Murine TNF(DeltaARE) Crohn's disease model displays diminished expression of intestinal Ca2+ transporters

BACKGROUND: Patients suffering from Crohn's disease (CD) show increased incidence of low bone mineral density. Investigating this complication is difficult because the exact etiology of CD remains elusive. Mice carrying a deletion in the tumor necrosis factor (TNF) AU-rich elements (ARE) are reported as a model for human CD and are characterized by elevated TNF-alpha levels and inflammations in the terminal ileum. To evaluate whether these mice have a Ca(2+) handling problem, this study analyzed the Ca(2+) homeostasis in heterozygous TNF(DeltaARE) mice (TNF(DeltaARE/+)) in comparison to wildtype littermates. METHODS: Beside serum Ca(2+) and vitamin D levels, the expression of Ca(2+) transporters was analyzed in intestine, kidney and bone using quantitative real-time PCR, Western blot and immunohistochemistry. Bone scans were performed to measure bone parameters. RESULTS: Ca(2+) transporters in duodenum (TRPV6, calbindin-D(9K), PMCA1b) and kidney (TRPV5, calbindin-D(28K), NCX1) showed significantly reduced mRNA expression levels in TNP(DeltaARE/+) mice, except for renal TRPV5. In bone, only calbindin-D(9K) mRNA displayed a significant down-regulation. These findings were supported by declined duodenal calbindin-D(9K) and renal calbindin-D(28K) protein values. Likely, this down-regulation of Ca(2+) transporters in TNP(DeltaARE/+) mice is mediated by the 58 +/- 9% reduction in serum 1,25(OH)(2)D(3) levels. Diminished expression of Ca(2+) transporters combined with unchanged serum Ca(2+) levels assumes Ca(2+) loss from bone to compensate for the body's overall Ca(2+) shortage. Indeed, microcomputed tomography scanning demonstrated reduced trabecular and corticol bone thickness and volume in TNF(DeltaARE/+) mice. This finding is further supported by increased total deoxypyridinoline in serum. CONCLUSIONS: Our results imply that TNF(DeltaARE/+) mice have a disturbed Ca(2+) homeostasis characterized by reduced duodenal and renal Ca(2+) transporters, diminished 1,25(OH)(2)D(3) levels, and increased bone resorption associated with profound bone abnormalities.     

Automated measurement of office,home and ambulatory blood pressure in atrial fibrillation

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On the protective mechanisms of nitric oxide in acute pancreatitis

Background—Ectopic proteaseactivation, microcirculatory changes, and leucocyte activation are themain events in the pathogenesis of acute pancreatitis. Nitric oxide(NO) is known to be a key mediator in the normal and inflamed pancreas.
Aims—To investigate the targets onwhich NO exerts its effect in caerulein induced pancreatitis.
Methods—Acute pancreatitis wasinduced in rats which additionally received either the NO synthasesubstrate, L-arginine; the NO donor, sodium nitroprusside;or the NO synthase inhibitor, N-nitro-L-arginine methylester (L-NAME). At six hours, pancreatic injury (oedema,leucocyte content, ectopic trypsinogen activation) was analysed andpancreatic oxygenation and perfusion were determined. A directinfluence of NO on amylase secretion and trypsinogen activation wasevaluated separately in vitro.
Results—Both NO donors reduced thegrade of inflammation. L-NAME increased the severity ofinflammation, while decreasing pancreatic tissue oxygenation. Althoughneither amylase secretion nor intracellular trypsinogen activation incaerulein stimulated pancreatic acini was influenced by either NOdonors or inhibitors, both NO donors decreased intrapancreatictrypsinogen activation peptide (TAP) and pancreatic oedema in vivo, andL-NAME increased TAP.
Conclusions—NO protects againstinjury caused by pancreatitis in the intact animal but has nodiscernible effect on isolated acini. It is likely that in pancreatitisNO acts indirectly via microcirculatory changes, including inhibitionof leucocyte activation and preservation of capillary perfusion.

Keywords:acute pancreatitis; nitric oxide; microcirculation; leucocytes; pancreatic secretion

     

Spontaneous inflammatory demyelinating disease in transgenic mice showing central nervous system-specific expression of tumor necrosis factor alpha.

Cytokines are now recognized to play important roles in the physiology of the central nervous system (CNS) during health and disease. Tumor necrosis factor alpha (TNF-alpha) has been implicated in the pathogenesis of several human CNS disorders including multiple sclerosis, AIDS dementia, and cerebral malaria. We have generated transgenic mice that constitutively express a murine TNF-alpha transgene, under the control of its own promoter, specifically in their CNS and that spontaneously develop a chronic inflammatory demyelinating disease with 100% penetrance from around 3-8 weeks of age. High-level expression of the transgene was seen in neurons distributed throughout the brain. Disease is manifested by ataxia, seizures, and paresis and leads to early death. Histopathological analysis revealed infiltration of the meninges and CNS parenchyma by CD4+ and CD8+ T lymphocytes, widespread reactive astrocytosis and microgliosis, and focal demyelination. The direct action of TNF-alpha in the pathogenesis of this disease was confirmed by peripheral administration of a neutralizing anti-murine TNF-alpha antibody. This treatment completely prevented the development of neurological symptoms, T-cell infiltration into the CNS parenchyma, astrocytosis, and demyelination, and greatly reduced the severity of reactive microgliosis. These results demonstrate that overexpression of TNF-alpha in the CNS can cause abnormalities in nervous system structure and function. The disease induced in TNF-alpha transgenic mice shows clinical and histopathological features characteristic of inflammatory demyelinating CNS disorders in humans, and these mice represent a relevant in vivo model for their further study.     

Peyer’s patch organogenesis is intact yet formation of B lymphocyte follicles is defective in peripheral lymphoid organs of mice deficient for tumor necrosis factor and its 55-kDa receptor

Targeted inactivation of genes in the tumor necrosis factor (TNF)/lymphotoxin (LT) ligand and receptor system has recently revealed essential roles for these molecules in lymphoid tissue development and organization. Lymphotoxin-αβ (LTαβ)/lymphotoxin-β receptor (LTβ-R) signaling is critical for the organogenesis of lymph nodes and Peyer’s patches and for the structural compartmentalization of the splenic white pulp into distinct B and T cell areas and marginal zones. Moreover, an essential role has been demonstrated for TNF/p55 tumor necrosis factor receptor (p55TNF-R) signaling in the formation of splenic B lymphocyte follicles, follicular dendritic cell networks, and germinal centers. In contrast to a previously described essential role for the p55TNF-R in Peyer’s patch organogenesis, we show in this report that Peyer’s patches are present in both TNF and p55TNF-R knockout mice, demonstrating that these molecules are not essential for the organogenesis of this lymphoid organ. Furthermore, we show that in the absence of TNF/p55TNF-R signaling, lymphocytes segregate normally into T and B cell areas and a normal content and localization of dendritic cells is observed in both lymph nodes and Peyer’s patches. However, although B cells are found to home normally within Peyer’s patches and in the outer cortex area of lymph nodes, organized follicular structures and follicular dendritic cell networks fail to form. These results show that in contrast to LTαβ signaling, TNF signaling through the p55TNF-R is not essential for lymphoid organogenesis but rather for interactions that determine the cellular and structural organization of B cell follicles in all secondary lymphoid tissues.     

Exclusive tumor necrosis factor (TNF) signaling by the p75TNF receptor triggers inflammatory ischemia in the CNS of transgenic mice

Tumor necrosis factor (TNF) is up-regulated in a variety of central nervous system (CNS) diseases with diverse etiology and pathologic manifestation. TNF mediates multiple biological activities through two membrane receptors, the p55 and p75 TNF receptors (TNFRs). We have shown previously that human transmembrane TNF (tmTNF)p55TNFR signaling in transgenic mice triggers oligodendrocyte apoptosis, endothelial cell activation, parenchymal inflammation, and primary demyelinating lesions similar to those of acute multiple sclerosis. To address the role of the p75TNFR in the CNS, we have generated "humanized" mice that express human tmTNF in astrocytes and a physiologically regulated human p75TNFR transgene, in the absence of the endogenous (murine) p55TNFR. Human tmTNFp75TNFR transgenic mice develop CNS vascular pathology, characterized by endothelial cell activation, meningeal inflammation, and vessel fibrosis. There is no evidence of oligodendrocyte apoptosis or primary demyelination in these mice. Late in disease, vasculitis can result in vessel occlusion and secondary, multifocal CNS ischemic injury. These results identify a proinflammatory role for the p75TNFR at the level of the CNS vascular endothelium, which correlates with the expression pattern of this receptor in the CNS, and indicate that the differential expression patterns of the two TNFRs within the CNS play a significant role in shaping the outcome of TNF signaling during neuroimmune interactions.     

Office blood pressure measurement types: Different methodology—Different clinical conclusions

The measurement of blood pressure in the office (OBP) remains the basis for hypertension diagnosis and management for more than half a century. Despite the increasing use of out‐of‐office blood pressure measurement using home and less so ambulatory monitoring and their endorsement by hypertension societies, at present and for some time to come it is likely that in many people the diagnosis and management of hypertension will be based on OBP measurement alone. OBP measurement is a very variable method affected by multiple factors, which have major impact on the OBP level, reproducibility and prognostic ability. Thus, there are several types of OBP measurement, depending on the device type, conditions, observer’s presence and the number of readings. The 4 main OBP types are: Type I: Auscultatory OBP in clinical practice; Type II: Automated attended OBP in clinical practice; Type III: Research setting OBP; Type IV: Unattended automated OBP. These OBP types have different standardization level, different reproducibility, different clinical relevance and different thresholds for hypertension diagnosis. Unless the methodological details of OBP measurement are reported, any conclusions based on such measurements in clinical research and in practice are questionable.

The measurement of blood pressure (BP) is the most common procedure performed in the doctor''s office. Moreover, the diagnosis of hypertension and the decisions for life‐time treatment are exclusively dependent on BP measurement. In 1964, Geoffrey Rose presented the categories of observer error in auscultatory BP measurement, including systematic error, terminal digit preference, and observer prejudice.¹ Recent guidelines in the USA and Europe recognized that office BP (OBP) measurement alone is often unable to diagnose hypertension accurately, mainly due to the white coat and masked hypertension phenomena.², ³ The US guidelines provide clear recommendations for using out‐of‐office BP monitoring (ambulatory [ABPM] or home [HBPM]) to confirm the need to initiate treatment in untreated or titrate in treated subjects.² The European guidelines provide very similar recommendations for out‐of‐office BP monitoring, yet they give two options for diagnosing hypertension: based on repeated OBP measurements using an auscultatory or electronic (oscillometric) device on several visits, or on ABPM or HBPM “provided that these measurements are logistically and economically feasible.”³ The recommendation to base decisions on OBP alone is scientifically problematic because it has been shown that, even with carefully measured OBP in repeated visits in a research setting, about 30% of untreated or treated subjects are misdiagnosed due to the white coat and masked hypertension phenomena.⁴ On the other hand, this recommendation presents a realistic approach given the limited use of HBPM and much less ABPM, even in Europe and North America. A recent study in 2221 primary care physicians in Spain showed that only 3% of them recommended ABPM always and 27% usually, and for HBPM 17% and 50%, respectively.⁵ Thus, the reality is that at present and for some time to come it is likely that in many people the diagnosis and management of hypertension will be based on OBP measurement alone. Hence, both the US and European guidelines provide detailed guidance for doctors to obtain standardized OBP measurements.There are two major methodological issues with OBP: (a) after half a century of wide application of the OBP measurement, the scientific community has failed to eliminate the observer related errors which remain very common in general practice, and (b) it is recognized that, even with standardized OBP measurement devoid of the observer errors (unattended automated OBP), the white coat and masked hypertension phenomena are still common and lead to misdiagnosis in a considerable proportion of untreated and treated subjects.⁶ In this issue of the Journal of Clinical Hypertension, Tang et al⁷ compared OBP measurements taken in a primary care clinic using a validated automated device and a standard protocol versus OBP taken in a research setting using the same automated device. The study showed that, despite the use of automated devices which eliminate the observer errors, primary care OBP was higher and more unstable than research setting OBP.⁷ More importantly, these differences were evident despite the implementation of an intervention program aiming to standardize OBP.⁷ These findings have important implications for clinical practice. It must be realized that OBP measurement is a very variable method affected by multiple factors, which have major impact on the OBP level, reproducibility, and prognostic value. Thus, there are several types of OBP measurement, depending on the device type, conditions, observer’s presence, and the number of readings, which have different standardization level, different reproducibility, different clinical relevance, and different thresholds for hypertension diagnosis. The four main OBP types and their characteristics and differences are summarized in Table ​Table11 and are the following:

1. Auscultatory OBP in clinical practice: This is a poorly standardized and highly variable method which considerably overestimates BP resulting in over‐diagnosis.⁸ Moreover, the auscultatory devices might induce a systematic error with time which is not evident without calibration.
2. Automated attended OBP in clinical practice: This method has the advantage of avoiding the observer errors and that automated devices are more likely to remain accurate for long time (or stop working). In the last 20 years, this method has been the most widely used in hypertension outcome trials, most of which obtained 2‐3 readings with automated devices.⁹ The Systolic Blood Pressure Intervention Trial (SPRINT) and other studies showed that when such measurements are taken in standardized conditions (few minutes resting, no talking, 3 or more measurements) they give similar OBP values as “unattended automated OBP” (see below type IV).¹⁰, ¹¹ However, in clinical practice issues with lack of resting period, inadequate body and arm position, and talking during or between the measurements are possible and might increase OBP.
3. Research setting OBP: This method taken with auscultatory or automated devices in the context of relatively small clinical trials performed in a single or few hypertension expert centers is “ideal” OBP as it is perfectly standardized. Such studies have investigated the OBP reproducibility, diagnostic value, drug effects, etc. Research setting OBP appears to have reproducibility¹² and association with indices of preclinical organ damage close to that of ABPM.¹³ However, it is unrealistic to achieve such measurements in clinical practice. It is important to note that the research OBP of outcome hypertension trials ⁹ has not been so carefully standardized, as it was performed in multiple centers without high expertise in hypertension research. Giorgini et al reviewed the OBP methodology in 64 mega‐trials in hypertension published from 1990 to 2014 and concluded that numerous aspects of OBP measurement often deviated from guideline recommendations and varied considerably across trials.⁹ They stated that “the lack of uniform methodologies in outcome studies that form the foundation of evidence‐based guidelines may have significant clinical implications.”⁹
4. Unattended automated OBP ⁶, ⁸: This is a unique method to achieve standardized OBP in primary care because (a) observer errors are avoided, (b) talking of the patient during and between measurements is avoided, and (c) it ensures that a standard protocol (eg, triplicate measurement) is followed.¹⁰ Disadvantages are that (a) it may not be applicable in all primary care settings as it requires a special device, more time, and office space, and (b) the threshold for hypertension diagnosis is lower and rather uncertain with scarce outcome data.¹⁰, ¹¹, ¹⁴

Table 1Types of office blood pressure measurement

Spectral responses of melanin to ultraviolet A irradiation

The purpose of this investigation was to study the ultraviolet A-induced effects on melanin pigmentation both in an in vitro model system and in vivo. Ultraviolet-Vis absorbance spectra of L-3,4-dihydroxyphenylalanine-melanin solutions at different concentrations were measured before and after ultraviolet A exposure (10-120 J per cm2). The difference spectra reveal that following ultraviolet A exposure the absorbance increases exponentially from 800 nm to 450 nm accompanied by a prominent decrease of absorbance in the ultraviolet A range. This change of spectral features depends on both ultraviolet A doses and melanin concentrations. The photo-bleaching effect observed in the ultraviolet A range also depends on oxygen. Human subjects were irradiated with ultraviolet A (40-80 J per cm2) on their back and diffuse reflectance spectra were collected at both irradiated and untreated sites. The absorption spectra of ultraviolet A-induced pigment were calculated as the difference of the two. The ultraviolet A-induced pigment in vivo has similar spectral characteristics and dose dependency as the in vitro system. Photo-oxidation of pheomelanin solutions presents distinctively different spectral and dose-response characteristics from eumelanin. After ultraviolet A irradiation pheomelanin absorbance decreases both in the visible and the ultraviolet A range. We conclude that irradiation with ultraviolet A induces significant photochemical alterations in the skin witnessed by increased photoprotection in the visible spectral range and reduced protection in the ultraviolet A range. We suggest that soluble melanin plays an important part in ultraviolet A-induced pigment in skin and two distinct absorption mechanisms of melanin may be involved in ultraviolet A photo-oxidation. We also propose that eumelanin and pheomelanin could be differentiated according to their spectral responses to ultraviolet A irradiation.