The TL MHC class Ib molecule has only marginal effects on the activation, survival and trafficking of mouse small intestinal intraepithelial lymphocytes

Thymus leukemia antigen (TL) is an MHC class Ib molecule that is highly conserved in rats and mice with no obvious human homolog. TL is expressed in mouse small intestinal epithelial cells and is known to interact with CD8alphaalpha homodimers, which are expressed by intraepithelial lymphocytes (IELs), some other T cell subsets and some non-T cells such as a subset of dendritic cells. We show here that TL is abundantly expressed on the basolateral surface of mouse small intestinal epithelial cells and that expression is abrogated in beta2m-/- mice but unaffected in TCR-/- mice or CD8alpha chain-/- mice. We demonstrate that the interaction between TL and CD8alphaalpha is not necessary for IEL survival in vitro or in vivo and does not modulate IEL trafficking in vivo. TL co-stimulation of alpha-CD3 antibody-activated IELs resulted in modestly enhanced production of IFN-gamma in one subset of IELs. The lack of effect on IEL survival and trafficking and the modest effect on IFN-gamma production suggest that the functional consequences of TL interaction with CD8alphaalpha as well as the more general biological role of TL in mucosal immunity remains to be discovered.     

Production of multiple murine CD2 receptor constructs using the baculovirus expression vector and a rapid dot-blot assay

The baculovirus expression system was used to produce three different constructs of the murine cell surface adhesion receptor CD2. One construct coded for a single, N-terminal, Ig-fold domain. It was inefficiently secreted and therefore primarily intracellular. The second construct coded for both extracellular, N-terminal Ig-fold domains. This was efficiently secreted into culture supernatant. The third construct coded for the full-length transmembrane molecule which localized to the cell surface. All constructs were monomers of predicted MW_r and were appropriately glycosylated. They retained epitopic specificity as demonstrated by binding to mAbs, and adhesion function as demonstrated by a rosetting assay.     

Complement-dependent synergistic effects of rat monoclonal IgG antibodies in vivo

We describe studies aimed at maximizing the effector mechanisms responsible for eliminating target erythrocytes from the circulation in a fully homologous opsonization system in vivo. The effects on the subsequent fate of target erythrocytes were examined in both normal and decomplemented rats preinjected with a variety of rat IgG monoclonal antibodies (mAb) directed against different epitopes on the RTlA^a, the classical class I major histocompatibiliy complex antigen of the DA rat. In general, the clearance of both DA and (DA × PVG)F₁ erythrocytes in normal rats preinjected with various pairs of noncompetitive mAb was very rapid when compared with the overall clearance patterns seen with individual antibodies. With all mAb combinations containing IgG2b or IgG2a, an intact complement system was an essential requirement for augmenting the initial clearance and promoting hepatic sequestration of these target cells. The removal of (DA × PVG)F₁ erythrocytes, expressing half as much antigen, was considerably slower than the DA cells for each antibody pair tested although a notable degree of heterogeneity was observed in the overall behavior of both types of target cells with different mAb combinations. Our results suggest that the limiting effects of low antigen density on the target cells combined with the use of mAb of an isotype like the rat IgG2a can be overcome using pairs of mAb that recognize different epitopes on the same target antigen.     

Glibenclamide treatment in a Cantú syndrome patient with a pathogenic ABCC9 gain‐of‐function variant: Initial experience

Cantú syndrome (CS), characterized by hypertrichosis, distinctive facial features, and complex cardiovascular abnormalities, is caused by pathogenic variants in ABCC9 and KCNJ8 genes. These genes encode gain‐of‐function mutations in the regulatory (SUR2) and pore‐forming (Kir6.1) subunits of K_ATP channels, respectively, suggesting that channel‐blocking sulfonylureas could be a viable therapy. Here we report a neonate with CS, carrying a heterozygous ABCC9 variant (c.3347G>A, p.Arg1116His), born prematurely at 32 weeks gestation. Initial echocardiogram revealed a large patent ductus arteriosus (PDA), and high pulmonary pressures with enlarged right ventricle. He initially received surfactant and continuous positive airway pressure ventilation and was invasively ventilated for 4 weeks, until PDA ligation. After surgery, he still had ongoing bilevel positive airway pressure (BiPAP) requirement, but was subsequently weaned to nocturnal BiPAP. He was treated for pulmonary hypertension with Sildenafil, but failed to make further clinical improvement. A therapeutic glibenclamide trial was commenced in week 11 (initial dose of 0.05 mg^–1 kg^–1 day^–1 in two divided doses). After 1 week of treatment, he began to tolerate time off BiPAP when awake, and edema improved. Glibenclamide was well tolerated, and the dose was slowly increased to 0.15 mg^?1 kg^?1day^?1 over the next 12 weeks. Mild transient hypoglycemia was observed, but there was no cardiovascular dysfunction. Confirmation of therapeutic benefit will require studies of more CS patients but, based on this limited experience, consideration should be given to glibenclamide as CS therapy, although problems associated with prematurity, and complications of hypoglycemia, might limit outcome in critically ill neonates with CS.     

Optimal field splitting for large intensity-modulated fields

The multileaf travel range limitations on some linear accelerators require the splitting of a large intensity-modulated field into two or more adjacent abutting intensity-modulated subfields. The abutting subfields are then delivered as separate treatment fields. This workaround not only increases the treatment delivery time but it also increases the total monitor units (MU) delivered to the patient for a given prescribed dose. It is imperative that the cumulative intensity map of the subfields is exactly the same as the intensity map of the large field generated by the dose optimization algorithm, while satisfying hardware constraints of the delivery system. In this work, we describe field splitting algorithms that split a large intensity-modulated field into two or more intensity-modulated subfields with and without feathering, with optimal MU efficiency while satisfying the hardware constraints. Compared to a field splitting technique (without feathering) used in a commercial planning system, our field splitting algorithm (without feathering) shows a decrease in total MU of up to 26% on clinical cases and up to 63% on synthetic cases.     

Immunophenotypic changes and clinical outcome in B-cell lymphomas treated with rituximab.

Rituximab is a chimeric monoclonal antibody that recognizes the CD20 antigen and is used to treat B-cell non-Hodgkin lymphoma (B-NHL). Few studies have been published examining the use of antibody panels to evaluate B-NHL treated with rituximab. The authors performed a retrospective analysis of immunophenotypic changes and clinical outcome in 18 patients with B-NHL following rituximab therapy. The intensity of CD20 expression was evaluated by flow cytometry and/or immunohistochemistry, before and after rituximab therapy; the latter samples were taken 5 to 12 months after initiating rituximab therapy (median 7 months). Nine of the 18 patients (50%) achieved complete or partial clinical remission and did not have morphologic evidence of lymphoma in the post-therapy samples. The other nine patients (50%) had persistent disease. Two patterns of CD20 expression were noted in the post-therapy samples: unchanged expression of CD20 in neoplastic cells (4/9 cases) and loss of or a significant decrease in detected CD20 expression in neoplastic cells (5/9 cases). These results show that in many cases of B-NHL persisting after rituximab therapy, CD20 expression decreases or is lost, raising the possibility of deletion or expression modulation of the CD20 gene in neoplastic cells. This study also underscores the importance of using a panel of antibodies to evaluate rituximab-treated B-NHL.     

Effect of glass composition on the degradation properties and ion release characteristics of phosphate glass--polycaprolactone composites

A series of polycaprolactone and ternary-based (Na(2)O)(0.55-x)(CaO)(x)(P(2)O(5))(0.45) glass composites were created, each containing 20% volume percentage of glass with various calcium compositions. A short-term degradation study was carried out to investigate the physical and ion release behaviour of these composites, utilising analytical techniques such as dynamical mechanical analysis, and ion chromatography. All the composites experienced significant loss of weight and stiffness throughout the study, with the 24 mol% calcium composites losing the greatest amount of weight and stiffness. The pH profile of the aqueous solutions in which the composites were placed were initially acidic, but began to neutralise mid-way through the study, with the 36 mol% solution achieving the most acidic conditions. The ion release behaviour mirrored the mass loss behaviour of the glass component of the composites. The cations (sodium and calcium ions) release was comparable with the initial stages of composite mass degradation, both of which exhibited almost immediate release when placed into solution. The 24 mol% composites underwent rapid rates of cation release, while the 36 mol% experienced the slowest rates of release. By contrast, anion (phosphates and polyphosphates) release showed a dissimilar trend, with rapid release of the P(2)O(7) and P(3)O(10) occurring during the first few hours in solution, whilst the P(3)O(9) structure released steadily during the first 48 h in solution. Finally, PO(4) release was at a constant rate over the duration of the study, releasing up to 300 ppm from the 32 and 36 mol% samples by the end of 200 h. To summarise, these results show that by combining phosphate glasses with biodegradable polymer, it is possible to create composites whose rate of degradation can be controlled to meet the needs of their end application.