Cross‐reactive adaptive immune response to oral commensal bacteria results in an induction of receptor activator of nuclear factor‐κB ligand (RANKL)‐dependent periodontal bone resorption in a mouse model

Introduction: The present study examined whether induction of an adaptive immune response to orally colonizing non‐pathogenic Pasteurella pneumotropica by immunization with the phylogenetically closely related bacterium, Actinobacillus actinomycetemcomitans, can result in periodontal bone loss in mice. Methods: BALB/c mice harboring P. pneumotropica (P. pneumotropica⁺ mice) in the oral cavity or control P. pneumotropica‐free mice were immunized with fixed A. actinomycetemcomitans. The animals were sacrificed on day 30, and the following measurements were carried out: (i) serum immunoglobulin G and gingival T‐cell responses to A. actinomycetemcomitans and P. pneumotropica; (ii) periodontal bone loss; and (iii) identification of receptor activator of nuclear factor‐κB ligand (RANKL) ‐positive T cells in gingival tissue. Results: Immunization with A. actinomycetemcomitans induced a significantly elevated serum immunoglobulin G response to the 29‐kDa A. actinomycetemcomitans outer membrane protein (Omp29), which showed strong cross‐reactivity with P. pneumotropica OmpA compared to results in the control non‐immunized mice. The A. actinomycetemcomitans‐immunized P. pneumotropica⁺ mice developed remarkable periodontal bone loss in a RANKL‐dependent manner, as determined by the abrogation of bone loss by treatment with osteoprotegerin‐Fc. The T cells isolated from the gingival tissue of A. actinomycetemcomitans‐immunized P. pneumotropica⁺ mice showed an in vitro proliferative response to both A. actinomycetemcomitans and P. pneumotropica antigen presentation, as well as production of soluble(s)RANKL in the culture supernatant. Double‐color confocal microscopy demonstrated that the frequency of RANKL⁺ T cells in the gingival tissue of A. actinomycetemcomitans‐immunized P. pneumotropica⁺ mice was remarkably elevated compared to control mice. Conclusion: The induction of an adaptive immune response to orally colonizing non‐pathogenic P. pneumotropica results in RANKL‐dependent periodontal bone loss in mice.     

Detection of Intimins α, β, γ, and δ, Four Intimin Derivatives Expressed by Attaching and Effacing Microbial Pathogens

Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions. A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides. Polyclonal antiserum was raised against Int280 from enteropathogenic Escherichia coli (EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic E. coli (EHEC) isolates, a rabbit diarrheagenic E. coli strain (RDEC-1), and Citrobacter rodentium. Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and C. rodentium. These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells. Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7). By using PCR primers designed on the basis of the intimin-encoding eae gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different eae gene derivatives. Accordingly, the different intimin types were designated α, β, δ, and γ, respectively.     

Liver transplantation with monosegment from a living donor

The shortage of organ donors for low-weight liver transplant recipients, especially for small children, has led to the development of new surgical techniques to increase the donor pool. Almost all of these techniques use the left lateral segment (Couinaud's segments II and III), but even this graft could be too large for children under 10 kg. We report here the case of an 8-month-old boy, weighing 6.1 kg, who received a monosegmental graft (segment III) from his grandmother weighing 68 kg. The graft was reduced at the donor surgery, before clamping of the vessels. The donor was discharged on the fourth post-operative day; the recipient had an uneventful post-operative period and was discharged after 22 days.     

Genetic aspects and microenvironment affect expression of CD18 and VLA-4 in experimental tuberculosis

Control of infection by Mycobacterium tuberculosis is dependent on macrophage activation and efficient migration of effector T-cell populations. Lymphocyte differentiation is associated with changes in cell surface phenotype and alterations in the migratory pattern of these cells. In this study, we investigated the expression of adhesion receptors involved in activation and migration process in experimental tuberculosis. We observed that susceptible BALB/c mice infected with virulent M. tuberculosis by intraperitoneal route presented downmodulation of very late antigen 4 (VLA-4) and unchanged levels of CD18 and CD44hi on peritoneal lymphocytes. On the other hand, lymphocytes from resistant C57BL/6 mice infected by the same route showed unchanged levels of VLA-4 and upregulation of CD18 and CD44hi. However, when BALB/c mice were infected by intratracheal route, lung lymphocytes presented a different pattern of CD18, CD44hi and VLA-4 expression from that observed on peritoneal cells, characterized by unchanged levels of VLA-4 and upregulation of CD18 and CD44hi- coincidentally the same phenotype found on peritoneal cells from C57BL/6. These results suggest that susceptibility and resistance to M. tuberculosis infection, depending on the experimental model, are related to the expression of CD18, CD44hi and VLA-4. Moreover, the microenvironment at the site of infection seems to differentially regulate the expression of these receptors. Thus, the up- or downmodulation of these adhesion receptors is probably associated with differential recruitment of T cells at the site of infection, which may or may not mediate protection in experimental tuberculosis.     

Pattern of T cell activation in absence of protective immunity against hepatitis B virus. Review

Hepatitis B is an important cause of morbidity and mortality around the world. One-third of the world's population has been estimated to be infected with hepatitis B virus (HBV). A significant amount of evidence suggests that both humoral and cellular immune responses are important to eliminate the virus and that, cellular immunity is involved in the pathogenesis of the disease. Vaccination with HBsAg is considered as the main strategy for effective control of the infection and viral transmission. However, approximately 5-10% of immunized individuals fail to elicit detectable specific antibodies and remain at risk for hepatitis B infection. In this work we have reviewed the current status in the pathogenesis of the disease and the mechanisms described to explain nonresponsiveness to the vaccine as well. Since nonresponders to the vaccine are at risk for the infection, a common mechanism to explain the absence or inappropriate immune response to virus components is proposed. Within the suggested model an impaired activation of T lymphocytes against viral antigens, both in nonresponders to vaccination and chronically infected patients, is described. These observations could be consistent with potential differences in the MHC/Ag presentation; therefore contributing to our understanding of the altered T helper response as an underlying mechanism for the lack of protective immunity against VHB.     

Cleavage of Osteopontin by Matrix Metalloproteinase-12 Modulates Experimental Autoimmune Encephalomyelitis Disease in C57BL/6 Mice

A role for osteopontin (OPN) in promoting disease activity of multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) has recently been suggested. As the biological activity of OPN is heavily influenced by posttranslational processing, we investigated the capacity of matrix metalloproteinase (MMP)-12 to cleave OPN and determined whether this influenced disease activity. We found that OPN mRNA and protein expression in the spinal cord increased with EAE disease in C57BL/6 mice concurrently with MMP-12 expression. A Western blot of EAE and control spinal cords revealed different OPN-immunoreactive bands, with a pattern that was similar to MMP-12 cleavage of recombinant OPN in vitro. In addition, OPN fragments in the spinal cord of EAE-afflicted mice were reduced in MMP-12^−/− mice compared with wild-type controls. However, examination of OPN^−/− mice in short- and long-term experiments revealed no difference in EAE outcomes from wild-type animals. OPN/MMP-12 double null mice were generated, and it was revealed that MMP-12^−/− mice had a worsening of disease compared with wild-type mice, which returned to wild-type levels in the OPN/MMP-12 double null mice. These results suggest that EAE disease activity may be modulated by the cleavage of OPN by MMP-12.Multiple sclerosis (MS) is a disease in which peripheral T cells infiltrate the central nervous system (CNS), where they become re-activated by presentation of CNS antigens by local antigen-presenting cells including microglia and dendritic cells to result in demyelination.^1,2,3,4 In attempts to find putative antigens and mediators of disease, studies using gene array analyses revealed that osteopontin (OPN) was highly up-regulated in lesions from patients with MS compared with control subjects.⁵ This result has prompted the investigations of OPN expression and function in MS and experimental autoimmune encephalomyelitis (EAE).OPN, also known as early T cell activation gene 1 (Eta-1), is a calcium binding phosphorylated acidic glycoprotein.^6,7 OPN has pluripotent activity and is involved in various biological roles such as extracellular matrix remodeling, tumor invasion, angiogenesis, cell-mediated immunity, and the regulation of urokinase and matrix metalloproteinase (MMP) production.⁸ Chabas et al⁵ found that OPN was expressed during EAE in various cell types and that OPN^−/− mice had reduced EAE clinical disease, which was associated with a shift toward a Th2 cytokine profile. Jansson et al⁹ also found that OPN^−/− mice had reduced mean maximal score, fewer days of paralyzing disease, and no spontaneous relapses on proteolipid protein-induced EAE. In that study, OPN^−/− mice showed reduced production of pro-inflammatory cytokines, interferon-γ (IFN-γ), and tumor necrosis factor-α. In contrast to these studies, Blom et al¹⁰ found no difference between OPN^−/− and wild-type mice for EAE outcomes.The EAE results sparked an interest in the expression of OPN in MS. Increased levels of OPN protein is reported in the serum and plasma in patients with relapsing-remitting MS compared with controls,^11,12,13,14 particularly during relapses, and in their cerebrospinal fluid.^15,16 However, there is little evidence for a genetic link between OPN and MS disease susceptibility and disease course.^{11,13,17,18,19,20} Elevated OPN levels have also been documented by immunohistochemistry around MS lesions^21,22 although this was not observed by others.²³ The role that OPN plays in EAE and MS remains uncertain, but a prevailing view is that OPN plays a destructive role during EAE since it reduces apoptosis of effector T cells and because the exogenous administration of recombinant OPN exacerbates EAE clinical disease.²⁴The biological functions of OPN are heavily influenced by posttranslational modifications such as phosphorylation, glycosylation, sulfation, and proteolytic cleavage.^25,26 Currently, there is very little knowledge about the role that proteolytic cleavage of OPN has on MS and EAE. In other disease states, the cleavage of OPN by MMPs and thrombin alters the biological functions of OPN.^27,28 For example, OPN that is cleaved by MMP-3 and MMP-7 significantly increases adhesion of tumor cells compared with full length OPN.²⁹ Recombinant MMP-12 has been demonstrated to cleave OPN, a protein that strongly influences osteoclasts activities, including attachment, spreading, and resorption.³⁰The family of MMPs is implicated in MS and EAE as several MMP members are elevated in biological samples from these conditions. These include MMP-2, -3, -8, -9, -10, -11, -12, -13, -14, and -25.^{31,32,33,34,35,36,37,38} Although the majority of these MMP members appear to have detrimental roles in MS and EAE,^39,40,41 MMP-12 is unique since its absence causes an exacerbation in EAE disease scores that is associated with up-regulation of pro-inflammatory cytokines.^31,42 Since the MMP-12^−/− mice phenotype is opposite to the reduced levels of pro-inflammatory cytokines in OPN^−/− mice with EAE,⁵ we considered the possibility that MMP-12 and OPN are inversely associated. In this article, we have tested the hypothesis that MMP-12 processes OPN to reduce its pro-inflammatory potential in EAE. We describe OPN mRNA and protein expression during EAE and have used a combination of MMP-12 and OPN single and OPN/MMP-12 double null mice to profile their EAE phenotypes.     

Early rapid growth,early birth: Accelerated fetal growth and spontaneous late preterm birth

The past two decades in the United States have seen a 24% rise in spontaneous late preterm delivery (34–36 weeks) of unknown etiology. This study tested the hypothesis that fetal growth was identical prior to spontaneous preterm (n = 221, median gestational age at birth 35.6 weeks) and term (n = 3706) birth among pregnancies followed longitudinally in Santiago, Chile. The hypothesis was not supported: Preterm‐delivered fetuses were significantly larger than their term‐delivered peers by mid‐second trimester in estimated fetal weight, head, limb, and abdominal dimensions, and they followed different growth trajectories. Piecewise regression assessed time‐specific differences in growth rates at 4‐week intervals from 16 weeks. Estimated fetal weight and abdominal circumference growth rates slowed at 20 weeks among the preterm‐delivered, only to match and/or exceed their term‐delivered peers at 24–28 weeks. After an abrupt growth rate decline at 28 weeks, fetuses delivered preterm did so at greater population‐specific sex and age‐adjusted birth weight percentiles than their peers from uncomplicated pregnancies (P < 0.01). Growth rates predicted birth timing: one standard score of estimated fetal weight increased the odds ratio for late preterm birth from 2.8 prior to 23 weeks, to 3.6 (95% confidence interval, 1.82–7.11, P < 0.05) between 23 and 27 weeks. After 27 weeks, increasing size was protective (OR: 0.56, 95% confidence interval, 0.38–0.82, P = 0.003). These data document, for the first time, a distinctive fetal growth pattern across gestation preceding spontaneous late preterm birth, identify the importance of mid‐gestation for alterations in fetal growth, and add perspective on human fetal biological variability. Am. J. Hum. Biol., 2009. © 2008 Wiley‐Liss, Inc.     

GM1 produces attenuation of short-term memory deficits in Hebb-Williams maze performance after unilateral entorhinal cortex lesions.

The Hebb-Williams maze was used to examine spatial abilities of adult male Sprague-Dawley rats with unilateral electrolytic entorhinal cortex lesions. The injured rats were treated for 14 days with either saline or ganglioside GM1. Testing was begun 7 weeks following injury, and involved 12 maze problems with independent configurations, with immediate starting replacement used for the six trials per problem. Compared to sham-operated counterparts, the rats with lesion plus saline treatment were impaired in total number of errors, initial entry errors, and repeat errors over 12 consecutive problems. GM1-treated rats showed improved performance, making significantly fewer total and repeat errors, indicating that this substance may be potentially useful as therapy after entorhinal cortex injury.     

Symptoms of ＂Heat＂ as a Pattern of Physiopathology

Objective： The guiding criteria are considered the backbone of Chinese medicine. They have previously been described as functional features （symptoms） leading to the overall assessment of human functions on the basis of a regulatory （cybernetic） model referring to the I Ging. Methods： The Heidelberg model can explain symptoms such as created by ＂heat＂ on a rational physiological level. Results ＆ Conclusion： The overall of physiological symptoms are shown as a schematic draft. The basis of ＂heat＂ is considered to be a general increase of microcirculation in the periphery. This leads to a couple of local pathophysiological consequences and sensations like 1） red tongue （the tongue is considered an embryological somatotopic system）. 2） Sensation of warmth （by increase of capillary flow）. 3） pre-inflammatory state, leading to pain modalities like ＂worse if pressed＂, as inflammations tend to be increasingly painful under pressure; 4） reddish skin, the mechanisms by which this is induced may include the release of substance P, therefore accompanied by burning sensation. Systemic pathophysiological consequences may include. Relative lack of fluid in the larger vessels, as fluid supplies peripheral capillary flow. This may lead to water saving mechanisms like thirst, dry mucosa with do, mouth, dry nose, dry lips, dry skin, and also dry stool, yellow and sparse urine.     

How nursing staff perceive the duration and quality of sleep and levels of alertness

This study was conducted among health care personnel (registered nurses and nurse aides) in a public hospital in S?o Paulo, Brazil. Work was organized in 12-hour daytime or nighttime shifts, followed by 36 hours off. The study aimed to evaluate how the nursing staff perceived the duration and quality of sleep both during and off work days, as well as their perception of alertness during working hours. There were significant differences between night and day in the duration of sleep (Student t test = 10.82; p < 0.000). Quality of daytime sleep after working night shifts was perceived as worse than nighttime sleep (Wilcoxon test, Z = 2.67; p < 0.007). Significant differences were detected in self-evaluation of alertness after the 2nd, 6th, and 10th hour of night shifts (Friedman = 63.0; p < 0.00). Alertness was perceived as worse during dawn hours. This is an indication of sleepiness at work and can have serious consequences for both health care workers and patients.