Paleopathology of Human Tuberculosis and the Potential Role of Climate

Both origin and evolution of tuberculosis and its pathogens (Mycobacterium tuberculosis complex) are not fully understood. The paleopathological investigation of human remains offers a unique insight into the molecular evolution and spread including correlative data of the environment. The molecular analysis of material from Egypt (3000–500 BC), Sudan (200–600 AD), Hungary (600–1700 AD), Latvia (1200–1600 AD), and South Germany (1400–1800 AD) urprisingly revealed constantly high frequencies of tuberculosis in all different time periods excluding significant environmental influence on tuberculosis spread. The typing of various mycobacteria strains provides evidence for ancestral M. tuberculosis strains in Pre- to early Egyptian dynastic material (3500–2650 BC), while typical M. africanum signatures were detected in a Middle Kingdom tomb (2050–1650 BC). Samples from the New Kingdom to Late Period (1500–500 BC) indicated modern M. tuberculosis strains. No evidence was seen for M. bovis in Egyptian material while M. bovis signatures were first identified in Siberian biomaterial dating 2000 years before present. These results contraindicates the theory that M. tuberculosis evolved from M. bovis during early domestication in the region of the “Fertile Crescent,” but supports the scenario that M. tuberculosis probably derived from an ancestral progenitor strain. The environmental influence of this evolutionary scenario deserves continuing intense evaluation

Paleopathology of Human Tuberculosis and the Potential Role of Climate

Both origin and evolution of tuberculosis and its pathogens (Mycobacterium tuberculosis complex) are not fully understood. The paleopathological investigation of human remains offers a unique insight into the molecular evolution and spread including correlative data of the environment. The molecular analysis of material from Egypt (3000–500 BC), Sudan (200–600 AD), Hungary (600–1700 AD), Latvia (1200–1600 AD), and South Germany (1400–1800 AD) urprisingly revealed constantly high frequencies of tuberculosis in all different time periods excluding significant environmental influence on tuberculosis spread. The typing of various mycobacteria strains provides evidence for ancestral M. tuberculosis strains in Pre- to early Egyptian dynastic material (3500–2650 BC), while typical M. africanum signatures were detected in a Middle Kingdom tomb (2050–1650 BC). Samples from the New Kingdom to Late Period (1500–500 BC) indicated modern M. tuberculosis strains. No evidence was seen for M. bovis in Egyptian material while M. bovis signatures were first identified in Siberian biomaterial dating 2000 years before present. These results contraindicates the theory that M. tuberculosis evolved from M. bovis during early domestication in the region of the “Fertile Crescent,” but supports the scenario that M. tuberculosis probably derived from an ancestral progenitor strain. The environmental influence of this evolutionary scenario deserves continuing intense evaluation

Development of a test system to analyze different hip fracture osteosyntheses under simulated walking

The mechanical complications of osteosyntheses after hip fractures are previously investigated by mostly static or dynamic uniaxial loading test systems. However, the physiologic loading of the hip joint during a normal gait is a multiplanar, dynamic movement. Therefore, we constructed a system to test osteosyntheses for hip fractures under physiologic multiplanar loading representative of normal gait. To evaluate the testing system, 12 femora pairs were tested under 25,000 cycles with two standard osteosyntheses (Proximal Femoral Nail Antirotation/Gamma3 Nail). For angular movement, the varus collapse to cut out (proportional to(CO)) (proportional to(CO) = 4.8 degrees +/- 2.1 degrees for blade and proportional to(CO) = 7.8 degrees +/- 3.8 degrees for screw) was the dominant failure mode, and only slight rotational angle shifts (proportional to(Rot)) (proportional to(Rot) = 1.7 degrees +/- 0.4 degrees for blade and proportional to(Rot) = 2.4 degrees +/- 0.3 degrees for screw) of the femoral head around the implant axis were observed. Angular displacements in varus direction and rotation were higher in specimens reinforced with screws. Hence, the cut out model and the migration directions showed a distinction between helical blade and hip screw. However, there were no significant differences between the different implants. The new setup is able to create clinical failures and allows to give evidence about the anchorage stability of different implant types under dynamic gait motion pattern

Biological treatment strategies for disc degeneration: potentials and shortcomings

Recent advances in molecular biology, cell biology and material sciences have opened a new emerging field of techniques for the treatment of musculoskeletal disorders. These new treatment modalities aim for biological repair of the affected tissues by introducing cell-based tissue replacements, genetic modifications of resident cells or a combination thereof. So far, these techniques have been successfully applied to various tissues such as bone and cartilage. However, application of these treatment modalities to cure intervertebral disc degeneration is in its very early stages and mostly limited to experimental studies in vitro or in animal studies. We will discuss the potential and possible shortcomings of current approaches to biologically cure disc degeneration by gene therapy or tissue engineering. Despite the increasing number of studies examining the therapeutic potential of biological treatment strategies, a practicable solution to routinely cure disc degeneration might not be available in the near future. However, knowledge gained from these attempts might be applied in a foreseeable future to cure the low back pain that often accompanies disc degeneration and therefore be beneficial for the patien

Internalization of Streptococcus pyogenes into human endothelial cells: Characterization of signal transduction mechanisms

Streptococcus pyogenes ist ein humanpathogener Erreger, der eine Reihe verschiedener Krankheiten verursachen kann. Über die Interaktion von Streptokokken mit humanen Endothelzellen während invasiver Streptokokkeninfektionen sind bisher nur wenige Daten vorhanden. In dieser Arbeit wurden deshalb die molekularen Mechanismen, die an der Aufnahme von S. pyogenes in primäre HUVEC beteiligt sind, eingehender charakterisiert. Es konnte gezeigt werden, dass Streptokokken des Serotyps M3 über cholesterinhaltige Membranmikrodomänen internalisiert werden. Die Internalisierung geht ultrastrukturell mit der Bildung von protrusiven Membranausstülpungen einher. Auf molekularer Ebene kommt es zu Reorganisationen des Aktin-Zytoskeletts, für die die Aktivität von Tyrosinkinasen der Src-Familie essentiell sind. Des Weiteren konnte gezeigt werden, dass die GTPase Rac1 während der Internalisierung aktiviert wird und für den Aufnahmeprozess essentiell ist. Darüber hinaus konnte das erste Mal eine Verbindung zwischen der Reorganisation des Aktin-Zytoskeletts während der Aufnahme von Streptokokken und der Rekrutierung des Arp2/3-Komplexes, der durch Rac1 aktiviert werden kann, nachgewiesen werden. Die Aktivierung von Rac1 erfolgt dabei auf einem Phosphatidylinositol 3-Kinase (PI3K)-unabhängigen Weg. Die Aktivität von PI3Ks spielt jedoch eine entscheidende Rolle für das intrazelluläre Schicksal der Streptokokken. Die pharmakologische Inhibierung der PI3Ks verhindert die Rekrutierung von LAMP1, einem Markerprotein für späte Endosomen/Lysosomen. Die Bakterien kolokalisieren transient mit EEA1 einem Markerprotein für frühe Endosomen, das über eine FYVE-Domäne an Phosphatidylinositol (3)-Phosphat (PtdIns(3)P) bindet. PtdIns(3)P wird durch Klasse III PI3Ks transient auf der endosomalen Membran gebildet. Diese Experimente zeigten das erste Mal eine Funktion dieser PI3K-Klasse bei der Internalisierung von S. pyogenes in eukaryontische Zellen.Streptococcus pyogenes (GAS) is a human pathogen that causes several diseases. There is little known about the interactions of streptococci with human endothelial cells during invasive streptococcal disease. In this work the molecular mechanisms involved in the uptake of S. pyogenes into primary HUVEC were characterized. It was shown that streptococci of serotype M3 are internalized via cholesterol containing membrane microdomains. On the ultrastructural level, the internalization is accompanied by the formation of membrane protrusions. On the molecular level the actin cytoskeleton becomes reorganized at streptococcal entry sites in a Src kinase-dependent manner. It was also shown that the GTPase Rac1 is activated in infected cells and is essential for the uptake process. Furthermore, a connection between reorganization of the actin cytoskeleton during GAS uptake and recruitment of the Arp2/3 complex, which can be activated by Rac1, was shown. The activation of Rac1 is phosphatidyinositol 3 kinase (PI3K)-independent whereas the activity of PI3Ks is crucial for the intracellular fate of GAS. It was shown that pharmacological inhibition of PI3K activity prevents recruitment of LAMP1, a marker protein of late endosomes/lysosomes. The bacteria co-localize transiently with EEA1, which is a marker for early endosomes that contains a FYVE domain. This domain binds to phosphatidylinositol (3)-phosphate that is transiently produced on endosomal membranes by class III PI3Ks. These experiments show for the first time a function of this PI3K class during internalization of S. pyogenes into eukaryotic cells

Analysis of Il36a induction by C/EBPβ via a half-CRE•C/EBP element in murine macrophages in dependence of its CpG methylation level

Interleukin-36α is a novel member of the IL-1 cytokine family that is highly expressed in epithelial tissues and several myeloid-derived cell types after induction. The transcription factor (TF) C/EBPβ binds specifically to an essential half-CRE•C/EBP motif in the Il36a promoter to induce Il36a expression upon LPS stimulation. C/EBPs regulate gene expression by binding to recognition sequences that can contain 5′-cytosine-phosphate-guanine-3′ dinucleotides (CpG), whose methylation can influence TF binding and gene expression. Herein we show that the half-CRE•C/EBP element in the Il36a promoter is differentially methylated in the murine RAW264.7 macrophage cell line and in primary murine macrophages. We demonstrate that C/EBPβ binding to the half-CRE•C/EBP element in the Il36a promoter following LPS stimulation is insensitive to CpG methylation and that methylation of the CpG in the half-CRE•C/EBP element does not alter LPS-induced Il36a promoter activity which correlated with similar Il36a mRNA copy numbers and pro-IL-36α protein amount in both cell types. Taken together, our data indicate that C/EBPβ binding to the half-CRE•C/EBP element and subsequent gene activation occurs independently of the CpG methylation status of the half-CRE•C/EBP motif and underlines the potential of C/EBPs to recognize methylated as well as unmethylated motifs

T-SP1: a novel serine protease-like protein predominantly expressed in testis

Here, we describe a novel member in the group of membrane-anchored chymotrypsin (S1)-like serine proteases, namely testis serine protease 1 (T-SP1), as it is principally expressed in testis tissue. The human T-SP1 gene encompasses 28.7 kb on the short arm of chromosome 8 and consists of seven exons. Rapid amplification of cDNA ends ( RACE) experiments revealed that due to alternative splicing three different variants (T-SP1/1, -2, -3) are detectable in testis tissue displaying pronounced heterogeneity at their 3'-end. T-SP1/1 consists of an 18 amino acid signal peptide and of a 49 amino acid propeptide. The following domain with the catalytic triad of His(108), Asp(156), and Ser(250) shares sequence identities of 42% and 40% with the blood coagulation factor XI and plasma kallikrein, respectively. Only T-SP1/1 contains a hydrophobic part at the C-terminus, which provides the basis for cell membrane anchoring. Using a newly generated polyclonal anti-T-SP1 antibody, expression of the T-SP1 protein was found in the Leydig and Sertoli cells of the testis and in the epithelial cells of the ductuli efferentes. Notably, T-SP1 protein was also detectable in prostate cancer and in some ovarian cancer tissues, indicating tumor-related synthesis of T-SP1 beyond testis tissue

Overexpression of the ATP binding cassette gene ABCA1 determines resistance to Curcumin in M14 melanoma cells

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Immunohistochemical localization of collagen VI in diabetic glomeruli

Immunohistochemical localization of collagen VI in diabetic glomeruli. Late stage diabetic nephropathy is histologically characterized by either diffuse or nodular expansion of the glomerular matrix. This is presumed to represent the morphological correlate for the functional impairment of the kidney. The exact matrix composition of the nodular glomerulosclerosis lesion of end-stage diabetic nephropathy is not known. Biochemical studies have provided evidence that the microfibrillar collagen type VI is increased in diabetic nephropathy. Consequently, this immunohistochemical study was designed to evaluate the extent and exact morphologic location of increased collagen VI deposition at various stages of diabetic glomerulosclerosis (GS). An irregular, sometimes spot-like staining of collagen VI was observed in diffuse GS in the mesangial portion. The uninterrupted staining which was evident along the glomerular basement membrane in normal glomeruli was discontinuous in diffusely sclerotic glomeruli. In nodular GS, the markedly increased deposition of collagen VI appeared to be evenly distributed throughout the entire nodular lesion. At the same time, mesangial staining for collagen IV was reduced in nodular GS, suggesting that in the expanded mesangial matrix collagen IV is progressively substituted by collagen VI during the transition from diffuse to nodular GS. The colocalization of PAS staining with collagen VI deposition in nodular GS suggests that the typical Kimmelstiel-Wilson lesions at least in part consist of collagen VI. Biochemical analysis confirmed the increased collagen VI deposition in glomeruli extracted from diabetic patients with nodular GS. Application of two antisera, recognizing primarily the α1(VI)- and α2(VI)-chains and the N-terminal part of α3(VI)-chain, respectively, revealed no difference in staining pattern. Comparison of the immunohistochemical results with clinical parameters of diabetic nephropathy suggested that increasing collagen VI deposition may be an indicator of the irreversible remodeling of the glomerular matrix to nodular GS which is associated with functional insufficiency. Our findings indicate striking differences of the mesangial matrix composition in diffuse and nodular GS. These observations together with earlier results provide evidence for a “switch” in the matrix protein production in association with the development of nodular GS in diabetic nephropathy

Matrix metalloproteinase expression levels suggest distinct enzyme roles during lumbar disc herniation and degeneration

The disruption of the extracellular disc matrix is a major hallmark of disc degeneration. This has previously been shown to be associated with an up-regulation of major matrix metalloproteinase (MMP) expression and activity. However, until now hardly any data are available for MMP/TIMP regulation and thereby no concept exists as to which MMP/TIMP plays a major role in disc degeneration. The objective of this study was, therefore, to identify and quantify the putative up-regulation of MMPs/TIMPs on the mRNA and protein level and their activity in disc material in relation to clinical data and histological evidence for disc degeneration. A quantitative molecular analysis of the mRNA expression levels for the MMPs (MMPs-1, -2, -3, -7, -8, -9, -13) and the MMP inhibitors (TIMPs-1 and -2) was performed on 37 disc specimens obtained from symptomatic disc herniation or degeneration. In addition, disc specimens from patients without disc degeneration/herniation (=controls) were analyzed. Expression of MMPs-1, -2, -3, -7, -8, -9, -13 and TIMPs-1, -2 was analyzed using quantitative RT-PCR, normalized to the expression level of a house keeping gene (GAPDH). Gene expression patterns were correlated with MMP activity (in situ zymography), protein expression patterns (immunohistochemistry), degeneration score (routine histology) and clinical data. MMP-3 mRNA levels were consistently and substantially up-regulated in samples with histological evidence for disc degeneration. A similar but less pronounced up-regulation was observed for MMP-8. This up-regulation was paralleled by the expression of TIMP-1 and to a lesser extent TIMP-2. In general, these findings could be confirmed with regard to protein expression and enzyme activity. This study provides data on the gene and protein level, which highlights the key role of MMP-3 in the degenerative cascade leading to symptomatic disc degeneration and herniation. Control of the proteolytic activity of MMP-3 may, therefore, come into the focus when aiming to develop new treatment options for early disc degeneratio