High resolution of heterogeneity among human neutrophil granules: physical, biochemical, and ultrastructural properties of isolated fractions

Previous studies on the fractionation of human neutrophil granules have identified two major populations: myeloperoxidase (MPO)-containing azurophil, or primary, granules and MPO-deficient specific, or secondary, granules. Peripheral blood neutrophils from individual donors were lysed in sucrose-free media by either hypotonic shock or nitrogen cavitation. Using a novel two-gradient Percoll density centrifugation system, the granule-rich postnuclear supernatant was rapidly (ten minutes) and reproducibly resolved into 13 granule fractions (L1 through L8 and H1 through H5). Granule flotation and recentrifugation experiments on both continuous, self-generated and multiple-step gradients using individual and mixed isolated fractions demonstrated that the banding patterns were isopycnic and nonartifactual. Isolated granules were intact based on the findings that biochemical latency of several granule enzymes was greater than 95%, and thin-sectioned electron micrographs demonstrated intact granule profiles. Biochemical analyses of the granule marker proteins MPO, beta-glucuronidase, lysozyme, and lactoferrin indicated that a number of the fractions were related to the major azurophil and specific granule populations. Lactoferrin was found in ten of 13 fractions (L1 through L8, H1 to H2), whereas MPO was found in every fraction. Consistent with these biochemical data, all fractions exhibited varying degrees of heterogeneity based on ultrastructural morphology and cytochemistry, including diaminobenzidine (DAB) reactivity for peroxidase and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining for complex glycoconjugates. A variable but significant percentage (23% to 70%) of the granules in fractions L1 through L8 and H1 and H2 showed DAB reactivity, while about 90% of the granules in fractions H3 through H5 were peroxidase positive. These results demonstrated that DAB-reactive granules spanned the entire range of granule size and density. Ultrastructural PA-TCH-SP staining of isolated granule fractions revealed patterns similar to those of granules in intact neutrophils at different stages of development. Granules from human acute promyelocytic leukemia cells (HL-60) exhibited a surprisingly low density compared with typical azurophil granules from normal, mature neutrophils. The data suggest that both functional and maturational differences contribute to granule heterogeneity, and provide a new practical and conceptual framework for further defining the phenomenon of neutrophil granule heterogeneity.     

Evaluation of lockdown effect on SARS-CoV-2 dynamics through viral genome quantification in waste water,Greater Paris,France, 5 March to 23 April 2020

IntroductionSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease (COVID-19). People infected with SARS-CoV-2 may exhibit no or mild non-specific symptoms; thus, they may contribute to silent circulation of the virus among humans. Since SARS-CoV-2 RNA can be detected in stool samples, monitoring SARS-CoV-2 RNA in waste water (WW) has been proposed as a complementary tool to investigate virus circulation in human populations.AimTo test if the quantification of SARS-CoV-2 genomes in WW correlates with the number of symptomatic or non-symptomatic carriers.MethodWe performed a time-course quantitative analysis of SARS-CoV-2 by RT-qPCR in raw WW samples collected from several major WW treatment plants in Greater Paris. The study period was 5 March to 23 April 2020, including the lockdown period in France (from 17 March).ResultsWe showed that the increase of genome units in raw WW accurately followed the increase of human COVID-19 cases observed at the regional level. Of note, the viral genome could be detected before the epidemic grew massively (around 8 March). Equally importantly, a marked decrease in the quantities of genome units was observed concomitantly with the reduction in the number of new COVID-19 cases, 29 days following the lockdown.ConclusionThis work suggests that a quantitative monitoring of SARS-CoV-2 genomes in WW could generate important additional information for improved monitoring of SARS-CoV-2 circulation at local or regional levels and emphasises the role of WW-based epidemiology.     

Amoxicillin for acute lower respiratory tract infection in primary care: subgroup analysis of potential high-risk groups

Background

Antibiotics are of limited overall clinical benefit for uncomplicated lower respiratory tract infection (LRTI) but there is uncertainty about their effectiveness for patients with features associated with higher levels of antibiotic prescribing.

Aim

To estimate the benefits and harms of antibiotics for acute LRTI among those producing coloured sputum, smokers, those with fever or prior comorbidities, and longer duration of prior illness.

Design and setting

Secondary analysis of a randomised controlled trial of antibiotic placebo for acute LRTI in primary care.

Method

Two thousand and sixty-one adults with acute LRTI, where pneumonia was not suspected clinically, were given amoxicillin or matching placebo. The duration of symptoms, rated moderately bad or worse (primary outcome), symptom severity on days 2–4 (0–6 scale), and the development of new or worsening symptoms were analysed in pre-specified subgroups of interest. Evidence of differential treatment effectiveness was assessed in prespecified subgroups by interaction terms.

Results

No subgroups were identified that were significantly more likely to benefit from antibiotics in terms of symptom duration or the development of new or worsening symptoms. Those with a history of significant comorbidities experienced a significantly greater reduction in symptom severity between days 2 and 4 (interaction term −0.28, P = 0.003; estimated effect of antibiotics among those with a past history −0.28 [95% confidence interval = −0.44 to −0.11], P = 0.001), equivalent to three people in 10 rating symptoms as a slight rather than a moderately bad problem. For subgroups not specified in advance antibiotics provided a modest reduction in symptom severity for non-smokers and for those with short prior illness duration (<7 days), and a modest reduction in symptom duration for those with short prior illness duration.

Conclusion

There is no clear evidence of clinically meaningful benefit from antibiotics in the studied high-risk groups of patients presenting in general practice with uncomplicated LRTIs where prescribing is highest. Any possible benefit must be balanced against the side-effects and longer-term effects on antibiotic resistance.     

The small heat‐shock protein αB‐crystallin is essential for the nuclear localization of Smad4: impact on pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by the proliferation of myofibroblasts and the accumulation of extracellular matrix (ECM) in the lungs. TGF‐β1 is the major profibrotic cytokine involved in IPF and is responsible for myofibroblast proliferation and differentiation and ECM synthesis. αB‐crystallin is constitutively expressed in the lungs and is inducible by stress, acts as a chaperone and is known to play a role in cell cytoskeleton architecture homeostasis. The role of αB‐crystallin in fibrogenesis remains unknown. The principal signalling pathway involved in this process is the Smad‐dependent pathway. We demonstrate here that αB‐crystallin is strongly expressed in fibrotic lung tissue from IPF patients and in vivo rodent models of pulmonary fibrosis. We also show that αB‐crystallin‐deficient mice are protected from bleomycin‐induced fibrosis. Similar protection from fibrosis was observed in αB‐crystallin KO mice after transient adenoviral‐mediated over‐expression of IL‐1β or TGF‐β1. We show in vitro in primary epithelial cells and fibroblasts that αB‐crystallin increases the nuclear localization of Smad4, thereby enhancing the TGF‐β1–Smad pathway and the consequent activation of TGF‐β1 downstream genes. αB‐crystallin over‐expression disrupts Smad4 mono‐ubiquitination by interacting with its E3–ubiquitin ligase, TIF1γ, thus limiting its nuclear export. Conversely, in the absence of αB‐crystallin, TIF1γ can freely interact with Smad4. Consequently, Smad4 mono‐ubiquitination and nuclear export are favoured and thus TGF‐β1–Smad4 pro‐fibrotic activity is inhibited. This study demonstrates that αB‐crystallin may be a key target for the development of specific drugs in the treatment of IPF or other fibrotic diseases. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Morphological integration in the forelimb of musteloid carnivorans

The forelimb forms a functional unit that allows a variety of behaviours and needs to be mobile, yet at the same time stable. Both mobility and stability are controlled, amongst others, at the level of the elbow joint. This joint is composed of the humero‐ulnar articulation, mainly involved during parasagittal movements; and the radio‐ulnar articulation, mainly allowing rotation. In contrast, the humero‐radial articulation allows both movements of flexion–extension and rotation. Here, we study the morphological integration between each bone of the forelimb at the level of the entire arm, as well as at the elbow joint, in musteloid carnivorans. To do so, we quantitatively test shape co‐variation using surface 3D geometric morphometric data. Our results show that morphological integration is stronger for bones that form functional units. Different results are obtained depending on the level of investigation: for the entire arm, results show a greater degree of shape co‐variation between long bones of the lower arm than between the humerus and either bone of the lower arm. Thus, at this level the functional unit of the lower arm is comprised of the radius and ulna, permitting rotational movements of the lower arm. At the level of the elbow, results display a stronger shape co‐variation between bones allowing flexion and stability (humerus and ulna) than between bones allowing mobility (ulna and radius and humerus and radius). Thus, the critical functional unit appears to be the articulation between the humerus and ulna providing the stability of the joint.     

De-escalating therapy in gastric aggressive lymphoma

The treatment of primary gastric diffuse large B-cell lymphoma (DLBCL) has changed radically over the last 10–15 years, with the abandonment of routine gastrectomy in favor of more conservative therapies. Low-level evidence suggests that consolidation radiotherapy could be avoided in patients with limited-stage DLBCL of the stomach who achieve complete remission after rituximab-CHOP combination. Small, recent prospective trials suggest that selected patients with limited-stage Helicobacter pylori (H. pylori)-positive DLBCL of the stomach and favorable prognostic factors can be managed with antibiotics alone, with excellent disease control and cure rates, keeping chemo-radiotherapy for unresponsive patients. This recommendation should equally regard patients with mucosa-associated lymphoid tissue-related or de novo DLBCL. Future studies should be focused on the establishment of reliable variables able to distinguish the best candidates for exclusive treatment with H. pylori eradication from those who need for conventional chemo-immunotherapy.     

An individual rehabilitation program: Evaluation by Parkinsonian patients and their physiotherapists

Objectives

The purpose of this work was to study the feasibility of an individual Parkinson disease (PD) rehabilitation program based on each patient's prevalent symptoms and to determine the effects of this program on patient's quality of life as well as the level of patient's and physiotherapist's satisfaction with the program.

Patients and methods

In association with physiotherapists with expertise in PD, a physical medicine and rehabilitation physician, we elaborated a physical therapy program based on the core areas for physical therapy in PD: transfers; posture; balance and falls; physical capacity and inactivity. Within this program, we selected exercises tailored to each patient's main impairment and proposed this selection to their local physiotherapist for three months. Quality of life was evaluated with PDQ-39 at baseline and after three months of the individualized physical therapy program. We built an anonymous satisfaction questionnaire for patients and physiotherapists that was filled out at the end of the program.

Results

One hundred and three individuals with moderately advanced but clinically stable idiopathic PD were included. Significant improvement was found for the emotional well-being, bodily discomfort and stigma domain (P ≤ 0.05). No significant improvement was found for the other PDQ-39 domains. The mean global satisfaction figures for this program were 6.0 ± 2.4 and 7.2 ± 2.1 for patients and physiotherapists respectively. Most of the patients felt improved by the physiotherapy program and especially for transfer, balance, gait, and mobility.

Conclusion

Our study found evidence of the potential benefits of a patient-tailored physiotherapy program. Such a program was feasible and had a favorable impact on patients’ quality of life and on physiotherapists’ practices for PD patients. Specific physiotherapy may be effective to limit physical mobility impairment. Our results also pointed out that physiotherapy may be efficient to confine the negative impact of social isolation, pain and emotional reactions. Such a program should be associated with a therapeutic education intervention such as encouraging patients to perform physical therapy exercises alone.