Estrogen receptors in skeletal metabolism: lessons from genetically modified models of receptor function

Estrogens have long been known to be important for skeletal homeostasis, but their precise mechanisms of action in bone are still unclear. Mice with targeted deletions of the estrogen receptors alpha (ERalpha) and beta (ERbeta) have been generated by two research groups and several studies performed characterizing the phenotype of ERalpha knockout (ERKOalpha), ERbeta knockout (ERKObeta), or double deletion of ERalpha and ERbeta (DERKO) mice. Initial studies reported a reduction in bone mineral density in male ERKOalpha mice. More extensive analyses have been puzzling, likely because of compensatory mechanisms in ERKO mice. Furthermore, the existence of a third ER continues to be a potential explanation for some actions of estrogen in bone. Other rodent models, including the testicular feminized mouse and rat, the aromatase knockout mouse, and a rat with a dominant negative ER mutation, have added information regarding estrogen's actions in bone. This review summarizes many reports characterizing available rodent models with genetic alterations relevant to estrogen action. The sum of these reports suggests that the ERbeta is not highly protective in bone because loss of its function results in minimal alterations in the skeleton. Furthermore, loss of both the ERalpha and the ERbeta does not account for loss of estrogen action in bone, because the impact of DERKO is seemingly not as great as the impact of gonadectomy on the skeleton. Finally, through studies of ERKO mice and other rodent models of altered sex steroid action, it appears that estrogen may be more protective in the skeleton than androgens.     

Production of serum antibodies that recognise epitopes located on the R3 region of Escherichia coli core lipopolysaccharides by patients infected with strains of enterohaemorrhagic E. coli

Antibody-antigen cross-reactions were examined with sera from patients with Escherichia coli O157 infection and lipopolysaccharide (LPS) purified from a range of enterohaemorrhagic E. coli (EHEC) including those belonging to serogroups O26, O103, O111, O145 and O157. Six of 10 patients infected with an O157 EHEC produced serum antibodies that cross-reacted with common LPS-core epitopes, which were expressed by 23 of 33 strains of EHEC examined. These common LPS-core epitopes were also present on strains of E. coli O26 which did not produce verocytotoxin. These cross-reacting antibodies did not influence the basic immunoblotting procedures used for the routine serodiagnosis of infections with E. coli O157.     

Distal Proctocolitis and n-3 Polyunsaturated Fatty Acids (n-3 PUFAs): The Mucosal Effect In Situ

It has been postulated that patients with ulcerative colitis (UC) have altered reactivity of gut-associated lymphoid tissue. In such cases there is intense infiltration of the mucosa with immune competent cells and associated tissue damage. We have shown previously that the dietary supplementation with the n-3 polyunsaturated fatty acids (n-3 PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) results in significant systemic immune suppression. The aim of this study, therefore, was to evaluate the in situ effect of n-3 PUFAs on distal proctocolitis. Each patient received either fish oil extract (EPA 3.2 g, DHA 2.4 g) (n = 9) or sunflower oil (n = 9) daily in a double blind manner for six months. Monthly assessment included: (1) disease activity using clinical, sigmoidoscopic, and histological scores and (2) immunohistochemical analysis (immunoglobulins, CD profiles) of rectal biopsy specimens (before and after six months supplementation) using monoclonal antibodies and quantitative computer-assisted video image analysis. Prior to receiving supplementation, patients with proctocolitis (n = 18) showed significantly higher numbers of cells expressing CD3 (pan T cells) and HLA-DR and IgM containing cells compared with non-colitic controls (n = 8). Six months supplementation with n-3 PUFAs resulted in significant reduction in the number of cells expressing CD3 and HLA and the percentage of cells containing IgM. There was no significant change in the CD20 nor the percentage of IgG or IgA containing cells in either group of patients with procto-colitis. In patients receiving n-3 PUFA supplementation, there was improvement in the disease activity and histological scores, compared with pretreatment evaluation. This study has demonstrated both evidence of suppression of in situ immune reactivity and concurrent reduction in disease activity in patients with proctocolitis receiving n-3 PUFA supplementation. This may have important implication for therapy in patients with ulcerative colitis.     

Effects of mutation and growth rates on patterns of microsatellite instability.

The detection of somatic microsatellite (MS) alterations in tumors is often interpreted as a sign of underlying genomic instability. However, it is unclear why the proportions of altered MS loci vary between different mutator phenotype tumors. We present a simple mathematical analysis that can account for some of these differences, recognizing that the mutations accumulated in a tumor reflect both its mutation rate and number of cell divisions. Only a small proportion of mutated MS loci are expected in tumors with normal or low mutation rates. In contrast, tumors with high mutation rates may or may not acquire mutations depending on the numbers of divisions that proceed the onset of the mutator phenotype. The majority of MS loci should accumulate mutations if high mutation rates are acquired early in tumor progression. Somatic MS mutations provide clues to both the mode and tempo of tumori-genesis.     

White Matter Fiber Tracking Computation Based on Diffusion Tensor Imaging for Clinical Applications

Fiber tracking allows the in vivo reconstruction of human brain white matter fiber trajectories based on magnetic resonance diffusion tensor imaging (MR-DTI), but its application in the clinical routine is still in its infancy. In this study, we present a new software for fiber tracking, developed on top of a general-purpose DICOM (digital imaging and communications in medicine) framework, which can be easily integrated into existing picture archiving and communication system (PACS) of radiological institutions. Images combining anatomical information and the localization of different fiber tract trajectories can be encoded and exported in DICOM and Analyze formats, which are valuable resources in the clinical applications of this method. Fiber tracking was implemented based on existing line propagation algorithms, but it includes a heuristic for fiber crossings in the case of disk-shaped diffusion tensors. We successfully performed fiber tracking on MR-DTI data sets from 26 patients with different types of brain lesions affecting the corticospinal tracts. In all cases, the trajectories of the central spinal tract (pyramidal tract) were reconstructed and could be applied at the planning phase of the surgery as well as in intraoperative neuronavigation.     

Novel mouse model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis

Pseudomonas aeruginosa causes a chronic infection in the lungs of cystic fibrosis (CF) patients by establishing an alginate-containing biofilm. The infection has been studied in several animal models; however, most of the models required artificial embedding of the bacteria. We present here a new pulmonary mouse model without artificial embedding. The model is based on a stable mucoid CF sputum isolate (NH57388A) with hyperproduction of alginate due to a deletion in mucA and functional N-acylhomoserine lactone (AHL)-based quorum-sensing systems. Chronic lung infection could be established in both CF mice (Cftr(tmlUnc-/-)) and BALB/c mice, as reflected by the detection of a high number of P. aeruginosa organisms in the lung homogenates at 7 days postinfection and alginate biofilms, surrounded by polymorphonuclear leukocytes in the alveoli. In comparison, both an AHL-producing nonmucoid revertant (NH57388C) from the mucoid isolate (NH57388A) and a nonmucoid isolate (NH57388B) deficient in AHL were almost cleared from the lungs of the mice. This model, in which P. aeruginosa is protected against the defense system of the lung by alginate, is similar to the clinical situation. Therefore, the mouse model provides an improved method for evaluating the interaction between mucoid P. aeruginosa, the host, and antibacterial therapy.     

Clostridium difficile vaccine and serum immunoglobulin G antibody response to toxin A

There is a strong association between serum antibody responses to toxin A and protection against Clostridium difficile diarrhea. A parenteral C. difficile toxoid vaccine induced very-high-level responses to anti-toxin A immunoglobulin G (IgG) in the sera of healthy volunteers. After vaccination, the concentrations of anti-toxin A IgG in the sera of all 30 recipients exceeded the concentrations that were associated with protection in previous clinical studies. Furthermore, the median concentration of serum anti-toxin A IgG in the test group was 50-fold higher than the previous threshold. These findings support the feasibility of using a vaccine to protect high-risk individuals against C. difficile-associated diarrhea and colitis.     

Microbial infections,immunomodulation, and drugs of abuse

The use of recreational drugs of abuse has generated serious health concerns. There is a long-recognized relationship between addictive drugs and increased levels of infections. Studies of the mechanisms of actions of these drugs became more urgent with the advent of AIDS and its correlation with abused substances. The nature and mechanisms of immunomodulation by marijuana, opiates, cocaine, nicotine, and alcohol are described in this review. Recent studies of the effects of opiates or marijuana on the immune system have demonstrated that they are receptor mediated, occurring both directly via specific receptors on immune cells and indirectly through similar receptors on cells of the nervous system. Findings are also discussed that demonstrate that cocaine and nicotine have similar immunomodulatory effects, which are also apparently receptor mediated. Finally, the nature and mechanisms of immunomodulation by alcohol are described. Although no specific alcohol receptors have been identified, it is widely recognized that alcohol enhances susceptibility to opportunistic microbes. The review covers recent studies of the effects of these drugs on immunity and on increased susceptibility to infectious diseases, including AIDS.     

PTEN mutational spectra,expression levels,and subcellular localization in microsatellite stable and unstable colorectal cancers

PTEN on 10q23.3 encodes a dual-specificity phosphatase that negatively regulates the phosphoinositol-3-kinase/Akt pathway and mediates cell-cycle arrest and apoptosis. Germline PTEN mutations cause Cowden syndrome and a range of several different hamartoma-tumor syndromes. Hereditary nonpolyposis colon cancer (HNPCC) syndrome is characterized by germline mutations in the mismatch repair (MMR) genes and by microsatellite instability (MSI) in component tumors. Although both colorectal carcinoma and endometrial carcinoma are the most frequent component cancers in HNPCC, only endometrial cancer has been shown to be a minor component of Cowden syndrome. We have demonstrated that somatic inactivation of PTEN is involved in both sporadic endometrial cancers and HNPCC-related endometrial cancers but with different mutational spectra and different relationships to MSI. In the current study, we sought to determine the relationship of PTEN mutation, 10q23 loss of heterozygosity, PTEN expression, and MSI status in colorectal cancers (CRCs). Among 11 HNPCC CRCs, 32 MSI+ sporadic cancers, and 39 MSI- tumors, loss of heterozygosity at 10q23.3 was found in 0%, 8%, and 19%, respectively. Somatic mutations were found in 18% (2 of 11) of the HNPCC CRCs and 13% (4 of 32) of the MSI+ sporadic tumors, but not in MSI- cancers (P = 0.015). All somatic mutations occurred in the two 6(A) coding mononucleotide tracts in PTEN, suggestive of the etiological role of the deficient MMR. Immunohistochemical analysis revealed 31% (14 of 45) of the HNPCC CRCs and 41% (9 of 22) of the MSI+ sporadic tumors with absent or depressed PTEN expression. Approximately 17% (4 of 23) of the MSI- CRCs had decreased PTEN expression, and no MSI- tumor had complete loss of PTEN expression. Among the five HNPCC or MSI+ sporadic CRCs carrying frameshift somatic mutations with immunohistochemistry data, three had lost all PTEN expression, one showed weak PTEN expression levels, and one had mixed tumor cell populations with weak and moderate expression levels. These results suggest that PTEN frameshift mutations in HNPCC and sporadic MSI+ tumors are a consequence of mismatch repair deficiency. Further, hemizygous deletions in MSI- CRCs lead to loss or reduction of PTEN protein levels and contribute to tumor progression. Finally, our data also suggest that epigenetic inactivation of PTEN, including differential subcellular compartmentalization, occurs in CRCs.