.

Application of PCR-SSCP in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis

Objective: To study the relationship between the polymorphism of drug resistant gene rpoB and drug resistance against rifampicin(RFP) of M. tuberculosis L-forms, and to evaluate its clinical application. Methods: A total of 52 clinical isolated strains of M. tuberculosis L-forms were collected. rpoB gene polymorphism was analyzed by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) and conventional antimicrobial susceptibility test (AST). Their results were compared. Results: AST results showed that 38 of 52 clinical isolated strains were drug resistance (73.08％),while PCR-SSCP indicated 65.38％ (32/52) rpoB gene polymorphism. There was no statistic significance(χ2= 2.4914) between the 2 methods. Conclusion:Combined the application of PCR-SSCP with AST in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis may have advantages at earlier diagnosis and guidance of clinical medications.

Detecting katG Drug Resistant Genetic Mutation among pneumoconiosis patients complicated with tuberculosis in Mycobacterium tuberculosis L-forms by PCR-SSCP

Objective：To study the relationship between mutation in the katG gene and drug resistance of INH in Mycobacterium tuberculosis L-forms among patients of pneumoconiosis complicated with pulmonary tuberculosis, and to explore the clinical application of PCR-SSCP. Methods： A total of 52 clinical isolated strains of Mycobacterium tuberculosis L-forms were collected. Mutation in the katG genes was detected by PCR-SSCP and traditional antimicrobial susceptibility test （AST）. Results： The results by AST showed that there were 40 persisters in 52 clinical isolated strains. The drug resistant rate was 76.92%（40/52）, and the gene mutation rate of katG was 57.70%（30/52）by PCR-SSCP, the difference was no quite significance （X^2 = 2.8507, P 〉 0.05）. The coincidence rate of two methods was 75.00% （30/40）. Conclusion： The detectionrate of katG resistant strains in Mycobacterium tuberculosis L-forms was high by PCR-SSCP. The combined application of PCR-SSCP and traditional antimicrobial susceptibility test can improve the detecting rate.

Detecting drug resistant genetic mutation among pneumoconiosis patients complicated with tuberculosis in Mycobacterium tuberculosis L-forms application of PCR-SSCP technique in Huainan mining district

Objective： To study the relationship between drug resistant genetic mutation and drug resistance in Mycobacterium tuberculosis L-form, discuss the internal relationship between drug resistances and drug-resistant related genes and explore the value of PCR- SSCP to clinical application. Methods： A total of 52 clinically isolated strains of tuberculosis L-form were collected among 97 pneumoconiosis patients complicated with tuberculosis. The gene mutations of katG, rpoB and rpsL were detected by PCR-SSCP, and the results were compared with those analyzed by traditional antimicrobial susceptibility test（AST）. Results： The gene muta- tion rates of katG, rpoB and rpsL by PCR-SSCP were respectively 57.70% （30/52）, 65.38% （32/52） and 40.38% （21/52）. The rate of reversion was 78.85%（41/52） and the result of drag-resistant genes was invariable. The results of AST showed that there were 40 （76.92%） multi-drug resistant strains in 52 clinically isolated strains. The number for three-drug resistant strain was 21 （40.38%） and that of two-drug resistant was 19（36.54%）, but only 12（23.08%） strains were one drug resistant. The rate of total drug-resistance was 100%, but there were 15 strains of allied mutation of three genes, 16 of two mutations and 6 of only one by PCR-SSCP. The coincidences were respectively 71.43%, 84.12% and 50.00%. Then there was no significant difference between the allied mutations of multi-drug resistant gene and the mutations of only one drug resistant gene （P 〉 0.05）. Conclusion： PCR-SSCP technique has a higher sensibility and specificity to detect the genes of katG, rpoB and rpsL in tuberculosis L-form among pneumoconiosis complicated with tuberculosis,and the detecting rate of two drug resistant strains and three drug resistant strains was higher. The combined application of PCR-SSCP and AST has advantages at earlier diagnosis and guidance of clinical medications.

Grand Zero Density Estimates of L-functions Associated with Cusp Forms

In this paper a zero-density estimate of the large sieve type is given for the automorphic L-function L f (s,χ),where f is a holomorphic cusp form and χ a Dirichlet character of mod q.

Hydrogen Atom and Equivalent Form of the Lévy-Leblond Equation

We discuss the equivalent form of the Ldvy-Leblond equation such that the nilpotent matrices are two-dimensional. We show that this equation can be obtained in the non-relativistic limit of the （2＋1）-dimensional Dirac equation. Phrthermore, we analyze the case with four-dimensional matrices, propose a Hamiltonian for the equation in （3＋1） dimensions, and solve it for a Coulomb potential The quantized energy levels for the hydrogen atom are obtained, and the result is consistent with the non-relativistic quantum mechanics.

基于自由曲面和L-system的红掌生长模型

针对红掌花卉生长可视化中参数难以确定的问题进行研究,提出推导控制点的方法,建立红掌器官模型:采用Bezier曲线结合扫描成型的方法来构造叶柄模型,采用双三次NURBS曲线对佛焰苞进行建模;运用红掌的相关表型数据拟合生长函数,提出基于红掌生长规律的微分L-system,可有效模拟红掌的拓扑结构和生长过程;通过虚拟器官表示红掌器官的几何属性,降低微分L-system的复杂度。试验验证提出的方法对红掌各项生长指标拟合度可达0.89以上,并可对每个生长阶段的红掌进行模拟,能有效地对红掌的生长过程进行建模。

L波段全国产化2.5 kW级脉冲模块设计

随着国防和民用技术的迅速发展,高性能的L波段脉冲模块成为雷达和通信领域的关键组件。文章介绍了一个全国产化的2.5 kW级L波段脉冲模块的设计流程。该模块采用先进的射频放大技术和脉冲形成网络设计,结合本土材料和工艺技术的优势,实现模块的高性能和低成本生产。仿真和实验验证表明,设计的模块满足了所有预期的技术参数,包括功率水平、脉冲宽度和效率,通过了严格的质量控制和可靠性评估。

<i>L^pp</i>-Harmonic 1-Forms on <i>δ</i>-Stable Hypersurface in Space Form with Nonnegative Bi-Ricci Curvature

In this paper, we investigate the space of Lp p-harmonic 1-forms on a complete noncompact orientable δ-stable hypersurface Mm that is immersed in space form with nonnegative BiRic curvature. We prove the nonexistence of Lp p-harmonic 1-forms on Mm. Moreover, we obtain some vanishing properties for this class of harmonic 1-forms.

胃癌中幽门螺杆菌L型感染与MIF、MMP9、VEGF表达的关系

目的探讨胃癌组织中幽门螺杆菌L型(Hp-L)感染与巨噬细胞移动抑制因子(MIF)、基质金属蛋白酶-9(MMP-9)和血管内皮生长因子(VEGF)表达之间的相互关系及临床意义。方法应用免疫组织化学染色法及革兰染色检测80例胃癌组织及50例癌旁正常组织中Hp-L感染情况,同时检测上述组织中MIF、MMP9、VEGF蛋白的表达;运用RT-PCR及Western blotting法检测30例新鲜胃癌组织及相应切缘组织中MIFmRNA及MIF、MMP9、VEGF蛋白的表达。结果 80例胃癌组织中经免疫组化和革兰染色同时阳性病例有57例,与对照组相比具有显著性差异(71.25%vs 14%,P<0.05);胃癌组织中Hp-L阳性组中MIF、MMP9、VEGF蛋白表达阳性率高于Hp-L阴性组(P<0.05),且Hp-L感染与MIF、MMP9和VEGF表达均呈正相关关系(r=0.598,r=0.292,r=0.341,P<0.05)。30例胃癌新鲜组织中MIF-mRNA表达(1.583±0.177)高于对照组(1.407±0.078),两者差异具有显著性(t=3.729,P=0.001);Western blotting法检测MIF、MMP9、VEGF蛋白在胃癌组织中的表达显著高于癌旁正常组织;胃癌组织中MIF、MMP9均与肿瘤浸润深度及淋巴结转移有关,VEGF蛋白表达与肿瘤浸润深度有关(P<0.05)。结论 Hp-L型感染是促进胃癌的侵袭与转移的重要因素之一,其机制与上调胃癌组织中MIF、MMP9、VEGF表达有关。

金葡菌及其L型感染荷瘤小鼠的实验研究

本文应用金黄色葡萄球菌及Ｌ型分别感染肿瘤和非肿瘤Ｃ＿（５７）ＢＬ／６Ｎ小鼠，病理学和细菌学检测发现细菌及Ｌ型感染种植肿瘤的小鼠组，肿瘤转移率和诱癌能力均显著提高。此外金葡菌及其Ｌ型感染还可引起小鼠多器官间质性炎，尤以肺、肝、肾、脑及生殖器官等为著。结果表明细菌及Ｌ型感染与肿瘤转移和肿瘤的发生可能有关。

结核分枝杆菌L型致病性的实验研究

以结核分枝杆菌稳定Ｌ型感染豚鼠，证明结核分枝杆菌变为Ｌ型后致病性减弱，发病时间延长，ＯＴ试验阴性，引起的病理变化主要表现为组织的间质性炎症，有干酪样坏死，而无典型结核结节形成。原因在于细菌变为Ｌ型后细胞壁缺损，磷脂减少，不足以刺激巨噬细胞转变为上皮样组胞与郎罕氏巨细胞，而形成结核结节。在诊断上容易造成误诊或漏诊。

人体病理组织中细菌L型的电镜观察

对9例细菌L型感染的人体病理组织进行透射电镜观察。结果显示:(1)L型可分布于组织间质或进入吞噬细胞、上皮细胞和癌细胞等细胞胞质;(2)L型具有多形态、大小不等、胞壁缺陷、电子密度低等特点,细胞胞质内的细菌L型尚有A、B两种形态区别;(3)L型在组织中形成了不完全箍缩分裂;(4)宿主细胞超微结构仅出现轻微改变。本结果与文献报道基本一致,提出了透射电镜下病理组织中细菌L型的形态特征及分布;并对L型感染与慢性炎症的关系等问题进行了讨论。

金黄色葡萄球菌L型和人乳头瘤病毒与宫颈癌关系的研究

应用改良革兰氏染色和免疫组化染色（ＡＢＣ法），对２７０例宫颈癌和３３例慢性宫颈炎进行了研究。结果发现，宫颈癌革兰氏染色切片中细菌Ｌ型感染率（７５．１％）明显高于慢性宫颈炎（４５．５％）（Ｐ＜０．０５），金黄色葡萄球菌Ｌ型抗原阳性率（７６．２％）明显高于人乳头瘤病毒（ＨＰＶ）抗原阳性率（５７．１％）。Ｌ型、ＨＰＶ混合感染率为４２．８％。上述结果表明宫颈癌中金葡菌Ｌ型与ＨＰＶ感染均常见，尤其金葡菌Ｌ型感染更为多见；金葡菌Ｌ型与ＨＰＶ感染的致病特征相似，两者均有不同程度的空泡细胞出现。提示金葡菌Ｌ型感染可能也是宫颈癌的致癌重要因素之一。

原位核酸杂交检测宫颈癌中金黄色葡萄球菌L型DNA

应用病原微生物培养、改良革兰氏染色、免疫组化染色及原位核酸杂交等方法，对５０例宫颈癌和３３例慢性宫颈炎进行了研究。结果发现宫颈癌病原微生物培养细菌及其Ｌ型阳性感染率为５８％（２９／５０例），以金黄色葡萄球菌（金葡菌）及其Ｌ型多见（１３／２９例）；宫颈癌和慢性宫颈炎组织切片革兰氏染色，细菌Ｌ型检出率分别为６６％和４５．５％；免疫组化染色（ＡＢＣ法）金葡菌Ｌ型抗原阳性率各为６８％和４８．５％。Ｌ型主要分布于癌间质、癌巢及宫颈上皮下结缔组织内。１２例宫颈癌金葡菌Ｌ型ＤＮＡ探针原位杂交显示，１０／１２例癌细胞核内有金葡菌Ｌ型ＤＮＡ杂交阳性信号，表明金葡菌Ｌ型ＤＮＡ已进入宫颈癌细胞内，可能会在基因水平上影响宫颈上皮恶变，提示宫颈癌发生与金葡菌Ｌ型感染关系密切，值得进一步研究。

肺结核患者痰标本中结核分枝杆菌L型的检测及其临床意义

目的 探讨肺结核患者结核分枝杆菌L型的检测及其临床意义。方法 对 12 4例活动性肺结核和 2 2例非活动性肺结核患者 ,分别作痰抗酸染色涂片、改良罗氏培养、BACTEC 960培养和结核分枝杆菌L型培养。结果 痰结核分枝杆菌L型培养阳性率显著高于改良罗氏培养 ,差异有显著性 (χ2 =4.6873 ,P <0 .0 5 ) ,也高于抗酸染色涂片和BACTEC 960培养 ,但差异无显著性 (P >0 .0 5 )。活动性肺结核患者L型培养阳性率为60 .48% ,显著高于非活动性肺结核患者 (χ2 =19.85 65 ,P <0 .0 0 1)。病程越长 ,痰结核分枝杆菌L型阳性率越高 ,但仅病程 >1年者与 <1个月者之间差异有显著性 (P <0 .0 5 )。复治病例L型培养阳性率显著高于初治病例 (χ2 =4.0 5 78,P <0 .0 5 )。有空洞患者L型培养阳性率为 71.19% ,显著高于无空洞患者 (χ2 =5 .3 941,P <0 .0 5 )。耐药和耐多药患者L型培养阳性率均显著高于敏感患者 (P <0 .0 5 )。化疗开始时痰结核分枝杆菌L型阳性者 ,化疗 6个月末痰菌阴转率显著低于L型阴性者 (χ2 =3 .913 8,P <0 .0 5 )。结论 结核分枝杆菌L型培养可提高结核分枝杆菌的阳性检出率 ,对结核病的早期、快速诊断具有重要价值。结核分枝杆菌L型感染X线表现特点为干酪样病变和空洞多。结核分枝杆菌L型耐药率高 ,临?

细菌L型感染荷瘤小鼠肿瘤组织中癌基因蛋白的表达

本文探讨了金葡菌Ｌ型和结核菌Ｌ型感染荷瘤小鼠癌基因Ｐ２１、Ｐ５３及Ｃ－ｅｒｂＢ－２产物的表达。结果发现感染小鼠上述癌基因产物水平较对照组小鼠明显升高（０．０１＜Ｐ＜０．０５）；实验组ＰＣＮＡ阳性细胞指数高于对照组（Ｐ＜０．０５）；荷瘤感染小鼠存活期缩短，自发性肿瘤增多（Ｐ＜０．０５）。提示细菌Ｌ型感染与肿瘤的发生、发展有关。

聚合酶链反应和改良抗酸染色诊断L型结核杆菌感染

目的 探讨 L 型抗酸菌的 IK染色和 PCR检测的临床应用价值 .方法 采用 IK染色和 PCR技术 ,对结核病患者(35例 )、非特异性淋巴结炎患者 (30例 )和正常胎儿 (1 0例 )的石蜡切片进行对比研究 .结果 两种方法 (PCR和 IK染色 )对上述 3组 75例的检测阳性率分别为 80 % ,83% ;6 7% ,6 0 %及 0 % .结论 PCR技术及 IK染色 ,检测抗酸菌 L

以VEGF、MMP-2检测验证幽门螺杆菌L型感染与胃癌的关系

目的研究幽门螺杆菌L型(Helicobacter pyloriL-form,Hp-L型)感染对胃癌的发生、发展和侵袭、转移等生物学行为的影响,并探讨其机制。方法应用革兰染色和免疫组化SP法检测130例胃癌和50例对照组的Hp-L型感染,应用免疫组化SP法检测上述组织的血管内皮生长因子(vascular endothelial growth factor,VEGF)、基质金属蛋白酶-2(matrixmet-alloproteinase-2,MMP-2)蛋白的表达。结果130例胃癌中免疫组化及革兰染色同时Hp-L型阳性的病例有88例。胃癌组与对照组的Hp-L型阳性率相比具有统计学意义(67.69%vs24%,P<0.05)。胃癌组中Hp-L型阳性组的MMP-2、VEGF表达阳性率高于其Hp-L型阴性组(P<0.05);胃癌组中Hp-L型阳性MMP-2和VEGF的表达阳性呈正相关(r=0.45,r=0.30,P<0.001)。结论Hp-L型感染是影响胃癌侵袭和转移的重要因素之一。其机制与胃癌细胞的VEGF、MMP-2的表达上调有关。