Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis

Several real-time PCR procedures for the detection and genotyping of oocysts of Cryptosporidium parvum were evaluated. A 40-cycle amplification of a 157-bp fragment from the C. parvum beta-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because C. parvum isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating C. parvum genotypes were developed. The first method used the same beta-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for C. parvum type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2 C. parvum on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of C. parvum oocysts.     

Development and application of genetic probes for detection of Enterocytozoon bieneusi in formalin-fixed stools and in intestinal biopsy specimens from infected patients.

The microsporidium Enterocytozoon bieneusi is closely linked to wasting and diarrhea in a high proportion of individuals with AIDS. However, its relative contribution to disease is uncertain because diagnosis until recently depended on procedures involving endoscopy. A sensitive PCR technique which amplifies a fragment of the small-subunit rRNA gene of E. bieneusi from formalin-fixed stool samples was developed. Of 80 formalin-fixed stool samples collected from 74 Zimbabweans and 6 U.S. patients who were human immunodeficiency virus positive, 50% tested positive for E. bieneusi by PCR, whereas 24% tested positive for E. bieneusi by light microscopy of trichrome-stained fecal smears. In addition, we describe an in situ hybridization technique which detected and identified E. bieneusi as the causative agent in all six intestinal biopsy specimens tested. Both the PCR and in situ hybridization procedures are sensitive diagnostic tools which will complement currently available techniques and enable the differentiation of E. bieneusi from other microsporidia to be made.     

Human neutrophils secrete gelatinase B in vitro and in vivo in response to endotoxin and proinflammatory mediators

Bacterial sepsis is characterized by a systemic inflammatory state, with activation of numerous cell types. Phagocytes participate in this phenomenon by secreting various proinflammatory cytokines and enzymes. Matrix metalloproteinases (MMPs) such as gelatinases are produced by phagocytes and are thought to play an important role in processes of cell transmigration and tissue remodeling. In this work, we show that endotoxin (lipopolysaccharide [LPS]) and other inflammatory mediators, such as tumor necrosis factor (TNF), interleukin-8, and granulocyte colony-stimulating factor, induce a rapid (within 20 min) release of gelatinase-B (MMP-9) zymogen in whole human blood, as determined by gelatin zymography. The polymorphonuclear neutrophil was identified as the cell responsible for this rapid secretion, as a result of the release of preformed enzymes stored in granules. Normal human subjects given LPS intravenously showed a similar pattern of proMMP-9 secretion, with maximum plasma levels reached 1.5 to 3 h after LPS administration (P = 0.0009). Prior administration of TNF receptor:Fc, a potent TNF antagonist, to subjects given LPS, only partially blunted the release of proMMP-9 (P = 0.033). Ibuprofen, a cyclooxygenase inhibitor, did not alter this pattern of release. Increased levels of proMMP-9 and proMMP-2, as well as activated forms of MMP-9, were found in plasma from two patients with gram-negative sepsis. The levels of MMPs paralleled the severity of clinical condition and a marker of the severity of sepsis, plasma procalcitonin. These data indicate that MMPs are released in whole blood in response to various inflammatory mediators and that they could serve as sensitive and early markers for cell activation during the course of bacterial sepsis.     

Intracellular calcium signalling in magnocellular neurones of the rat supraoptic nucleus: understanding the autoregulatory mechanisms

Oxytocin and vasopressin, released at the soma and dendrites of neurones, bind to specific autoreceptors and induce an increase in [Ca²⁺]₁. In oxytocin cells, the increase results from a mobilisation of Ca²⁺ from intracellular stores, whereas in vasopressin cells, it results mainly from an influx of Ca²⁺ through voltage-dependent channels. The response to vasopressin is coupled to phospholipase C and adenylyl-cyclase pathways which are activated by V₁ (V_1a and V_1b)- and V₂-type receptors respectively. Measurements of [Ca²⁺]₁ in response to V_1a and V₂ agonists and antagonists suggest the functional expression of these two types of receptors in vasopressin neurones. The intracellular mechanisms involved are similar to those observed for the action of the pituitary adenylyl-cyclase-activating peptide (PACAP). Isolated vasopressin neurones exhibit spontaneous [Ca²⁺]₁ oscillations and these are synchronised with phasic bursts of electrical activity. Vasopressin modulates these spontaneous [Ca²⁺]₁ oscillations in a manner that depends on the initial state of the neurone, and such varied effects of vasopressin may be related to those observed on the electrical activity of vasopressin neurones in vivo.     

HMGB1 promotes CXCL12-dependent egress of murine B cells from Peyer's patches in homeostasis

High mobility group box-1 protein (HMGB1) is an alarmin that, once released, promotes inflammatory responses, alone and as a complex with the chemokine CXCL12. Here, we report that the HMGB1–CXCL12 complex plays an essential role also in homeostasis by controlling the migration of B lymphocytes. We show that extracellular HMGB1 is critical for the CXCL12-dependent egress of B cells from the Peyer's patches (PP). This promigratory function of the complex was restricted to the PPs, since HMGB1 was not required for B-cell migratory processes in other locations. Accordingly, we detected higher constitutive levels of the HMGB1–CXCL12 complex in PPs than in other lymphoid organs. HMGB1–CXCL12 in vivo inhibition was associated with a reduced basal IgA production in the gut. Collectively, our results demonstrate a role for the HMGB1–CXCL12 complex in orchestrating B-cell trafficking in homeostasis, and provide a novel target to control lymphocyte migration in mucosal immunity.     

Correlation between in vivo and in vitro efficacy of antimicrobial agents against foreign body infections

Implant-associated infections are often resistant to antibiotic therapy. Routine sensitivity tests fail to predict therapeutic success. Therefore experimental in vitro tests were sought that would better correlate with drug efficacy in device-related infections. The activity of six different antibiotics against methicillin-resistant Staphylococcus epidermidis was investigated. In vivo studies were performed with the guinea pig tissue-cage animal model; in vitro studies with minimum inhibiting and bactericidal concentrations, time-kill studies of growing and stationary-phase microorganisms, the killing of glass-adherent S. epidermidis. Drug efficacy on stationary and adherent microorganisms, but not minimum inhibiting concentrations, predicted the outcome of device-related infections. Rifampin cured 12 of 12 infections and was also the most efficient drug in any experimental in vitro test. Similarly, the failure of ciprofloxacin to eradicate foreign body infections correlated with its low efficacy on stationary-phase and adherent S. epidermidis.     

Minimal mechanical load and tissue culture conditions preserve native cell phenotype and morphology in tendon—a novel ex vivo mouse explant model

Appropriate mechanical load is essential for tendon homeostasis and optimal tissue function. Due to technical challenges in achieving physiological mechanical loads in experimental tendon model systems, the research community still lacks well‐characterized models of tissue homeostasis and physiological relevance. Toward this urgent goal, we present and characterize a novel ex vivo murine tail tendon explant model. Mouse tail tendon fascicles were extracted and cultured for 6 days in a load‐deprived environment or in a custom‐designed bioreactor applying low magnitude mechanical load (intermittent cycles to 1% strain, at 1 Hz) in serum‐free tissue culture. Cells remained viable, as did collagen structure and mechanical properties in all tested conditions. Cell morphology in mechanically loaded tendon explants approximated native tendon, whereas load‐deprived tendons lost their native cell morphology. These losses were reflected in altered gene expression, with mechanical loading tending to maintain tendon specific and matrix remodeling genes phenotypic of native tissue. We conclude from this study that ex vivo load deprivation of murine tendon in minimal culture medium results in a degenerative‐like phenotype. We further conclude that onset of tissue degeneration can be suppressed by low‐magnitude mechanical loading. Thus a minimal explant culture model featuring serum‐free medium with low mechanical loads seems to provide a useful foundation for further investigations. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1383–1390, 2018.

     

Real-time assessment of anteroposterior stability of spinal segments

Purpose

While anteroposterior instability of spinal segments is regarded as an important biomechanical aspect in the clinical evaluation of lumbar pathologies, the reliability of the available diagnostic tools is limited and an intraoperative method to quantify stability is lacking. The aim of this study was to develop and validate an instrument to measure the anteroposterior stability of a spinal segments in real-time.

Methods

Torsi of five fresh-frozen human cadavers were used for this study. After pedicle screw insertion, a specifically modified reposition tool composed with load and linear sensors was used to measure the segmental anteroposterior motion caused by 100 N anterior and posterior force during 5 loading cycles on either side of the instrumentation by two different operators. The spinal segments were then resected from the torsi and anteroposterior loading with ± 100 N was repeated in an advanced biomechanical spine testing setup as a reference measurement. The Inter-correlation coefficient (ICC) was used for validation of the “intraoperative” device.

Results

Inter-operator repeatability of the measurements showed an ICC of 0.93 (p < 0.0001) and the bilateral (left–right) comparison had an ICC of 0.73 (p < 0.0001). The ICC resulting from the comparison to the reference measurement was 0.82 (p < 0.0001) without offset correction, and 0.9 (p < 0.0001) with offset correction. The ICC converged at this value already after two of the five performed loading cycles.

Conclusion

An accurate and reliable measurement tool is developed and validated for real-time quantification of anteroposterior stability of spinal segments and serves as a basis for future intraoperative use.

     

Family beyond parents? An exploration of family configurations and psychological adjustment in young adults with intellectual disabilities

This research explores the family configurations of young adults with intellectual disability. Based on a sample of 40 individuals interviewed two times in a year, we found as many as four types of family configurations, with distinct compositions, and different types of social capital. This diversity is not without consequences for individual psychological adjustment. The results are discussed in the light of the configurational approach to families.