XLP1 inhibitory effect by 2B4 does not affect DNAM‐1 and NKG2D activating pathways in NK cells

X‐linked lymphoproliferative disease 1 (XLP1) is a rare congenital immunodeficiency caused by SH2D1A (Xq25) mutations resulting in lack or dysfunction of SLAM‐associated protein adaptor molecule. In XLP1 patients, upon ligand (CD48) engagement, 2B4 delivers inhibitory signals that impair the cytolytic activity of NK (and T) cells. This causes the selective inability to control EBV infections and the occurrence of B‐cell lymphomas. Here, we show that in the absence of SLAM‐associated protein, co‐engagement of 2B4 with different activating receptors, either by antibodies or specific ligands on target cells, inhibits different ITAM‐dependent signaling pathways including activating killer Ig‐like receptors. In XLP1 NK cells, 2B4 affected both the cytolytic and IFN‐γ production capabilities, functions that were restored upon disruption of the 2B4/CD48 interactions. Notably, we provide evidence that 2B4 dysfunction does not affect the activity of DNAM‐1 and NKG2D triggering receptors. Thus, while CD48⁺ B‐EBV and lymphoma B cells devoid of NKG2D and DNAM‐1 ligands were resistant to lysis, the preferential usage of these receptors allowed XLP1 NK cells to kill lymphomas that expressed sufficient amounts of the specific ligands. The study sheds new light on the XLP1 immunological defect and on the cross‐talk of inhibitory 2B4 with triggering NK (and T) receptors.     

IL‐4‐induced gene 1 maintains high Tob1 expression that contributes to TCR unresponsiveness in human T helper 17 cells

Human Th17 cells have a limited proliferative capacity compared to other T‐cell subsets. We have shown that human Th17 cells display impaired IL‐2 production due to IL‐4‐induced gene 1 (IL4I1) upregulation. Here, we show that in human Th17 cells, IL4I1 also maintains high levels of Tob1, a member of the Tob/BTG (B‐cell traslocation gene) antiproliferative protein family, which prevents cell‐cycle progression mediated by TCR stimulation. Indeed, Th17 cells exhibited higher levels of Tob1 than Th1 cells in both resting and TCR‐activated conditions. Accordingly, the expression of positive regulators of the cell cycle (cyclin A, B, C, and E and Cdk2), as well as of Skp2, which promotes Tob1 degradation, was lower in Th17 cells than in Th1 cells. Tob1 expression in human Th17 cells correlated with both RAR (retinoic acid receptor)‐related orphan receptor C (RORC) and IL4I1 levels. However, RORC was not directly involved in the regulation of Tob1 expression, whereas IL4I1 silencing in Th17 cells induced a substantial decrease of Tob1 expression. These data suggest that IL4I1 upregulation in human Th17 cells limits their TCR‐mediated expansion not only by blocking the molecular pathway involved in the activation of the IL‐2 promoter, but also by maintaining high levels of Tob1, which impairs entry into the cell cycle.     

IsdC from Staphylococcus lugdunensis Induces Biofilm Formation under Low-Iron Growth Conditions

Staphylococcus lugdunensis is a coagulase-negative staphylococcus that is a commensal of humans and an opportunistic pathogen. It can cause a spectrum of infections, including those that are associated with the ability to form biofilm, such as occurs with endocarditis or indwelling medical devices. The genome sequences of two strains revealed the presence of orthologues of the ica genes that are responsible for synthesis of poly-N-acetylglucosamine (PNAG) that is commonly associated with biofilm in other staphylococci. However, we discovered that biofilm formed by a panel of S. lugdunensis isolates growing in iron-restricted medium was susceptible to degradation by proteases and not by metaperiodate, suggesting that the biofilm matrix comprised proteins and not PNAG. When the iron concentration was raised to 1 mM biofilm formation by all strains tested was greatly reduced. A mutant of strain N920143 lacking the entire locus that encodes iron-regulated surface determinant (Isd) proteins was defective in biofilm formation under iron-limited conditions. An IsdC-null mutant was defective, whereas IsdK, IsdJ, and IsdB mutants formed biofilm to the same level as the parental strain. Expression of IsdC was required both for the primary attachment to unconditioned polystyrene and for the accumulation phase of biofilm involving cell-cell interactions. Purified recombinant IsdC protein formed dimers in solution and Lactococcus lactis cells expressing only IsdC adhered to immobilized recombinant IsdC but not to IsdJ, IsdK, or IsdB. This is consistent with a specific homophilic interaction between IsdC molecules on neighboring cells contributing to accumulation of S. lugdunensis biofilm in vivo.     

Balanced and unbalanced solutions modulate the release of Matrix Metalloproteinase-9 (MMP-9) from neutrophils in response to inflammatory stimuli: an in vitro study

Objectives and design

We investigated the effect of balanced (BS) and unbalanced (UBS) solutions in the absence or presence of hydroxyethyl starch (HES) on neutrophil functionality, evaluating the release of matrix metalloproteinase (MMP)-9, myeloperoxidase (MPO), and MMP-8.

Materials and methods

Neutrophils were isolated by gradient centrifugation and dextran sedimentation and incubated in BS or UBS without or with HES, in the absence or presence of Interleukin-8 (IL-8) or Lipopolysaccharide (LPS). MMP-9, MPO, and MMP-8 were assayed by commercially available ELISA kits.

Results

There was not any influence of volume replacement solutions on the release of the enzymes from resting neutrophils. After IL-8 stimulation, the release of MMP-9 was higher in BS than in UBS or RPMI-1640, whereas HES enhanced its release regardless of the composition. After LPS stimulation, the release of MMP-9 was higher in both UBS and BS than RPMI-1640, but HES brought its release back to physiological conditions. No difference was found in the release of MPO and MMP-8 after stimulation with IL-8 or LPS.

Conclusion

Volume replacement solutions might have an impact on the release of MMP-9 depending on the inflammatory milieu, suggesting that the use of balanced or unbalanced solutions is not a neutral choice.     

Classification of Lower Extremity Movement Patterns Based on Visual Assessment: Reliability and Correlation With 2-Dimensional Video Analysis

Context:

Abnormal movement patterns have been implicated in lower extremity injury. Reliable, valid, and easily implemented assessment methods are needed to examine existing musculoskeletal disorders and investigate predictive factors for lower extremity injury.

Objective:

To determine the reliability of experienced and novice testers in making visual assessments of lower extremity movement patterns and to characterize the construct validity of the visual assessments.

Design:

Cross-sectional study.

Setting:

University athletic department and research laboratory.

Patients or Other Participants:

Convenience sample of 30 undergraduate and graduate students who regularly participate in athletics (age = 19.3 ± 4.5 years). Testers were 2 experienced physical therapists and 1 novice postdoctoral fellow (nonclinician).

Main Outcome Measure(s):

We took videos of 30 athletes performing the single-legged squat. Three testers observed the videos on 2 occasions and classified the lower extremity movement as dynamic valgus, no change, or dynamic varus. The classification was based on the estimated change in frontal-plane projection angle (FPPA) of the knee from single-legged stance to maximum single-legged squat depth. The actual FPPA change was measured quantitatively. We used percentage agreement and weighted κ to examine tester reliability and to determine construct validity of the visual assessment.

Results:

The κ values for intratester and intertester reliability ranged from 0.75 to 0.90, indicating substantial to excellent reliability. Percentage agreement between the visual assessment and the quantitative FPPA change category was 90%, with a κ value of 0.85.

Conclusions:

Visual assessments were made reliably by experienced and novice testers. Additionally, movement-pattern categories based on visual assessments were in excellent agreement with objective methods to measure FPPA change. Therefore, visual assessments can be used in the clinic to assess movement patterns associated with musculoskeletal disorders and in large epidemiologic studies to assess the association between lower extremity movement patterns and musculoskeletal injury.Key Words: movement analysis, screening, athletic injuries, knee valgus

Key Points

• With training and the use of standardized techniques, both experienced and novice testers reliably classified lower extremity movement patterns based on visual assessment.
• Movement-pattern category-based visual assessments were in excellent agreement with objective methods to measure changes in frontal-plane projection angle.
• Visual assessment based on the methods described in this study may be used in the clinical setting, as well as in large epidemiologic studies and screening assessments for sport participation, to identify distinct categories of lower extremity movement patterns.

Abnormal movement patterns of the lower extremity have been implicated in noncontact anterior cruciate ligament (ACL) injuries¹ and other musculoskeletal problems, such as patellofemoral pain^2–4 and acetabular labral tears.⁵ In addition, correcting these abnormal movement patterns has been shown to prevent ACL injury⁶ and is proposed to reduce symptoms in people with preexisting pain conditions.^5,7,8 Thus, assessment of lower extremity movement patterns may be a way to guide treatment of existing musculoskeletal pain problems and to identify people at risk for future injury or musculoskeletal pain. To facilitate the examination of existing musculoskeletal disorders and the investigation of predictive factors of lower extremity injury, reliable, valid, and feasible methods to assess lower extremity movement patterns are needed.One method to assess lower extremity movement patterns is the Landing Error Scoring System (LESS).^9–11 The LESS uses a standard technique to make visual assessments of movement patterns during a drop vertical jump. The LESS is reliable and valid^9–11; however, the drop vertical jump is a relatively high-level activity and may not be the best way to assess movement patterns in patients with existing injury or in athletes whose sports do not involve landing from a jump. In addition, the drop vertical jump is a bilateral activity that may allow the participant to use 1 limb to compensate for the other. Visual assessment of the single-legged squat (SLSquat), a unilateral limb task, may provide an alternative to the LESS.We have developed standardized methods using a visual assessment of the frontal-plane projection angle (FPPA) to classify the lower extremity movement pattern during an SLSquat. The FPPA is a 2-dimensional (2-D) representation of the lower extremity position¹² and has been used to identify differences between men and women¹² and between women with patellofemoral pain and control participants^4,13 and to detect change in movement patterns after specific training.¹⁴ We established specific criteria to define the categories of lower extremity movement pattern based on the change in FPPA (FPPA change) during motion. The tester observes the angle formed between a line that bisects the thigh and a line that bisects the lower leg. During movement tests, the tester compares the FPPA at the start position with the FPPA at the end position. For example, to assess an SLSquat, the examiner compares the FPPA during the start position of single-legged stance with the end position of maximum squat depth. The difference observed in FPPA from the start to the end position can then be classified as dynamic valgus (change in the valgus direction), no change, or dynamic varus (change in the varus direction). We have used this assessment extensively in the clinical setting, but we have not assessed the rater reliability or the construct validity of our visual assessments.The purpose of this study was to assess the intratester and intertester reliability of 3 testers (2 experienced, 1 novice) categorizing the lower extremity movement pattern demonstrated during an SLSquat. A standardized protocol was used to assess videos of healthy participants performing the SLSquat. We hypothesized that the testers, both experienced and novice, would demonstrate good to excellent reliability using the standardized methods. In addition, we used the objective measure of quantifying FPPA as described by Willson and Davis¹² to determine the construct validity of our visual assessments. We hypothesized that we would see good to excellent agreement between our visual assessments and the quantitative FPPA change.     

Loss of Miro1-directed mitochondrial movement results in a novel murine model for neuron disease

Defective mitochondrial distribution in neurons is proposed to cause ATP depletion and calcium-buffering deficiencies that compromise cell function. However, it is unclear whether aberrant mitochondrial motility and distribution alone are sufficient to cause neurological disease. Calcium-binding mitochondrial Rho (Miro) GTPases attach mitochondria to motor proteins for anterograde and retrograde transport in neurons. Using two new KO mouse models, we demonstrate that Miro1 is essential for development of cranial motor nuclei required for respiratory control and maintenance of upper motor neurons required for ambulation. Neuron-specific loss of Miro1 causes depletion of mitochondria from corticospinal tract axons and progressive neurological deficits mirroring human upper motor neuron disease. Although Miro1-deficient neurons exhibit defects in retrograde axonal mitochondrial transport, mitochondrial respiratory function continues. Moreover, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or mitochondrial calcium buffering. Our findings indicate that defects in mitochondrial motility and distribution are sufficient to cause neurological disease.Motor neuron diseases (MNDs), including ALS and spastic paraplegia (SP), are characterized by the progressive, length-dependent degeneration of motor neurons, leading to muscle atrophy, paralysis, and, in some cases, premature death. There are both inherited and sporadic forms of MNDs, which can affect upper motor neurons, lower motor neurons, or both. Although the molecular and cellular causes of most MNDs are unknown, many are associated with defects in axonal transport of cellular components required for neuron function and maintenance (1–6).A subset of MNDs is associated with impaired mitochondrial respiration and mitochondrial distribution. This observation has led to the hypothesis that neurodegeneration results from defects in mitochondrial motility and distribution, which, in turn, cause subcellular ATP depletion and interfere with mitochondrial calcium ([Ca²⁺]_m) buffering at sites of high synaptic activity (reviewed in ref. 7). It is not known, however, whether mitochondrial motility defects are a primary cause or a secondary consequence of MND progression. In addition, it has been difficult to isolate the primary effect of mitochondrial motility defects in MNDs because most mutations that impair mitochondrial motility in neurons also affect transport of other organelles and vesicles (1, 8–11).In mammals, the movement of neuronal mitochondria between the cell body and the synapse is controlled by adaptors called trafficking kinesin proteins (Trak1 and Trak2) and molecular motors (kinesin heavy chain and dynein), which transport the organelle in the anterograde or retrograde direction along axonal microtubule tracks (7, 12–24). Mitochondrial Rho (Miro) GTPase proteins are critical for transport because they are the only known surface receptors that attach mitochondria to these adaptors and motors (12–15, 18, 25, 26). Miro proteins are tail-anchored in the outer mitochondrial membrane with two GTPase domains and two predicted calcium-binding embryonic fibroblast (EF) hand motifs facing the cytoplasm (12, 13, 25, 27, 28). A recent Miro structure revealed two additional EF hands that were not predicted from the primary sequence (29). Studies in cultured cells suggest that Miro proteins also function as calcium sensors (via their EF hands) to regulate kinesin-mediated mitochondrial “stopping” in axons (15, 16, 26). Miro-mediated movement appears to be inhibited when cytoplasmic calcium is elevated in active synapses, effectively recruiting mitochondria to regions where calcium buffering and energy are needed. Despite this progress, the physiological relevance of these findings has not yet been tested in a mammalian animal model. In addition, mammals ubiquitously express two Miro orthologs, Miro1 and Miro2, which are 60% identical (12, 13). However, the individual roles of Miro1 and Miro2 in neuronal development, maintenance, and survival have no been evaluated.We describe two new mouse models that establish the importance of Miro1-mediated mitochondrial motility and distribution in mammalian neuronal function and maintenance. We show that Miro1 is essential for development/maintenance of specific cranial neurons, function of postmitotic motor neurons, and retrograde mitochondrial motility in axons. Loss of Miro1-directed retrograde mitochondrial transport is sufficient to cause MND phenotypes in mice without abrogating mitochondrial respiratory function. Furthermore, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or [Ca²⁺]_m buffering. These findings have an impact on current models for Miro1 function and introduce a specific and rapidly progressing mouse model for MND.     

Community Health Workers and Stand-Alone or Integrated Case Management of Malaria: A Systematic Literature Review

A systematic literature review was conducted to assess the effectiveness of strategies to improve community case management (CCM) of malaria. Forty-three studies were included; most (38) reported indicators of community health worker (CHW) performance, 14 reported on malaria CCM integrated with other child health interventions, 16 reported on health system capacity, and 13 reported on referral. The CHWs are able to provide good quality malaria care, including performing procedures such as rapid diagnostic tests. Appropriate training, clear guidelines, and regular supportive supervision are important facilitating factors. Crucial to sustainable success of CHW programs is strengthening health system capacity to support commodity supply, supervision, and appropriate treatment of referred cases. The little evidence available on referral from community to health facility level suggests that this is an area that needs priority attention. The studies of integrated CCM suggest that additional tasks do not reduce the quality of malaria CCM provided sufficient training and supervision is maintained.