Learning and implementation of TransREctus sheath PrePeritoneal procedure for inguinal hernia repair.

PURPOSE: The TransREctus sheath PrePeritoneal procedure (TREPP) was introduced as an alternative open and preperitoneal technique for inguinal hernia mesh repair, demonstrating safety and efficacy in retro- and prospective studies. However, little is known about the technique's inherent learning curve. In this study, we aimed to determine TREPP learning curve effects after its implementation in high-volume surgical practice. METHODS: All primary, unilateral TREPP procedures performed in the first three years after implementation (between January 2016 and December 2018) were included out of a large preconstructed regional inguinal hernia database. Data were analyzed on outcome (i.e., surgical complications, hernia recurrences, postoperative pain). Learning curve effects were analyzed by assessing outcome in relation to surgeon experience. RESULTS: In total, 422 primary, unilateral TREPP procedures were performed in 419 patients. In three patients a unilateral TREPP procedure was performed on both sides separated in time. A total of 99 surgical complications were registered in 83 procedures (19.6% of all procedures), most commonly inguinal postoperative pain (8%) and bleeding complications (7%). Hernia recurrences were observed in 17 patients (4%). No statistically significant differences on outcome were found between different surgeon experience (< 40 procedures, 40-80 procedures, > 80 procedures). CONCLUSION: Implementation of TREPP seems not to be associated with a notable increase of adverse events. We were not able to detect a clear learning curve limit, potentially suggesting a relatively short learning curve among already experienced hernia surgeons compared to other guideline techniques.

Transrectus sheath pre-peritoneal (TREPP) procedure versus totally extraperitoneal (TEP) procedure and Lichtenstein technique: a propensity-score-matched analysis in Dutch high-volume regional hospitals.

PURPOSE: Results of the most commonly used inguinal hernia repair techniques often originate from expert centers or from randomized controlled studies. In this study, we portray daily-practice results of a high-volume, regional surgical group in the Netherlands, comparing TREPP (open (posterior) transrectus sheath pre-peritoneal) with Lichtenstein (open anterior) and TEP (endoscopic (posterior) totally extraperitoneal). We hypothesize that the TREPP shows more favorable outcome compared to the current gold standard procedures: TEP and Lichtenstein. METHODS: Between January 2016 and December 2018, 3285 consecutive patients underwent surgical treatment and were included for analysis. The outcome measures were postoperative pain, recurrence rate and other surgical complications. Propensity-score matching was used to address potential selection bias. RESULTS: After propensity-score matching, there was no statistically significant difference in postoperative pain in the TREPP group compared to the Lichtenstein group (TREPP 7.3% versus Lichtenstein 6.3%; p = 0.67) nor in TREPP compared to TEP (TREPP 7.4% versus TEP 4.1%; p = 0.064). There was no statistically significant difference in recurrences in the TREPP group compared to Lichtenstein (3.8% vs 2.5%; p = 0.42), nor in the TREPP versus TEP comparison (3.9% vs 2.8%; p = 0.55) CONCLUSION: This study compares TREPP with Lichtenstein and TEP in the presence of postoperative pain, recurrences and other adverse outcomes. After propensity-score matching, no statistically significant difference in postoperative pain or recurrences remained between either TREPP compared to Lichtenstein, or TREPP compared to TEP. Based on these results, TREPP, Lichtenstein and TEP showed comparable results in postoperative pain, recurrences and other surgical site complications.

Nodal promotes invasive phenotypes via a mitogen-activated protein kinase-dependent pathway.

The progression of cancer from localized to invasive disease is requisite for metastasis, and is often characterized by epithelial-to-mesenchymal transition (EMT) and alterations in cellular adhesion and migration. Studies have shown that this transition is associated with an upregulation of embryonic stem cell-associated genes, resulting in a dedifferentiated phenotype and poor patient prognosis. Nodal is an embryonic factor that plays a critical role in promoting early invasive events during development. Nodal is silenced as stem cells differentiate; however, it re-emerges in adult life during placentation and mammary gland development, and is aberrantly expressed in many cancers. Here, we show that Nodal overexpression, in poorly invasive breast cancer and choriocarcinoma cells, causes increased invasion and migration in vitro. Furthermore, we show that Nodal overexpression in these epithelial cancer types induces an EMT-like event concomitant with the internalization of E-Cadherin. This ability of Nodal to promote cellular invasion and EMT-like phenomena is dependent upon the phosphorylation of ERK1/2. As Nodal normally signals through SMADs, these findings lend insight into an alternative pathway that is hijacked by this protein in cancer. To evaluate the clinical implications of our results, we show that Nodal inhibition reduces liver tumor burden in a model of spontaneous breast cancer metastasis in vivo, and that Nodal loss-of-function in aggressive breast cancer lines results in a decrease in invasive phenotypes. Our results demonstrate that Nodal is involved in promoting invasion in multiple cellular contexts, and that Nodal inhibition may be useful as a therapeutic target for patients with progressive disease.

GSK-3ß function in bone regulates skeletal development, whole-body metabolism, and male life span.

Glycogen synthase kinase 3 ß (GSK-3ß) is an essential negative regulator or "brake" on many anabolic-signaling pathways including Wnt and insulin. Global deletion of GSK-3ß results in perinatal lethality and various skeletal defects. The goal of our research was to determine GSK-3ß cell-autonomous effects and postnatal roles in the skeleton. We used the 3.6-kb Col1a1 promoter to inactivate the Gsk3b gene (Col1a1-Gsk3b knockout) in skeletal cells. Mutant mice exhibit decreased body fat and postnatal bone growth, as well as delayed development of several skeletal elements. Surprisingly, the mutant mice display decreased circulating glucose and insulin levels despite normal expression of GSK-3ß in metabolic tissues. We showed that these effects are due to an increase in global insulin sensitivity. Most of the male mutant mice died after weaning. Prior to death, blood glucose changed from low to high, suggesting a possible switch from insulin sensitivity to resistance. These male mice die with extremely large bladders that are preceded by damage to the urogenital tract, defects that are also seen type 2 diabetes. Our data suggest that skeletal-specific deletion of GSK-3ß affects global metabolism and sensitizes male mice to developing type 2 diabetes.

Intrapancreatic delivery of human umbilical cord blood aldehyde dehydrogenase-producing cells promotes islet regeneration.

AIMS/HYPOTHESIS: We sought to investigate the stimulation of islet regeneration by transplanted human umbilical cord blood (UCB) cells purified according to high aldehyde dehydrogenase (ALDH) activity (ALDH(hi)), a conserved characteristic of multiple progenitor lineages. We hypothesised that direct intrapancreatic (iPan) delivery of ALDH(hi) progenitors would augment islet regeneration via timely and localised exposure to islet-regenerative stimuli. METHODS: Cells were purified from UCB based on flow cytometry for low ALDH activity (ALDH(lo)) vs ALDH(hi). UCB ALDH(lo) or ALDH(hi) cells were compared for surface marker expression, as well as haematopoietic, endothelial and multipotent stromal progenitor content in vitro. UCB ALDH(lo) or ALDH(hi) cells were i.v. or iPan injected into streptozotocin-treated non-obese diabetic/severe combined immune-deficient mice temporally monitored for blood glucose, serum insulin and glucose tolerance. Human cell recruitment and survival in the pancreas, insulin content, islet-associated cell proliferation and islet vascularisation were documented in situ. RESULTS: UCB-derived ALDH(hi) cells were highly enriched for haematopoietic and endothelial progenitor frequency, and showed increased expression of progenitor and myeloid cell surface markers. Although i.v. transplantation of ALDH(hi) cells demonstrated low pancreas engraftment and only transient blood glucose lowering capacity, iPan injected ALDH(hi) cells reversed established hyperglycaemia, increased serum insulin and improved the response to a glucose challenge. iPan injected ALDH(hi) cells surrounded damaged islets at early time points and increased islet-associated cell proliferation, resulting in the recovery of beta cell mass. CONCLUSIONS/INTERPRETATION: iPan delivery of UCB ALDH(hi) cells potentiated islet-associated cell proliferation, insulin production and islet revascularisation, resulting in the recovery of host islet function. Elucidation of the progenitor-specific pathways stimulated during islet regeneration may provide new approaches to promote islet expansion during diabetes.

Coculture and transplant of purified CD34(+)Lin(-) and CD34(-)Lin(-) cells reveals functional interaction between repopulating hematopoietic stem cells.

The human hematopoietic stem cell compartment is comprised of repopulating CD34(+) and CD34(-) cells. The interaction between these subsets with respect to their reconstitution capacity in vivo remains to be characterized. Here, lineage-depleted (Lin(-)) human CD34(+) and CD34(-) hematopoietic cells were isolated from human male and female umbilical cord blood (CB) and transplanted into immune-deficient NOD/SCID EMV(null) mice, thereby allowing the use of human and Y-chromosome-specific DNA sequences to discriminate human reconstitution contributed by CD34(+) vs CD34(-) repopulating stem cells. Although cultured human CB CD34(-)Lin(-) cells transplanted alone possessed only minimal repopulating capacity, with 15% of mice achieving low levels of engraftment, transplantation of cocultured male CD34(-)Lin(-) cells with female CD34(+)Lin(-) cells demonstrated human repopulation with a contribution from CD34(-)Lin(-)-derived progeny in 80% of the recipients. After coculture and transplantation, male CD34(-)Lin(-) cells gave rise to primitive CD34(+)CD38(-) cells isolated in vivo, which demonstrated clonogenic progenitor function into multiple lineages. Taken together, our study indicates that the presence of CD34(+)Lin(-) cells in coculture enhanced the low repopulating function of human CD34(-)Lin(-) cells in vivo. We propose that CD34(+)Lin and CD34(-)Lin cells represent phenotypically distinct, but related cell types that exhibit unique and previously unappreciated functional interaction.

Ambulatory sentinel node biopsy under local anaesthesia for patients with early breast cancer.

AIMS: Sentinel lymph node biopsy (SLNB) may permit reliable identification of patients with axillary node involvement. The aim of this study was to report our experience with this procedure under local anaesthesia. METHODS: One hundred and sixty-two patients underwent a sentinel node procedure under local anaesthesia without sedation. The SLN was identified by (99m)Tc-nano-colloid and patent blue. Immediate histopathologic examination and immunohistochemistry was performed. Patients with positive SLNs proceded to axillary dissection under general anaesthesia. RESULTS: In all 162 patients the SLN ('s) were found using blue dye and gamma-probe. The SLN was positive in 55/162 patients (34%). Five of these were detected using immunohistochemistry only. CONCLUSIONS: A 100% detection rate of sentinel nodes in early breast cancer harvested under local anaesthesia was achieved without serious morbidity. This allows the surgeon to select preoperatively the treatment given to the patient.

The human hematopoietic stem cell compartment is heterogeneous for CXCR4 expression.

The chemokine stromal derived factor-1alpha (SDF-1alpha) has been implicated recently in the chemotaxis of primitive human hematopoietic cells, suggesting that pluripotent human stem cells express the SDF-1alpha receptor, CXCR4. By using flow cytometry and confocal microscopy, we have identified and isolated primitive subsets of human CXCR4(+) and CXCR4(-) cells. Distinctions in the progenitor content and response to SDF-1alpha in vitro indicate that CXCR4(+) and CXCR4(-) cells represent discrete populations of primitive blood cells. The i.v. transplantation of these subfractions into immune-deficient mice established that both possess comparable engraftment capacity in vivo. Human myeloid, lymphoid, and primitive CD34(+) CXCR4(+) cells were present in chimeric animals transplanted with either subset, indicating that CXCR4(+) and CXCR4(-) stem cells have equivalent proliferative and differentiative abilities. Our study indicates that the human stem cell compartment is heterogeneous for CXCR4 expression, suggesting that the relationship between CXCR4 expression and stem cell repopulating function is not obligatory.

Cytotoxicity of sulfonamide reactive metabolites: apoptosis and selective toxicity of CD8(+) cells by the hydroxylamine of sulfamethoxazole.

Treatment with sulfonamide antibiotics in HIV-infected patients is associated with a high incidence (> 40%) of adverse drug events, including severe hypersensitivity reactions. Sulfonamide reactive metabolites have been implicated in the pathogenesis of these adverse reactions. Sulfamethoxazole hydroxylamine (SMX-HA) induces lymphocyte toxicity and suppression of proliferation in vitro; the mechanism(s) of these immunomodulatory effects remain unknown. We investigated the cytotoxicity of SMX-HA via apoptosis on human peripheral blood mononuclear cells and purified cell subpopulations in vitro. CD19(+), CD4(+), and CD8(+) cells were isolated from human peripheral blood by positive selection of cell surface molecules by magnetic bead separation. SMX-HA induced significant CD8(+) cell death (67 +/- 7%) at 100 microM SMX-HA, with only minimal CD4(+) cell death (8 +/- 4%). No significant subpopulation toxicity was shown when incubated with parent drug (SMX). Flow cytometry measuring phosphatidylserine externalization 24 h after treatment with 100 microM and 400 microM SMX-HA revealed 14.1 +/- 0.7% and 25. 6 +/- 4.2% annexin-positive cells, respectively, compared to 3.7 +/- 1.2% in control PBMCs treated with 400 microM SMX. Internucleosomal DNA fragmentation was observed in quiescent and stimulated PBMCs 48 h after incubation with SMX-HA. Our data show that CD8(+) cells are highly susceptible to the toxic effects of SMX-HA through enhanced cell death by apoptosis.

Pretreatment with glucocorticoids enhances T-cell effector function: possible implication for immune rebound accompanying glucocorticoid withdrawal.

Glucocorticoids (GCs) exert their immunosuppressive/antiproliferative effects largely through inhibition of cytokine expression, and paradoxically upregulate the expression of (proinflammatory) cytokine receptors on select nonlymphoid cells. Clinically, withdrawal of GCs was frequently associated with worsening of the outcome of heightened immunity disorders, thereby implicating enhanced cytokine and cytokine receptor expression as a possible consequence of acute/short-term GCs withdrawal. In view of the significance of this complication of GC therapy, we addressed the effect of GC withdrawal on cytokine receptor expression and subsequent T-cell effector function, using the proliferation of human T cells as biological readout. To mimic GC withdrawal, T cells were treated with GCs or controls, stimulated, and incubated for 16-20 h at 37 degrees C, washed, and reactivated for a further 4-48 h. Surface marker expression was assessed by FACS analysis, and proliferation was determined by measuring the cellular uptake of tritiated thymidine. Dexamethasone (DEX) and prednisolone (PRED), in a concentration-dependent manner, inhibited T-cell proliferation induced by anti-CD28 Ab + PMA. However, pretreatment of T cells activated with mitogens, cross-linking antibodies, or PMA + ionomycin ("CD3-bypass" stimulation regimen), but not resting T cells, with DEX or PRED resulted in a marked increase in IL-IR, IL-2R alpha, and IL-6R expression, which was accompanied by a significant enhancement in T-cell proliferation. This effect of GCs was neither stimulus specific nor did it result from alteration in cell viability, and was paralleled by augmentation in cytokine (rIL-2) effects on DEX-pretreated and preactivated T cells. Taken together, our results underline the dual effects of GCs in regulating T-cell activation and cytokine expression. In essence, GCs directly inhibited T-cell proliferation by suppressing cytokine production, and, by enhancing cytokine receptor expression, pretreatment with GCs augmented T-cell proliferation.

Multiplicity of glucocorticoid action in inhibiting allograft rejection.

Glucocorticoids (GCs) are used as immunosuppressive and antiinflammatory agents in organ transplantation and in treating autoimmune diseases and inflammatory disorders. GCs were shown to exert their antiproliferative effects directly through blockade of certain elements of an early membrane-associated signal transduction pathway, modulation of the expression of select adhesion molecules, and by suppression of cytokine synthesis and action. GCs may act indirectly by inducing lipocortin synthesis, which in turn, inhibits arachidonic acid release from membrane-bound stores, and also by inducing transforming growth factor (TGF)-beta expression that subsequently blocks cytokine synthesis and T cell activation. Furthermore, by preferentially inhibiting the production of Th1 cytokines, GCs may enhance Th2 cell activity and, hence, precipitate a long-lasting state of tolerance through a preferential promotion of a Th2 cytokine-secreting profile. In exerting their antiproliferative effects, GCs influence both transcriptional and posttranscriptional events by binding their cytosolic receptor (GR), which subsequently binds the promoter region of cytokine genes on select DNA sites compatible with the GCs responsible elements (GRE) motif. In addition to direct DNA binding, GCs may also directly bind to, and hence antagonize, nuclear factors required for efficient gene expression, thereby markedly reducing transcriptional rate. The pleiotrophy of the GCs action, coupled with the diverse experimental conditions employed in assessing the GCs effects, indicate that GCs may utilize more than one mechanism in inhibiting T cell activation, and warrant careful scrutiny in assigning a mechanism by which GCs exert their antiproliferative effects.

The role of reactive drug metabolites in immune-mediated adverse drug reactions.

OBJECTIVE: To highlight recent advances in the understanding of adverse drug reactions (ADRs), with a focus on models outlining interactions between drug metabolism, disease processes, and immunity. Specific mechanisms that identify the metabolic pathways responsible for drug bioactivation to reactive drug metabolites (RDMs) involved in the initiation and propagation of specific immune-mediated hypersensitivity reactions are discussed. Drug classes well known to be associated with immune-mediated ADRs are reviewed and the clinical implications of current research are discussed. DATA SOURCES: Original experimental research and immunologic review articles relevant to ADR diagnosis and etiology. DATA EXTRACTION: Results of relevant in vitro experiments and clinical reactions to drug therapy were compiled and reviewed. Critical discoveries concerning the identification of RDMs involved in ADRs were highlighted, with respect to RDM involvement in the production of an immune response to drug haptens. DATA SYNTHESIS: Drug adverse effects are classified according to clinical characteristics, immune interactions, and mechanistic similarities. Cytochrome P450 bioactivation of drug molecules to RDMs is a prerequisite to many ADRs. An electrophilic metabolite may react with cellular macromolecules (i.e., lipids, proteins, nucleic acids), resulting in direct cellular damage and organ toxicity. Covalent binding of an RDM to cellular macromolecules may also result in the formation of a hapten that is capable of eliciting a cellular or humoral immune response against drug or protein epitopes, culminating in the characteristic symptoms of hypersensitivity reactions. Mechanistic details concerning the identification of stable protein-metabolite conjugates and their interaction with the immune system remain unclear. Genetic imbalance between bioactivation and detoxification pathways, as well as reduced cellular defense against RDMs due to disease or concomitant drug therapy, act as risk factors to the onset and severity of ADRs. CONCLUSIONS: Adverse reactions to drug therapy cause significant morbidity and mortality. Identification of the pathways involved in drug bioactivation and detoxification may elucidate the potential of chemical agents to induce immune-mediated ADRs. Understanding the mechanisms of ADRs to current xenobiotics is helpful in the prevention and management of ADRs, and may prove useful in the design of novel therapeutic agents with reduced incidence of severe adverse events.

The hydroxylamine of sulfamethoxazole synergizes with FK506 and cyclosporin A, inhibiting T-cell proliferation.

We previously demonstrated the capacity of the hydroxylamine metabolite of sulfamethoxazole (SMX-HA) to inhibit mitogen-induced T-cell proliferation. We studied the interaction of SMX-HA with the immuno-suppressants cyclosporin A (CsA), FK506 and rapamycin. Human peripheral blood mononuclear leukocytes were treated with SMX-HA and combined in culture with CsA or FK506 or rapamycin. The cells were stimulated with phytohaemaglutinin, and phorbol myristate acetate and proliferation was determined by cellular uptake of 3H-thymidine. Using median-effect analysis and concentration reduction index calculations to assess immunosuppressive drug interactions, we produced synergistic immunosuppression by SMX-HA/CsA and SMX-HA/FK506. Concentration reductions at the 50% inhibitory level of over 46-fold and 64-fold with CsA and FK506, respectively, were observed with 25 microM SMX-HA, and this effect was not associated with reduced cell viability. SMX-HA failed to augment the suppressive capacity of rapamycin in inhibiting mitogen-induced cellular proliferation. SMX-HA at immunosuppressive concentrations also failed to interfere with interleukin-2 mRNA transcription and interleukin-2 protein production, which suggests that signaling events proximal to cytokine production are not affected by the metabolite. Synergy between SMX-HA/FK506 and SMX-HA/CsA suggests that the mechanism(s) of action of reactive sulfonamide metabolites may occur in later stages of lymphocyte activation.

Integration of clinical and administrative strategies to reduce expenditures for antimicrobial agents.

A comprehensive program of clinical and administrative strategies to reduce expenditures for antimicrobial agents is described. Clinical intervention strategies include the use of antimicrobial order sheets, standardized dosage regimens, restriction policies for certain antimicrobial agents, and position statements on the use of restricted agents. A cornerstone of the program is the support for cost-reduction interventions offered by the pharmacy and therapeutics committee and its subcommittee on therapeutics; that support is demonstrated through endorsement and enforcement of pharmacy programs. Physicians are reminded of the cost-reduction programs through periodic articles in the pharmacy newsletter and an "antibiogram" card supplied by the division of epidemiology. The effectiveness of these interventions has been demonstrated by progressive decreases in expenditures for antimicrobial agents during 1987 and 1988. Antimicrobial agents also account for increasingly smaller percentages of the total drug budget. This combination of clinical and administrative strategies reduced expenditures for antimicrobial agents by more than $700,000 over two years without the use of clinical specialists or any apparent sacrifice in the quality of patient care.

Comparison of aspartame- and fructose-sweetened layer cakes: importance of panels of users for evaluation of alternative sweeteners.

Panelists with and without known carbohydrate metabolic diseases evaluated layer cakes sweetened with aspartame, alone or in combination with low levels of fructose, for texture and flavor. Panelists used a 5-point, descriptive rating scale to evaluate flavor and texture of lemon, orange, spice, and chocolate layer cakes baked in conventional and microwave ovens. Panelists judged that aspartame alone was not suitable in layer cakes. In general, healthy panelists evaluated the cakes as sweeter, crust bitterness as greater, and overall eating quality as higher than the panel members with carbohydrate metabolic disorders. Panelists did not differ in their evaluation of textural qualities.

A new experimental approach to assessment of velopharyngeal adequacy: nasal manometric bleed testing.

A new experimental method for assessment of velopharyngeal adequacy/inadequacy, using nasal manometric pressure readings for three types of speech conditions under controlled incremental bleed conditions, is described. Cleft palate speakers (N = 30) tended to have higher nasal pressure readings than non-flect palate speakers (N = 56), with or without bleed conditions. Reduction in nasal pressure under controlled incremental bleed for non-cleft palate speakers and cleft palate adequate (CPA) speakers is compatible with an aerodynamic quadratic equation. Unlike cleft palate inadequate (CPI) speakers, non-cleft palate speakers and CPA speakers exhibit similar ability to achieve 0 cm nasal monometric pressure under a bleed bore diameter of 4 mm for a circular bleed orifice. Results for cleft palate marginal (CPM) speakers are less conclusive. Reliability of nasal manometric bleed testing was limitedly tested but not conclusively established in the present report. Further study of reliability and validity is necessary.