Laparoscopic versus robotic TAPP/TEP inguinal hernia repair: a multicenter, propensity score weighted study.

PURPOSE: The objective of this retrospective study was to assess safety and comparative clinical effectiveness of laparoscopic inguinal hernia repair (LIHR) and robot-assisted inguinal hernia repair (RIHR) from multi-institutional experience in Taiwan. METHODS: Medical records from a total of eight hospitals were retrospectively collected and analyzed. Patients primarily diagnosed of inguinal hernia, recurrent inguinal hernia or incarceration groin hernia patients who either underwent laparoscopic or robot-assisted inguinal hernia repair between January 2018 and December 2022 were included in the study. Baseline characteristics, intra-operative and post-operative results were analyzed. To compare two cohorts, overlap weighting was employed to balance the significant inter-group differences. We also conducted subgroup analyses by state of a hernia (primary or recurrent/incarceration) and laterality (unilateral or bilateral) that indicated complexity of surgery. RESULTS: A total of 1,080 patients who underwent minimally invasive inguinal hernia repair from 8 hospitals across Taiwan were collected. Following the application of inclusion criteria, there were 279 patients received RIHR and 763 patients received LIHR. In the baseline analysis, RIHR was more often performed in recurrent/incarceration (RIHR 18.6% vs LIHR 10.3%, p = 0.001) and bilateral cases (RIHR 81.4 vs LIHR 58.3, p < 0.001). Suturing was dominant mesh fixation method in RIHR (RIHR 81% vs LIHR 35.8%, p < 0.001). More overweight patients were treated with RIHR (RIHR 58.8% vs LIHR 48.9%, p = 0.006). After overlap weighting, there were no significant difference in intraoperative and post-operative complications between RIHR and LIHR. Reoperation and prescription rates of pain medication (opioid) were significantly lower in RIHR than LIHR in overall group comparison (reoperation: RIHR 0% vs. LIHR 2.9%, p = 0.016) (Opioid prescription: RIHR 3.34 mg vs LIHR 10.82 mg, p = 0.001) while operation time was significantly longer in RIHR (OR time: RIHR 155.27 min vs LIHR 95.30 min, p < 0.001). CONCLUSIONS: This real-world experience suggested that RIHR is a safe, and feasible option with comparable intra-operative and post-operative outcomes to LHIR. In our study, RIHR showed technical advantages in more complicated hernia cases with yielding to lower reoperation rates, and less opioid use.

Immunogenicity of the mRNA-1273 and ChAdOx1 nCoV-19 vaccines in Asian patients with autoimmune rheumatic diseases under biologic and/or conventional immunosuppressant treatments.

OBJECTIVE: Nucleic acid-based vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection are effective in the general population. However, it is unknown whether this is true in Asian patients with autoimmune rheumatic diseases (ARDs) who have received various combinations of disease-modifying anti-rheumatic drugs (DMARDs). METHOD: We designed a large prospective observational study recruiting 228 patients with ARDs in a tertiary rheumatology centre in Taiwan. Altogether, 142 received biological or targeted synthetic DMARDs and 86 received only conventional synthetic (cs) DMARDs. Serum levels of immunoglobulin G antibody against SARS-CoV-2 spike proteins were measured 2-6 weeks after COVID-19 vaccination with mRNA-1273 (Moderna®) or ChAdOx1 nCoV-19 (Oxford/AstraZeneca®). The immunomodulatory therapies were not modified before or after vaccination. RESULTS: Overall, 194 patients (85.09%) exhibited antibodies (758.33 ± 808.43 ng/mL) but 34 patients did not (103.24 ± 41.08 ng/mL). Patients with systemic lupus erythematosus or rheumatoid arthritis had significantly lower humoral responses to COVID-19 vaccination than those with other ARDs (p < 0.05). There was no significant difference in immunogenicity among patients on different csDMARD treatments. Compared to patients treated with only csDMARDs, those on rituximab or abatacept therapy had significantly lower immune response to the vaccination (p = 0.008 and p = 0.035, respectively). Patients who were treated with anti-tumour necrosis factor-α or interleukin-6 inhibitor exhibited higher titres of vaccination antibodies than those treated with direct lymphocyte inhibitors. CONCLUSIONS: mRNA-1273 and ChAdOx1 nCoV-19 vaccines were immunogenic in the majority of ARD patients. Rituximab and abatacept were associated with significantly diminished COVID-19 vaccination immunogenicity.

Protective role of γδ T cells in cigarette smoke and influenza infection.

Airborne pathogens commonly trigger severe respiratory failure or death in smokers with lung disease. Cigarette smoking compromises the effectiveness of innate immunity against infections but the underlying mechanisms responsible for defective acquired immune responses in smokers remains less clear. We found that mice exposed to chronic cigarette smoke recovered poorly from primary Influenza A pneumonia with reduced type I and II interferons (IFNs) and viral-specific immunoglobulins, but recruited γδ T cells to the lungs that predominantly expressed interleukin 17A (IL-17A). Il-17a-/- mice exposed to smoke and infected with Influenza A also recruited γδ T cells to the lungs, but in contrast to wild-type mice, expressed increased IFNs, made protective influenza-specific antibodies, and recovered from infection. Depletion of IL-17A with blocking antibodies significantly increased T-bet expression in γδ T cells and improved recovery from acute Influenza A infection in air, but not smoke-exposed mice. In contrast, when exposed to smoke, γδ T cell deficient mice failed to mount an effective immune response to Influenza A and showed increased mortality. Our findings demonstrate a protective role for γδ T cells in smokers and suggest that smoke-induced increase in IL-17A inhibits the transcriptional programs required for their optimal anti-viral responses. Cigarette smoke induces IL-17A expression in the lungs and inhibits γδ T-cell-mediated protective anti-viral immune responses.

New strategy for the analysis of phenotypic marker antigens in brain tumor-derived neurospheres in mice and humans.

OBJECT: Brain tumor stem cells (TSCs) hypothetically drive the malignant phenotype of glioblastoma multiforme (GBM), and evidence suggests that a better understanding of these TSCs will have profound implications for treating gliomas. When grown in vitro, putative TSCs grow as a solid sphere, making their subsequent characterization, particularly the cells within the center of the sphere, difficult. Therefore, the purpose of this study was to develop a new method to better understand the proteomic profile of the entire population of cells within a sphere. METHODS: Tumor specimens from patients with confirmed GBM and glioma models in mice were mechanically and enzymatically dissociated and grown in traditional stem cell medium to generate neurospheres. The neurospheres were then embedded in freezing medium, cryosectioned, and analyzed with immunofluorescence. RESULTS: By sectioning neurospheres as thinly as 5 mum, the authors overcame many of the problems associated with immunolabeling whole neurospheres, such as antibody penetration into the core of the sphere and intense background fluorescence that obscures the specificity of immunoreactivity. Moreover, the small quantity of material required and the speed with which this cryosectioning and immunolabeling technique can be performed make it an attractive tool for the rapid assessment of TSC character. CONCLUSIONS: This study is the first to show that cryosectioning of neurospheres derived from glioma models in mice and GBM in humans is a feasible method of better defining the stem cell profile of a glioma.

Activation of protein phosphatase-2A1 by HIV-1 Vpr cell death causing peptide in intact CD(4+) T cells and in vitro.

HIV-1, the etiologic agent of human AIDS, causes cell death in host and non-host cells via HIV-1 Vpr, one of its auxiliary gene product. HIV-1 Vpr can also cause cell cycle arrest in several cell types. The cellular processes that link HIV-1 Vpr to the cell death machinery are not well characterized. Here, we show that the C terminal portion of HIV-1 Vpr which encompasses amino acid residues 71-96 (HIV-1 Vpr(71-96)), also termed HIV-1 Vpr cell death causing peptide, is an activator of protein phosphatase-2A(1) when applied extracellularly to CD(4+) T cells. HIV-1 Vpr(71-96) is a direct activator of protein phosphatase-2A(1) that has been purified from CD(4+) T cells. Full length HIV-1 Vpr by itself does not cause the activation of protein phosphatase-2A(1) in vitro. HIV-1 Vpr(71-96) also causes the activation of protein phosphatase-2A(0) and protein phosphatase-2A(1) from brain, liver, and adipose tissues. These results indicate that HIV-1 can cause cell death of infected cells and non-infected host and non-host cells via HIV-1 Vpr derived C terminal peptide(s) which act(s) by cell penetration and targeting of a key controller of the cell death machinery, namely, protein phosphatase-2A(1). The activation of other members of the protein phosphatase-2A subfamily of enzymes which are involved in the control of several metabolic pathways in brain, liver, and adipose tissues by HIV-1 Vpr derived C terminal peptide(s) may underlie various metabolic disturbances that are associated with HIV-1 infection.

Direct activation of protein phosphatase-2A0 by HIV-1 encoded protein complex NCp7:vpr.

The effects of HIV-1 encoded proteins NCp7, vpr and NCp7:vpr complex on the activity of protein phosphatase-2A0 have been tested. We report that NCp7 is an activator of protein phosphatase-2A0 and that vpr activated protein phosphatase-2A0 only slightly. We also report that NCp7 and vpr form a tight complex which becomes a more potent activator of protein phosphatase-2A0 than NCp7 alone. The ability of NCp7 to activate protein phosphatase-2A0 is regulated by vpr. The C-terminal portion of vpr prevents NCp7 from activating protein phosphatase-2A0 while the N-terminal portion of vpr potentiates the effect of NCp7 on the activity of protein phosphatase-2A0. Our findings indicate that vpr may be acting as a targeting subunit which directs NCp7 to activate protein phosphatase-2A0. In view of the fact that protein phosphatase-2A functions as an inhibitor of G0 to M transition of the cell cycle and is involved in other key cellular processes such as the control of RNA transcription, the results presented in this report may explain how HIV-1 causes cell cycle arrest which may lead to CD4+ T cell depletion and also how it disturbs normal cellular processes of its host cell.

The v-rel oncogene product is complexed to a 40-kDa phosphoprotein in transformed lymphoid cells.

The transforming protein encoded by the v-rel oncogene of avian reticuloendotheliosis virus (REV-T) is a very low copy number molecule in the cytosol of transformed cells. Analysis of cytosolic extracts from a REV-T-transformed lymphoid cell line by gel filtration on Sephacryl S-300 indicated that most of the v-rel oncogene product, pp59v-rel, eluted with an apparent molecular mass of 400 kDa. The size of this complex was confirmed by analysis on a fast-protein liquid chromatography gel filtration column. A 40-kDa cellular protein copurified with pp59v-rel on sequential gel filtration on Sephacryl S-200 and immunoaffinity chromatography with a monoclonal antibody directed against pp59v-rel. The 40-kDa cellular protein could also be immunoprecipitated together with pp59v-rel from cell extracts of [35S]methionine-labeled cells, suggesting that pp59v-rel is complexed with the 40-kDa protein in transformed lymphoid cells. Both the 59- and 40-kDa proteins were phosphorylated when the highly purified preparation containing pp59v-rel was incubated with [gamma-32P]ATP and 10 mM MgCl2 in vitro. The identity of the kinase in the highly purified preparation containing pp59v-rel, however, is unknown. Immune complexes recovered from extracts of REV-T-transformed lymphoid cells labeled with [32P]orthophosphate also contained the 59- and 40-kDa phosphoproteins. These observations suggest that pp59v-rel is complexed with a 40-kDa cellular phosphoprotein to form a 400-kDa heteropolymer in the cytoplasm of transformed lymphoid cells.

Serine phosphorylation of the v-rel oncogene product/pp40 complex.

The transforming protein encoded by the v-rel oncogene of reticuloendotheliosis virus has been purified from REV-T transformed lymphoid cells by sequential DEAE-Sepharose and immunoaffinity chromatography. The purified preparation consisted of pp59v-rel and the 40 kDa cellular protein which is complexed with the v-rel oncogene product in transformed cells as well as some minor proteins. Incubation of this purified preparation in the presence of Mg2+ and (gamma-32P)ATP resulted in phosphorylation of both pp59v-rel and the 40 kDa protein. This preparation was also able to phosphorylate casein on serine residues. Immunoprecipitates obtained from extracts of REV-T transformed lymphoid cells labeled with 32P-orthophosphate contained 59 and 40 kDa phosphoproteins. Both pp59v-rel and the 40 kDa protein were phosphorylated on serine residues in transformed cells.

The protein phosphatases involved in cellular regulation. Comparison of native and reconstituted Mg-ATP-dependent protein phosphatases from rabbit skeletal muscle.

The 'native' Mg-ATP-dependent protein phosphatase was isolated from rabbit skeletal muscle by a procedure that avoided the use of organic solvents or heating at 90-100 degrees C. The purified enzyme was composed of two major proteins (molecular mass 37 kDa and 31 kDa) that were present in a 1:1 molar ratio, and accounted for 70-80% of the material. The 37-kDa component comigrated with the catalytic subunit of protein phosphatase-1, and its identity with this protein was established by peptide mapping, and by its cleavage to the characteristic 34-kDa and 33-kDa fragments following incubation with chymotrypsin. The 31-kDa protein comigrated with inhibitor-2, and its identity with this protein was established by its heat stability, ability to inhibit protein phosphatase-1 at nanomolar concentrations, and its phosphorylation on a threonine residue by glycogen synthase kinase 3. It is therefore concluded that the 'native' Mg-ATP-dependent protein phosphatase is composed of the catalytic subunit of protein phosphatase-1 (37 kDa) and inhibitor-2 (31 kDa) in a 1:1 molar ratio. The 'native' Mg-ATP-dependent protein phosphatase had virtually identical properties to the enzyme reconstituted from inhibitor-2 and the 37-kDa catalytic subunit of protein phosphatase-1. Each preparation had a similar specific activity and was inhibited by identical concentrations of inhibitor-1. Both enzymes could be activated by incubation with glycogen synthase kinase-3 and Mg-ATP, or by Mn2+ and trypsin (or chymotrypsin). However, Mn2+ alone, or proteinase digestion in the absence of Mn2+, failed to activate either preparation. Incubation with glycogen synthase kinase-3 and Mg-ATP did not dissociate the 'native' or 'reconstituted' enzymes, whereas treatment with Mn2+ and trypsin decreased their apparent molecular masses from 70 kDa to 35 kDa. Incubation with chymotrypsin converted the 'native' and 'reconstituted' enzymes to forms that required preincubation with glycogen synthase kinase-3, Mg-ATP and inhibitor-2, in order to exhibit catalytic activity. The Mg-ATP-dependent protein phosphatase reconstituted from the 'nicked' 33-kDa catalytic subunit dissociated upon activation, in contrast to the enzyme reconstituted from the undegraded 37-kDa catalytic subunit. The results suggest that a 3-4-kDa fragment at one end of the polypeptide is involved in strengthening interaction between the undegraded 37-kDa catalytic subunit and the phosphorylated form of inhibitor-2.

DARPP-32, a dopamine-regulated neuronal phosphoprotein, is a potent inhibitor of protein phosphatase-1.

The neurotransmitter dopamine has been demonstrated by biochemical, histochemical and immunocytochemical techniques to be unevenly distributed in the mammalian central nervous system. DARPP-32 (dopamine- and cyclic-AMP-regulated phosphoprotein of molecular weight 32,000) is a neuronal phosphoprotein that displays a regional distribution in the mammalian brain very similar to that of dopamine-containing nerve terminals, being highly concentrated in the basal ganglia. The state of phosphorylation of DARPP-32 can be regulated by dopamine and by cyclic AMP in intact nerve cells, suggesting a role for this phosphoprotein in mediating certain of the effects of dopamine on dopaminoceptive cells. The observation that many of the physical and chemical properties of purified DARPP-32 resemble those of phosphatase inhibitor-1 (inhibitor-1), a widely distributed inhibitor of protein phosphatase-1, suggests that DARPP-32 might also function as a phosphatase inhibitor. We report here that DARPP-32 inhibits protein phosphatase-1 at nanomolar concentrations. Moreover, like inhibitor-1, DARPP-32 is effective as an inhibitor in its phosphorylated but not its dephosphorylated form. Thus, the basal ganglia of mammalian brain contain a region-specific neuronal phosphoprotein that is a protein phosphatase inhibitor.

The catalytic subunits of protein phosphatase-1 and protein phosphatase 2A are distinct gene products.

Three forms of protein phosphatase-1 were isolated from rabbit skeletal muscle that had Mr values of 37 000, 34 000 and 33 000 determined by sodium dodecyl sulphate (SDS) gel electrophoresis. Each species dephosphorylated the beta-subunit of phosphorylase kinase very much faster than the alpha-subunit, was inhibited by inhibitors 1 and 2 with equal potency, and was converted to a form dependent on glycogen synthase kinase-3 and Mg-ATP for activity by incubation with inhibitor-2. Digestion with cyanogen bromide or Staphylococcus aureus proteinase followed by SDS gel electrophoresis showed a very similar pattern of cleavage products for all three forms. The Mr-37 000 and Mr-34 000 species were converted to the Mr-33 000 form by incubation with chymotrypsin. It is concluded that the Mr-33 000 and Mr-34 000 forms are derived from the Mr-37 000 component by limited proteolysis. Conversion of the Mr-37 000 to the Mr-33 000 form was accompanied by a two-fold increase in activity, indicating that an Mr-4000 fragment at one end of the polypeptide is an inhibitory domain that decreases enzyme activity. The catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle had an Mr of 36 000 determined by SDS gel electrophoresis and its specific activity (3 kU/mg) was much lower than that of the Mr-37 000 (15-20 kU/mg) or Mr-33/34 000 (40-50 kU/mg) forms of protein phosphatase-1. It dephosphorylated the alpha-subunit of phosphorylase kinase 4-5-fold faster than the beta-subunit, was unaffected by inhibitor-1 or inhibitor-2, and preincubation with the latter protein did not result in the production of a glycogen synthase kinase-3 and Mg-ATP-dependent form of the enzyme. Digestion with chymotrypsin did not alter the electrophoretic mobility of protein phosphatase 2A under conditions that caused quantitative conversion of the Mr-37 000 form of protein phosphatase-1 to the Mr-33 000 species. Digestion with cyanogen bromide or S. aureus proteinase, followed by SDS gel electrophoresis, showed a quite different pattern of cleavage products to those observed with protein phosphatase 1. Antibody to protein phosphatase-2A raised in sheep did not cross-react with any of the forms of protein phosphatase-1, as judged by immunoelectrophoretic and immunotitration experiments. It is concluded that protein phosphatase-1 and protein phosphatase-2A are distinct gene products.

Characterisation of a reconstituted Mg-ATP-dependent protein phosphatase.

Homogenous preparations of the catalytic subunit of protein phosphatase-1 and inhibitor-2 can be combined to produce an inactive enzyme that consists of a 1:1 complex between these two proteins. This species is indistinguishable from the Mg-ATP-dependent protein phosphatase in that preincubation with glycogen synthase kinase-3 and Mg-ATP is required to generate activity. Activation results from the phosphorylation of inhibitor-2. The molar concentrations of protein phosphatase-1 and inhibitor-2 in rabbit skeletal muscle (0.25-0.5 microM) are similar. Incubation of the reconstituted Mg-ATP-dependent protein phosphatase with chymotrypsin is accompanied by limited proteolysis of inhibitor-2 and the loss of its phosphorylation site(s). This species can be activated by glycogen synthase kinase-3 and Mg-ATP provided that inhibitor-2 is added. This exogenous inhibitor-2 appears to displace the fragments of inhibitor-2 from the enzyme that were generated by chymotryptic digestion. These experiments may explain the report [Yang, S.D., Vandenheede, J.R. and Merlevede, W. (1981) J. Biol. Chem. 256, 10231-10234] that inhibitor-2 can function as an 'activator' as well as an inhibitor of the Mg-ATP-dependent protein phosphatase. Incubation of the catalytic subunit of protein phosphatase-1 with sodium fluoride or sodium pyrophosphate converted the enzyme to an inactive form that could be partially reactivated by manganese ions, but not by glycogen synthase kinase-3 and Mg-ATP. Conversely, the reconstituted Mg-ATP-dependent protein phosphatase could only be activated by glycogen synthase kinase-3 and Mg-ATP, and not by manganese ions. It is concluded that the conversion of protein phosphatase-1 to a manganese-ion dependent form is a quite separate phenomenon from the formation of the Mg-ATP-dependent protein phosphatase. Inhibitor-2 can inactivate protein phosphatase-1 by a second mechanism that is not reversed by preincubation with glycogen synthase kinase-3 and Mg-ATP. This occurs at higher concentrations of inhibitor-2 than those required to form the Mg-ATP-dependent protein phosphatase, and appears to result from the binding of inhibitor-2 to a distinct site on the enzyme.