ERK-mediated NF-κB activation through ASIC1 in response to acidosis.

Acidic microenvironment is a common feature of solid tumors. We have previously shown that neuron specific acid-sensing ion channel 1 (ASIC1) is expressed in breast cancer, and it is responsible for acidosis-induced cellular signaling through AKT, leading to nuclear factor-κB (NF-κB) activation, and cell invasion and metastasis. However, AKT is frequently activated in cancer. Thus, a key question is whether ASIC1-mediated cell signaling still takes place in the cancer cells carrying constitutively active AKT. In the present study, we show that among four prostate cancer cell lines tested, 22Rv1 cells express the highest level of phosphorylated AKT that is not impacted by acidosis. However, acidosis can still induce NF-κB activation during which extracellular signal-regulated kinase (ERK) serves as an alternative pathway for ASIC-mediated cell signaling. Inhibition of ERK by chemical inhibitors or small interfering RNAs suppresses the acidosis-induced NF-κB activity through regulation of the inhibitory subunit IκBα phosphorylation. Furthermore, suppression of ASIC1-mediated generation of reactive oxygen species (ROS) by ROS scavengers, such as glutathione or N-acetyl-cysteine causes a decrease in ERK phosphorylation and degradation of IκBα. Finally, ASIC1 is upregulated in a subset of prostate cancer cases and ASIC1 knockout by CRISPR/Cas9 significantly suppresses cell invasion, and castration resistance both in vitro and in vivo. Together, these results support the significance of ASIC1-ROS-ERK-IκBα-NF-κB axis in prostate tumorigenesis, especially in the constitutively active AKT background.

Red blood cell transfusions increase fecal calprotectin levels in premature infants.

OBJECTIVE: We hypothesized that red blood cell (RBC) transfusions influence intestinal inflammation in very low birth weight (VLBW) infants. We also suspected that hematocrit (Hct) at transfusions and RBC storage time correlate with intestinal inflammation. STUDY DESIGN: VLBW infants, without major congenital defects, intestinal perforation or necrotizing enterocolitis, were enrolled prospectively. Fecal calprotectin (FC) levels were measured from stool samples collected before and after RBC transfusions. Data on Hct and RBC storage time were collected. RESULT: Data from 42 RBC transfusions given to 26 infants revealed that FC levels increased faster than baseline after RBC transfusions (P=0.018) and were higher in multiple-transfused infants (0 to 48 and >48 h post transfusion, P=0.007 and P=0.005, respectively). Lower Hct and RBC storage >21 days correlated with higher FC levels (P=0.044 and P=0.013, respectively). CONCLUSION: RBC transfusions, anemia and prolonged RBC storage were associated with an increase in intestinal inflammation.

Involvement of polypyrimidine tract-binding protein (PTBP1) in maintaining breast cancer cell growth and malignant properties.

We have investigated some roles of splicing factor polypyrimidine tract-binding protein (PTBP1) in human breast cancer. We found that PTBP1 was upregulated in progressively transformed human mammary epithelial cells (HMECs), as well as in breast tumor cell lines compared with HMECs with finite growth potential and found that the level of PTBP1 correlated with the transformation state of HMECs. Knockdown of PTBP1 expression substantially inhibited tumor cell growth, colony formation in soft agar and in vitro invasiveness of breast cancer cell lines, a result similar to what we have reported in ovarian cancer. However, ectopic expression of PTBP1 (as a PTBP1-EGFP fusion protein) did not enhance the proliferation of immortalized HMEC. Rather, PTBP1 expression promoted anchorage-independent growth of an immortalized HMEC as assessed by increased colony formation in soft agar. In addition, we found that knockdown of PTBP1 expression led to upregulation of the expression of the M1 isoform of pyruvate kinase (PKM1) and increase of the ratio of PKM1 vs PKM2. PKM1 has been reported to promote oxidative phosphorylation and reduce tumorigenesis. Correspondingly, we observed increased oxygen consumption in PTBP1-knockdown breast cancer cells. Together, these results suggest that PTBP1 is associated with breast tumorigenesis and appears to be required for tumor cell growth and maintenance of transformed properties. PTBP1 exerts these effects, in part, by regulating the splicing of pyruvate kinase, and consequently alters glucose metabolism and contributes to the Warburg effect.

Rho proteins crosstalk via RhoGDIalpha: At random or hierarchically ordered?

The small GTPases of the Rho family are key signaling molecules regulating a plethora of biological pathways. They can exert diverse, sometimes opposite, contributions to specific cellular processes explaining why their regulation and their crosstalk must be finely tuned. Several mechanisms driving crosstalk between Rho GTPases have been described in the literature. They implicate proteins regulating their activity or common downstream effectors. Among the proteins regulating Rho GTPases cycling, RhoGDIs were viewed until very recently as passive inhibitors. Here, we will focus on recent data supporting a role for RhoGDIalpha in the crosstalk between RhoGTPases and present our results suggesting that "preferential" RhoGDIalpha-mediated crosstalk takes place between closely related Rho GTPases.

Clostridium perfringens hip arthritis in a haemodialysis patient.

Haemodialysis patients have acquired immunity disturbances, co-morbidities and a vascular access, factors predisposing them to infection and bacteraemia. Clostridium perfringens is an anaerobic bacterium potentially causing severe infections, including rarely septic arthritis. We report the first case of Clostridium perfringens septic arthritis in a haemodialysis patient and suggest a haematogenous spread. After rapid joint lavage combined with appropriate anti-microbial therapy, the patient recovered.

RhoGDIα-dependent balance between RhoA and RhoC is a key regulator of cancer cell tumorigenesis.

RhoGTPases are key signaling molecules regulating main cellular functions such as migration, proliferation, survival, and gene expression through interactions with various effectors. Within the RhoA-related subclass, RhoA and RhoC contribute to several steps of tumor growth, and the regulation of their expression affects cancer progression. Our aim is to investigate their respective contributions to the acquisition of an invasive phenotype by using models of reduced or forced expression. The silencing of RhoC, but not of RhoA, increased the expression of genes encoding tumor suppressors, such as nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1), and decreased migration and the anchorage-independent growth in vitro. In vivo, RhoC small interfering RNA (siRhoC) impaired tumor growth. Of interest, the simultaneous knockdown of RhoC and NAG-1 repressed most of the siRhoC-related effects, demonstrating the central role of NAG-1. In addition of being induced by RhoC silencing, NAG-1 was also largely up-regulated in cells overexpressing RhoA. The silencing of RhoGDP dissociation inhibitor α (RhoGDIα) and the overexpression of a RhoA mutant unable to bind RhoGDIα suggested that the effect of RhoC silencing is indirect and results from the up-regulation of the RhoA level through competition for RhoGDIα. This study demonstrates the dynamic balance inside the RhoGTPase network and illustrates its biological relevance in cancer progression.

Knockdown of splicing factor SRp20 causes apoptosis in ovarian cancer cells and its expression is associated with malignancy of epithelial ovarian cancer.

Our previous study revealed that two splicing factors, polypyrimidine tract-binding protein (PTB) and SRp20, were upregulated in epithelial ovarian cancer (EOC) and knockdown of PTB expression inhibited ovarian tumor cell growth and transformation properties. In this report, we show that knockdown of SRp20 expression in ovarian cancer cells also causes substantial inhibition of tumor cell growth and colony formation in soft agar and the extent of such inhibition appeared to correlate with the extent of suppression of SRp20. Massive knockdown of SRp20 expression triggered remarkable apoptosis in these cells. These results suggest that overexpression of SRp20 is required for ovarian tumor cell growth and survival. Immunohistochemical staining for PTB and SRp20 of two specialized tissue microarrays, one containing benign ovarian tumors, borderline/low malignant potential (LMP) ovarian tumors as well as invasive EOC and the other containing invasive EOC ranging from stage I to stage IV disease, reveals that PTB and SRp20 are both expressed differentially between benign tumors and invasive EOC, and between borderline/LMP tumors and invasive EOC. There were more all-negative or mixed staining cases (at least two evaluable section cores per case) in benign tumors than in invasive EOC, whereas there were more all-positive staining cases in invasive EOC than in the other two disease classifications. Among invasive EOC, the majority of cases were stained all positive for both PTB and SRp20, and there were no significant differences in average staining or frequency of positive cancer cells between any of the tumor stages. Therefore, the expression of PTB and SRp20 is associated with malignancy of ovarian tumors but not with stage of invasive EOC.

RhoA-GDP regulates RhoB protein stability. Potential involvement of RhoGDIalpha.

RhoA plays a significant role in actin stress fibers formation. However, silencing RhoA alone or RhoA and RhoC did not completely suppress the stress fibers suggesting a residual "Rho-like" activity. RhoB, the third member of the Rho subclass, is a shortlived protein barely detectable in basal conditions. In various cell types, the silencing of RhoA induced a strong up-regulation of both total and active RhoB protein levels that were rescued by re-expressing RhoA and related to an enhanced half-life of the protein. The RhoA-dependent regulation of RhoB does not depend on the activity of RhoA but is mediated by its GDP-bound form. The stabilization of RhoB was not dependent on isoprenoid biosynthesis, Rho kinase, extracellular signal-regulated kinase, p38 mitogen-activated kinase, or phosphatidylinositol 3'-OH kinase pathways but required RhoGDIalpha. The forced expression of RhoGDIalpha increased RhoB half-life, whereas its knock-down antagonized the induction of RhoB following RhoA silencing. Moreover, a RhoA mutant (RhoAR68E) unable to bind RhoGDIalpha was significantly less efficient as compared with wild-type RhoA in reversing RhoB up-regulation upon RhoA silencing. These results suggest that, in basal conditions, RhoGDIalpha is rate-limiting and the suppression of RhoA makes it available to stabilize RhoB. Our results highlight RhoGDIalpha-dependent cross-talks that regulate the stability of RhoGTPases.

Establishment of stable human fibroblast cell lines constitutively expressing active Rho-GTPases.

Small GTP-binding proteins of the Rho family (RhoA, Cdc42, Rac1) regulate the organisation and the turnover of the cell's cytoskeleton and adhesion structures. A significant function of these cellular structures is to translate and counterbalance forces applied to, or generated by, cells in order to maintain homeostasis and control cell movement. We therefore hypothesised that Rho-GTPases are directly involved in cellular gravity perception and may participate in the alterations induced in microgravity. To define an adequate cellular model allowing to investigate this issue, we have established stable cell lines constitutively expressing active forms of either RhoA, Cdc42, or Rac1. The three cell lines differ by morphology and by their ability to form filopodia, lamellipodia, and bundles of actin stress fibers. Overexpression of the active form of either RhoA, Cdc42, or Rac1 is compatible with cell viability and does not affect cell population doubling time. Thus, our series of mutant cells appear well suited to gain further knowledge on the molecular mechanisms of cellular gravity perception.

Cdc42 downregulates MMP-1 expression by inhibiting the ERK1/2 pathway.

The small GTPases of the Rho family are key intermediates in cellular signalling triggered by activated cell-adhesion receptors. In this study, we took advantage of RNA interference (RNAi) using small interfering RNAs (siRNAs) to define the roles of the best-characterized members of the RhoGTPase family, RhoA, Rac1 and Cdc42, in the control of MMP-1, MMP-2 and type-I-collagen expression in normal human skin fibroblasts (HSFs). A specific and long-lasting repression, up to 7 days after transfection, of the three GTPases was achieved by transient transfection of specific siRNA. The silencing of Cdc42, but not that of RhoA or Rac1, induced a 15-fold increase in MMP-1 secretion. This upregulation was confirmed at the mRNA level and observed with two different siRNAs targeting Cdc42. Such a regulation was also observed in various human cell lines and was rescued by re-expressing wild-type Cdc42 encoded by a construct bearing silent mutations impeding its recognition by the siRNA. By contrast, MMP-2 and type-I-collagen expression was not affected by the individual silencing of each Rho GTPase. Cytokine protein array, enzyme-linked immunosorbent assays and reverse-transcription PCR measurements revealed that ablation of Cdc42 induced an overexpression of interleukin 8 and MCP-1. Although these cytokines are known to induce the expression of MMP-1, we showed that they were not involved in the Cdc42-mediated upregulation of MMP-1. Silencing of Cdc42 also induced an increased phosphorylation of ERK1/2 and p38 MAP kinase. The use of chemical inhibitors on Cdc42-ablated cells revealed that the upregulation of MMP-1 is dependent on the ERK1/2 pathways, whereas the p38 MAP kinase pathway displayed an inhibitory role. Simultaneous knock-down of two or three Rho GTPases allowed us to demonstrate that the RhoA-ROCK pathway was not involved in this regulation but that the silencing of Rac1 reduced the effect of Cdc42 suppression. These data suggest that, in vivo, when cell/extracellular-matrix interactions via integrins induce cytoskeleton organization, MMP-1 expression is maintained at a low level by Cdc42 via a repression of the Rac1 and ERK1/2 pathways. Therefore, Cdc42 contributes to ECM homeostasis and connective tissue integrity.

Low yield of chest radiography in screening for active pulmonary tuberculosis in HIV-infected patients in Hong Kong.

This study assessed the usefulness of routine chest radiography for detecting active pulmonary tuberculosis in persons infected with human immunodeficiency virus (HIV) without suggestive symptoms in Hong Kong. Tuberculosis is common in this locality and tuberculosis/HIV co-infection has been a frequent and significant problem. Records of patients attending the largest HIV clinic were reviewed. Three hundred and eleven routine chest radiographs were performed among 191 HIV-infected patients with a total follow-up period of 792 person years. Of the 22 routine chest radiographs that had abnormalities in the lungs or hilar region, only one had led to the diagnosis of pulmonary tuberculosis. No patient with a normal chest radiograph was diagnosed to have tuberculosis within the following 2 months. The low yield (0.32%) suggests that routine chest radiography is not useful in screening for active pulmonary tuberculosis in asymptomatic HIV-infected patients even in a locality where the tuberculosis rate is high.

Indinavir-associated facial lipodystrophy in HIV-infected patients.

The objectives of this study were to document the prevalence of facial lipodystrophy in patients with HIV infection receiving protease inhibitors and to identify associated factors. All patients with HIV infection receiving protease inhibitors seen at an HIV clinic in Hong Kong during a 2-month period, from August to October 1997, were assessed for facial lipodystrophy. Among 29 patients who had been receiving indinavir for 3 months or more, facial lipodystrophy was found in 7 (24%). Facial lipodystrophy in these patients was found to be an isolated event and was not associated with noticeable wasting elsewhere. The development of facial lipodystrophy was not found to be associated with age, sex, ethnicity, route of HIV transmission, CD4 cell count, history of AIDS-defining illness, or concurrent anti-retroviral treatment. Facial lipodystrophy was not observed in patients who had received indinavir for less than 3 months. The condition was also not found in patients taking other protease inhibitors, although this could be due to the small sample size. Prospective study of this condition with a larger sample and with objective anthropomorphic measurements would be desirable. In conclusion, facial lipodystrophy is a common occurrence among patients receiving indinavir, and physicians should be alerted to this condition.

HIV-associated eosinophilic folliculitis in a Chinese woman: a case report and a survey in Hong Kong.

This is the first survey of eosinophilic folliculitis (EF) in patients infected with the human immunodeficiency virus (HIV) in Hong Kong. The present report provides the local data on HIV-associated eosinophilic folliculitis (HIV-EF) and includes the first Chinese, heterosexual female patient with this condition. This is a retrospective study on all HIV-positive patients (n = 451) attending the outpatient clinic of the AIDS Unit in Hong Kong. Patients diagnosed as having EF with histological support were included for analysis. The data were presented by descriptive method. Three patients were identified, all of them had skin biopsies done which confirmed the diagnosis; including the female case. Recognition of HIV-EF is important because it is indicative of significant immunosuppression with risk of opportunistic infection. We concluded that HIV-EF is no longer an exclusive male disease in homosexual patients only. We expect more female patients or heterosexual subjects who are HIV positive developing this disease in the future.

PIP: A retrospective record review of HIV-associated eosinophilic pustular folliculitis (EF) cases in 451 HIV-positive patients attending an outpatient AIDS clinic in Hong Kong revealed the first female case of HIV-EF in a Chinese heterosexual woman. A 45-year-old HIV-positive woman presented with a 2-week history of pruritic papulopustular eruption on the face. Clinical examination revealed multiple erythematous follicular papules and pustules on her face and neck. Histologic examination of a skin biopsy indicated spongiosis and exocytosis in the epidermis involving infundibulum and sebaceous glands with micropustule formation. This patient's EF responded well to treatment with ultraviolet B phototherapy. This record review also revealed 2 HIV-EF cases in Chinese men (1 homosexual and 1 heterosexual). In previous series, HIV-EF has been found only in homosexual men.

Evaluation of leukocyte differential counts on the QBC centrifugal hematology analyzer according to NCCLS standard H20-T.

A performance study of the QBC centrifugal hematology system was conducted according to reference and analytical procedures defined in NCCLS Tentative Standard for Leukocyte Differential Counting, H20-T. A complex mathematical transformation and analysis of variance (ANOVA) were applied to the test data. This statistical technique is intended to segregate and emphasize differences in samples and methods. The comparative study of leukocyte differential counts show that QBC is equivalent in performance to the NCCLS reference method when the latter counts are grouped according to the WBC subpopulations reported by QBC. Within these grouped subpopulations, QBC counts were more precise than manual reference counts and otherwise comparable in terms of results. Similarly, QBC compared favorably with the reference method in its clinical sensitivity to abnormals, based on between-method versus within-method studies of hospital specimens.

Pre-precipitated and solid-phase second antibody compared in radioimmunoassay.

We describe simple methods for preparing and determining the titer of pre-precipitated second antibody. We compared the performance of this type of insolubilized second antibody with that of a commercially available solid-phase second antibody (DASP) for separation of reverse triiodothyronine (rT3) and for thyrotropin radioimmunoassays. Equilibrium time for both reagents was about 30 min, considerably briefer than for the usual second-antibody procedure. Within-assay precision was improved and percentages of nonspecific binding (mean and SD) were lower if pre-precipitated second antibody was used in both assays [rT3 pre-precipitate = 1.48 (SD 0.16), rT3 DASP = 3.76 (SD 0.22); thyrotropin pre-precipitate = 1.45 (SD 0.18), thyrotropin DASP = 3.45 (SD 0.26)]. The cost of DASP was three to 10 times that of pre-precipitated second antibody prepared with commercially available products.

Improved radioimmunoassay of platelet factor 4 and beta-thromboglobulin in plasma.

We describe rapid radioimmunoassays for both platelet factor 4 (PF-4) and beta-thromboglobulin (beta-TG) on the same plasma sample with use of pre-precipitated second antibody for the separation step. The assays are sensitive (B/B0 of 90% is obtained with 30 pg PF-4 and 100 pg beta-TG), specific, accurate, and reproducible. Labeled antigens are highly immunogenic, stable, and require no initial or periodic purification. Both assays require less than five hours for precount incubation times and can easily be completed in one day. Normal plasma values for PF-4 and beta-TG were 2.98 ng/ml and 25.83 ng/ml, respectively.