Analysis of tissue distribution of TNF-alpha, TNF-alpha-receptors, and the activating TNF-alpha-converting enzyme suggests activation of the TNF-alpha system in the aging intervertebral disc.

We immunohistochemically analyzed the expression and localization of TNF-alpha, its receptors TNF-RI and -RII, and the TNF-alpha-activating enzyme TACE in human autopsy (n=63) and surgical (n=35) lumbar intervertebral disc samples. In parallel, the TNF-alpha-mRNA was quantified by reverse transcriptase-polymerase chain reaction (RT-PCR). All samples were morphometrically evaluated for the proportion of positively labeled cells in the different anatomical regions of the disc. We detected a significant and comparable expression of all four parameters beginning in young adult age (c. 18 years) and being most extensive in the nucleus pulposus. This level was slightly reduced in older age discs. The annulus fibrosus contained significantly less labeled cells. In accord, the number of TNF-alpha-transcripts was elevated in most cases with immunohistochemical TNF-alpha expression. We provide clear evidence that TNF-alpha is expressed in discs of increasing age, which correlates with histomorphological signs of disc degeneration. In consequence, TNF-alpha seems to be activated (by the converting enzyme TACE) and biologically active through its receptors in human lumbar disc tissue.

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 patient.

The course of macroscopic degeneration in the human lumbar intervertebral disc.

STUDY DESIGN: Assessment of age-related macroscopic changes in human lumbar intervertebral discs (IVD) and vertebral bodies. OBJECTIVES: To determine the sequence of macroscopic changes during aging/degeneration. SUMMARY OF BACKGROUND DATA: Descriptive studies on macroscopic alterations of the IVD during aging/degeneration are readily available, but quantitative analyses are sparse. METHODS: A total of 248 mid-/parasagittal sections of lumbar IVD and vertebral bodies from 41 routine autopsies (range, 7 months to 88 years) were semiquantitatively assessed for macroscopic parameters and correlated with age. RESULTS: Nuclear fibrous transformation, anular disorganization, endplate, and vertebral body alterations progress predominantly in the first two and in the fifth to seventh decades. In the third and fourth decade, little progression occurs. Nuclear clefts and anular tears appear later, mostly starting in the second decade, with clefts preceding tear formation. Radial and concentric tears develop similarly over time, whereas rim lesions mostly develop after the sixth decade. Significant differences are observed between upper and lower lumbar spine. CONCLUSION: Our data show that fibrous nuclear transformation during aging/degeneration precedes cleft formation. The temporal sequence suggests a strong correlation of cleft and tears formation starting with clefts in the second decade. Our results support the hypothesis that disc degeneration starts in the nucleus. Extensive macroscopic alterations already apparent in the second life decade present a challenge to any tissue engineering and repair attempt.

Chronic Salmonella enterica serovar Typhimurium-induced colitis and cholangitis in streptomycin-pretreated Nramp1+/+ mice.

Salmonella enterica subspecies 1 serovar Typhimurium is an enteric bacterial pathogen infecting a broad range of hosts. In susceptible Nramp1(-/-) (Slc11alpha1(-/-)) mice, serovar Typhimurium cannot efficiently colonize the intestine but causes a systemic typhoid-like infection. However, after pretreatment with streptomycin, these susceptible (C57BL/6 and BALB/c) mice develop acute serovar Typhimurium-induced colitis (M. Barthel et al., Infect. Immun. 71:2839-2858, 2003). It was not clear whether resistant Nramp1(+/+) (Slc11alpha1(+/+)) mouse strains would similarly develop colitis. Here we compared serovar Typhimurium infection in streptomycin-pretreated susceptible (C57BL/6) and resistant (DBA/2 and 129Sv/Ev) mouse strains: We found that acute colitis (days 1 and 3 postinfection) is strikingly similar in susceptible and resistant mice. In 129Sv/Ev mice we followed the serovar Typhimurium infection for as long as 6 weeks. After the initial phase of acute colitis, these animals developed chronic crypt-destructive colitis, including ulceration, crypt abscesses, pronounced mucosal and submucosal infiltrates, overshooting regeneration of the epithelium, and crypt branching. Moreover, we observed inflammation of the gall duct epithelium (cholangitis) in the 129Sv/Ev mice between days 14 and 43 of infection. Cholangitis was not attributable to side effects of the streptomycin treatment. Furthermore, chronic infection of 129Sv/Ev mice in a typhoid fever model did not lead to cholangitis. We propose that streptomycin-pretreated 129Sv/Ev mice provide a robust murine model for chronic enteric salmonellosis including complications such as cholangitis.

Genes in the Salmonella pathogenicity island 2 and the Salmonella virulence plasmid are essential for Salmonella-induced apoptosis in intestinal epithelial cells.

Intestinal epithelial cells are an important site of the host's interaction with enteroinvasive bacteria. Genes in the chromosomally encoded Salmonella pathogenicity island 2 (SPI 2) that encodes a type III secretion system and genes on the virulence plasmid pSDL2 of Salmonella enteritica serovar Dublin (spv genes) are thought to be important for Salmonella dublin survival in host cells. We hypothesized that genes in those loci may be important also for prolonged Salmonella growth and the induction of apoptosis induced by Salmonella in human intestinal epithelial cells. HT-29 human intestinal epithelial cells were infected with wild-type S. dublin or isogenic mutants deficient in the expression of spv genes or with SPI 2 locus mutations. Neither the spv nor the SPI 2 mutations affected bacterial entry into epithelial cells or intracellular proliferation of Salmonella during the initial 8 h after infection. However, at later periods, bacteria with mutations in the SPI 2 locus or in the spv locus compared to wild-type bacteria, manifested a marked decrease in intracellular proliferation and a different distribution pattern of bacteria within infected cells. Epithelial cell apoptosis was markedly increased in response to infection with wild-type, but not the mutant Salmonella. However, apoptosis of epithelial cells infected with wild-type S. dublin was delayed for approximately 28 h after bacterial entry. Apoptosis was preceded by caspase 3 activation, which was also delayed for approximately 24 h after infection. Despite its late onset, the cellular commitment to apoptosis was determined in the early period after infection as inhibition of bacterial protein synthesis during the first 6 h after epithelial cell infection with wild-type S. dublin, but not at later times, inhibited the induction of apoptosis. These studies indicate that genes in the SPI 2 and the spv loci are crucial for prolonged bacterial growth in intestinal epithelial cells. In addition to their influence on intracellular proliferation of Salmonella, genes in those loci determine the ultimate fate of infected epithelial cells with respect to caspase 3 activation and undergoing death by apoptosis.

Ubiquitous production of macrophage migration inhibitory factor by human gastric and intestinal epithelium.

BACKGROUND & AIMS: Macrophage migration inhibitory factor (MIF) inhibits macrophage migration and has pleiotropic activities on immune and inflammatory responses, cell growth, and glucose metabolism. MIF is produced by T cells, macrophages, and endothelial cells. Because intestinal epithelial cells produce mediators important for regulating mucosal immune and inflammatory responses, we sought to determine if these cells produce MIF. METHODS: MIF expression was determined by immunostaining of human intestinal mucosa, intestinal xenografts, and cultured cells. MIF protein levels were quantitated by enzyme-linked immunosorbent assay and immunoblot analysis, messenger RNA was assessed by real-time reverse-transcription polymerase chain reaction, and functional activity was assessed by enzymatic and migration assays. RESULTS: MIF was abundantly expressed in vivo in gastric, small intestinal, and colonic epithelium and in epithelium lining human intestinal xenografts. MIF was also constitutively expressed at the messenger RNA and protein level by several cultured colon and gastric epithelial cell lines, and its expression in those cells was not up-regulated by the proinflammatory cytokines interleukin 1alpha, tumor necrosis factor alpha, or interferon gamma. Epithelial MIF from cultured cells was released predominantly from the apical side after Salmonella infection, had tautomerase activity, and arrested macrophage migration. CONCLUSIONS: Human intestinal epithelial cells are a major source of MIF, a molecule that can regulate macrophage migration, inflammation, and cell metabolism.

Modulation of chloride secretory responses and barrier function of intestinal epithelial cells by the Salmonella effector protein SigD.

The Salmonella effector protein SigD is an inositol phosphate phosphatase that inhibits phosphatidylinositol 3-kinase-dependent signaling. Because epidermal growth factor (EGF) inhibits chloride secretion via phosphatidylinositol 3-kinase, we explored whether Salmonella infection might modify the inhibitory effect of EGF. As expected, EGF inhibited chloride secretion induced by carbachol in T(84) epithelial cells. Infection with wild-type (WT) but not sigD(-) mutant S. typhimurium SL1344 decreased CCh-stimulated chloride secretion. Moreover, WT but not sigD(-) Salmonella reduced the inhibitory effect of EGF on carbachol-stimulated chloride secretion. Complementation of sigD restored the ability of mutant Salmonella to reverse the inhibitory effect of EGF. EGF-induced EGF receptor phosphorylation was similar in cells infected with either WT or mutant Salmonella, and neither WT nor sigD(-) Salmonella altered recruitment of the p85 subunit of phosphatidylinositol 3-kinase to EGF receptor, implying that SigD acts downstream of these signaling events. Furthermore, transepithelial resistance fell more rapidly in cells infected with WT vs. sigD(-) Salmonella, indicating an early role for SigD in reducing barrier function, perhaps via activation of protein kinase C. We conclude that the Salmonella bacterial effector protein SigD may play critical roles in the pathogenesis of disease caused by this microorganism.