Kinase domain of the muscle-specific receptor tyrosine kinase (MuSK) is sufficient for phosphorylation but not clustering of acetylcholine receptors: Required role for the MuSK ectodomain?

Formation of the neuromuscular junction (NMJ) depends upon a nerve-derived protein, agrin, acting by means of a muscle-specific receptor tyrosine kinase, MuSK, as well as a required accessory receptor protein known as MASC. We report that MuSK does not merely play a structural role by demonstrating that MuSK kinase activity is required for inducing acetylcholine receptor (AChR) clustering. We also show that MuSK is necessary, and that MuSK kinase domain activation is sufficient, to mediate a key early event in NMJ formation—phosphorylation of the AChR. However, MuSK kinase domain activation and the resulting AChR phosphorylation are not sufficient for AChR clustering; thus we show that the MuSK ectodomain is also required. These results indicate that AChR phosphorylation is not the sole trigger of the clustering process. Moreover, our results suggest that, unlike the ectodomain of all other receptor tyrosine kinases, the MuSK ectodomain plays a required role in addition to simply mediating ligand binding and receptor dimerization, perhaps by helping to recruit NMJ components to a MuSK-based scaffold.     

IgMs produced by two acquired immune deficiency syndrome lymphoma cell lines: Ig binding specificity and VH-gene putative somatic mutation analysis [published erratum appears in Blood 1994 Aug 1;84(3):995]

Two B-cell lines, 2F7 and 10C9, were established by single cell cloning from biopsies obtained from two acquired immune deficiency syndrome patients with Burkitt's lymphoma. Representation of the original tumors was verified by demonstration of (1) identical biallelic rearrangement of Ig genes for 2F7 and (2) shared idiotype for 10C9. Both cell lines displayed cell-surface Ig and secreted Ig (IgM lambda for 2F7, IgM kappa for 10C9). IgMs from both cell lines immunoprecipitated actin; in addition, 2F7 IgM lambda immunoprecipitated recombinant human immunodeficiency virus type 1 (HIV-1) gp 160. 2F7 IgM lambda did not react with other autoantigens (double-stranded and single-stranded DNA, actin, bovine serum albumin, IgG), whereas 10C9 IgM kappa reacted with human IgG. The 2F7 IgM heavy-chain variable region (VH) showed a 95% nucleotide homology with a previously sequenced VHIII germline gene, hv3019b9, whereas the 10C9 IgM VH showed a 95% homology with a previously sequenced VHIV germline gene, VH4.21. Use of minimally modified VH genes and demonstration of reactivity with chronically present antigens (ie, actin, HIV-1 gp 160, or human IgG) suggests that B cells in HIV-1-infected individuals proliferating in response to chronic antigenic stimulation may be at increased risk for lymphomagenesis.     

Protection against methylglyoxal-derived AGEs by regulation of glyoxalase 1 prevents retinal neuroglial and vasodegenerative pathology

Aims/hypothesis

Methylglyoxal (MG) is an important precursor for AGEs. Normally, MG is detoxified by the glyoxalase (GLO) enzyme system (including component enzymes GLO1 and GLO2). Enhanced glycolytic metabolism in many cells during diabetes may overpower detoxification capacity and lead to AGE-related pathology. Using a transgenic rat model that overexpresses GLO1, we investigated if this enzyme can inhibit retinal AGE formation and prevent key lesions of diabetic retinopathy.

Methods

Transgenic rats were developed by overexpression of full length GLO1. Diabetes was induced in wild-type (WT) and GLO1 rats and the animals were killed after 12 or 24?weeks of hyperglycaemia. N ^??-(Carboxyethyl)lysine (CEL), N ^??-(carboxymethyl)lysine (CML) and MG-derived-hydroimidazalone-1 (MG-H1) were determined by immunohistochemistry and by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MSMS). Müller glia dysfunction was determined by glial fibrillary acidic protein (GFAP) immunoreactivity and by spatial localisation of the potassium channel Kir4.1. Acellular capillaries were quantified in retinal flat mounts.

Results

GLO1 overexpression prevented CEL and MG-H1 accumulation in the diabetic retina when compared with WT diabetic counterparts (p?GLO1 overexpression (p?GLO1 diabetic animals showed fewer acellular capillaries than WT diabetic animals (p?Conclusions/interpretation Detoxification of MG reduces AGE adduct accumulation, which, in turn, can prevent formation of key retinal neuroglial and vascular lesions as diabetes progresses. MG-derived AGEs play an important role in diabetic retinopathy.     

Active immunization of murine allogeneic bone marrow transplant donors with B-cell tumor-derived idiotype: a strategy for enhancing the specific antitumor effect of marrow grafts

Persistence of the underlying malignancy remains the major obstacle limiting the success of high-dose chemoradiotherapy with allogeneic bone marrow transplantation (BMT) for lymphomas and multiple myeloma. We used the C3H 38C13 murine B-cell lymphoma, which expresses and secretes clonally derived Ig, the idiotype of which can serve as a tumor-specific antigen, to test the principle of transfer of tumor idiotype-specific immunity with BM. BALB/c marrow donors were twice immunized with 38C13-derived Ig, or with an isotype-matched control Ig, conjugated to keyhole limpet hemocyanin. Lethally irradiated C3H recipients reconstituted with marrow from idiotype immune, but not nonspecifically immune, donors demonstrated protection against subsequent lethal tumor challenge. The immunoprotective effect of immune allogeneic marrow was abrogated by T-cell depletion of the marrow graft before infusion. Low levels of serum anti-idiotypic antibody remained unaltered in recipients of T-cell-depleted immune marrow, consistent with a primary role for T-cell immunity in the cellular mechanism of this phenomenon. A modest therapeutic effect of immune allogeneic marrow was observed against 10 day, 1 cm established subcutaneous tumors, but only in combination with a booster immunization of the recipient post-BMT. These results provide the rationale for a novel strategy for enhancing the specific antitumor effect of allogeneic marrow grafts.     

Epidemiology of tuberculosis and HIV coinfections in Singapore, 2000–2014

Cross‐matching of records between Singapore's tuberculosis and HIV registries showed that 3.3% of individuals with tuberculosis (TB) were coinfected with HIV (2000?2014), the TB incidence among individuals with HIV infection was 1.65 per 100 person‐years, and 53% of coinfections were diagnosed within 1 month of each other. The findings supported joint prevention programmes for early diagnosis and treatment.     

The adverse effect of treatment prolongation in cervical carcinoma

: Proliferation of surviving tumor clonogens during a course of protracted radiation therapy may be a cause of local failure in cervical carcinoma. The effect of total treatment time was analyzed retrospectively in relation to pelvic control and overall survival for squamous cell carcinomas of the uterine cervix.

: Two hundred and nine patients (Stage IB-IIIB) treated with a combination of external beam and low dose rate intracavitary irradiation were evaluable for study. Multivariate analysis and Kaplan-Meier statistical methods were used to determine the effect of treatment time on pelvic control and survival at 5 years.

: The median treatment duration was 55 days. For all stages combined, the 5-year survival and pelvic control rates were significantly different with treatment times < days vs. ≥ 55 days 65 and 54% (p = 0.03), 87 and 72% (p = 0.006), respectively. By stage, a shorter treatment duration (i.e., < 55 days vs. ≥ 55 days) was significant for 5-year overall survival and pelvic control for Stages IB/IIA and III, but not for Stage IIB: Stage IB/IIA (81 and 67%, and 84%), Stage III disease (52 and 42%, 76 and 55%) and Stage IIB (43 and 50%, 74 and 80%, respectively). Survival decreased 0.6%/day and pelvic control decreased 0.7%/day for each additional day of treatment beyond 55 days for all stages of disease. Additionally, significantly late complications were not influenced by treatment time.

: These results that prolongation of treatment time is associated with decreased local control and survival in patients with cervical carcinoma. This is consistent with emerging data from other institutions. Therapeutic implications include avoidance of unnecessary treatment breaks, the design of fractionation schemes that decrease treatment duration, and possibly the use of tumor cytostatic drugs during conventional radiation.     

Effect of off-pump coronary artery bypass grafting on risk-adjusted and cumulative sum failure outcomes after coronary artery surgery

BACKGROUND AND AIM: We have shown that cumulative sum (CUSUM) failure analysis may be more sensitive than standard statistical methods in detecting a cluster of adverse patient outcomes after cardiac surgical procedures. We therefore applied CUSUM, as well as standard statistical techniques, to analyze a surgeon's experience with off-pump coronary artery bypass grafting (OPCAB) and on-pump procedures to determine whether the two techniques have similar or different outcomes. METHODS: In 320 patients undergoing nonemergent, first time coronary artery bypass grafting, preoperative patient characteristics, rates of mortality and major complications, and ICU and hospital lengths of stay were compared between the on-pump and OPCAB cohorts using Fisher's exact tests and Wilcoxon two sample tests. Predicted mortality and length of stay were determined using previously validated models of the Cardiac Care Network of Ontario. Observed versus expected ratios of both variables were calculated for the two types of procedures. Furthermore, CUSUM curves were constructed for the on-pump and OPCAB cohorts. A multivariable analysis of the predictors of hospital length of stay was also performed to determine whether the type of coronary artery bypass procedure had an independent impact on this variable. RESULTS: The predicted mortality risk and predicted hospital length of stay were almost identical in the 208 on-pump patients (2.2 +/- 3.9%; 8.2 +/- 2.5 days) and the 112 OPCAB patients (2.0 +/- 2.2%; 7.8 +/- 2.1 days). The incidence of hospital mortality and postoperative stroke were 2.9% and 2.4% in on-pump patients versus zero in OPCAB patients (p = 0.09 and 0.17, respectively). Mechanical ventilation for greater than 48 hours was significantly less common in OPCAB (1.8%) than in on-pump patients (7.7%, p = 0.04). The rate of 10 major complications was 14.9% in on-pump versus 8.0% in OPCAB patients (p = 0.08). OPCAB patients experienced a hospital length of stay that was a median of 1.0 day shorter than on-pump patients (p = 0.01). The observed versus expected ratio for length of stay was 0.78 in OPCAB patients versus 0.95 in on-pump patients. On CUSUM analysis, the failure curve in OPCAB patients was negative and was flatter than that of on-pump patients throughout the duration of the study. Furthermore, OPCAB was an independent predictor of a reduced hospital length of stay on multivariable analysis. CONCLUSIONS: OPCAB was associated with better outcomes than on-pump coronary artery bypass despite a similar predicted risk. This robust finding was documented on sensitive CUSUM analysis, using standard statistical techniques and on a multivariable analysis of the independent predictors of hospital length of stay.