Identification of class I major histocompatibility complex encoded molecules in the amphibian Xenopus

Class I-like molecules have been immunoprecipitated from Xenopus leukocytes and erythrocytes with alloantisera directed against major histocompatibility complex (MHC)-linked antigens. The heavy chains, depending on the allele examined, have molecular weights of 40 000–44 000 of which 3000 daltons are asparagine-linked carbohydrates, probably present as one N-linked glycan. The presumed analogue of ₂-microglobulin has a molecular weight of 13 000 and bears no asparagine-linked glycans. Family studies show that the heavy chains are encoded by genes residing in or closely linked to the MHC.Abbreviations used in this paper MHC major histocompatibility complex - CML cell-mediated lympholysis - MLR mixed leukocyte reaction - APBS amphibian phosphate-buffered saline - kd kilodalton - LG Xenopus laevis — Xenopus gilli species hybrids - IEF isoelectric focusing Founded and supported by F. Hoffmann-La Roche & Co., Limited Company, CH-4005 Basel, Switzerland.     

Air travel and incidence of pneumothorax in lymphangioleiomyomatosis

Background

Pulmonary lymphangioleiomyomatosis (LAM) is a rare disease of women characterized by multiple lung cysts leading to respiratory insufficiency and frequent pneumothorax (PT). Air travel (AT) could increase the risk of PT in LAM through rupture of subpleural cysts induced by atmospheric pressure changes in aircraft cabin. To determine whether AT increases the risk of PT in LAM, we performed a retrospective survey of members of European LAM patient associations. A flight-related PT was defined as occurring ≤30?days after AT.

Results

145 women reported 207 PT. In 128 patients with available data, the annual incidence of PT was 8% since the first symptoms of LAM and 5% since LAM diagnosis, compared to 0.006% in the general female population. Following surgical or chemical pleurodesis, the probability of remaining free of PT recurrence was respectively 82, 68, and 59% after 1, 5 and 10?years, as compared to only 55, 46 and 39% without pleurodesis (p?=?0.026). 70 patients with available data performed 178 AT. 6 flight-related PT occurred in 5 patients. PT incidence since first symptoms of LAM was significantly higher ≤30?days after AT as compared to non-flight periods (22 versus 6%, risk ratio 3.58, confidence interval 1.40–7.45).

Conclusions

The incidence of PT in LAM is about 1000 times higher than in the general female population, and is further increased threefold after AT. Chemical or surgical pleurodesis partly reduces the risk of PT recurrence in LAM.

     

Immunogenetic studies on the cell-mediated cytotoxicity in the clawed toadXenopus laevis

In order to test for further homologies between the MHC of mammals and amphibians, experiments were conducted to assess whether lower vertebrates such as anurans were able to generate killer cells after allogeneic stimulation. The generation of cytotoxic effector cells could be obtained in outbred families and clones of isogenic frogs after in vivo priming with either irradiated allogeneic lymphocytes or with an allogeneic skin graft, provided that the immune spleen cells were restimulated in vitro with the specific irradiated cells used for priming. Effector cells generated against a definedMHC haplotype could lyse targets having one of their two haplotypes in common with the stimulators. In contrast, no lysis was observed when the target cells differed from the specific stimulators by twoMHC haplotypes.The cytotoxic activity of the MLR-restimulated lymphocytes appeared to be mediated by T cells since passage of the effector spleen cells through a nylon wool column, under conditions which removedXenopus B lymphocytes, improved killing on a per cell basis. It therefore appears that the genes responsible for the highly specialized function of T killer cells have emerged early in evolution at least at the time of the emergence of the amphibians (300 million years ago) and that they were already linked to the MHC of this species. The MHC polymorphism inXenopus seems to be lower than in mammals as evidenced by the high frequency of cross-killing observations paralleled by the high frequency of MLR identical animals found in a large outbred population.     

tBid mediated activation of the mitochondrial death pathway leads to genetic ablation of the lens in Xenopus laevis

Xenopus is a well proven model for a wide variety of developmental studies, including cell lineage. Cell lineage in Xenopus has largely been addressed by injection of tracer molecules or by micro-dissection elimination of blastomeres. Here we describe a genetic method for cell ablation based on the use of tBid, a direct activator of the mitochondrial apoptotic pathway. In mammalian cells, cross-talk between the main apoptotic pathways (the mitochondrial and the death domain protein pathways) involve the pro-death protein BID, the active form of which, tBID, results from protease truncation and translocation to mitochondria. In transgenic Xenopus, restricting tBID expression to the lens-forming cells enables the specific ablation of the lens without affecting the development of other eye structures. Thus, overexpression of tBid can be used in vivo as a tool to eliminate a defined cell population by apoptosis in a developing organism and to evaluate the degree of autonomy or the inductive effects of a specific tissue during embryonic development.     

Fine needle aspiration of breast carcinoma: a preliminary cytoprognostic study

A cytologic grading method for fine needle aspiration smears was applied to 178 histologically confirmed breast carcinomas. Grade I defined a well-differentiated carcinoma, grade II a carcinoma with pleomorphic tumor cells and grade II an anaplastic carcinoma. The cell-to-cell relationship (topography) and the cytologic criteria contributed to the grading. Special attention was paid to grade III tumors, which have an unfavorable prognosis. The correlation of grading with the clinical course of the disease was evaluated after a 12-month followup. In 4% of the patients classified as having grade I disease, 8.9% as having grade II and 66% as having grade III, local recurrence of disease, metastasis or death was observed within one year. The contribution of cytologic grading to the prognosis of breast cancers is discussed.     

Patterns of residual HIV-1 RNA shedding in the seminal plasma of patients on effective antiretroviral therapy

Background

More and more HIV-1-infected men on effective antiretroviral treatment (ART) have unprotected sex in order to procreate. The main factor influencing transmission is seminal HIV shedding. While the risk of HIV transmission is very low, it is difficult to assess in individuals. Nevertheless, it should be quantified.

Results

We retrospectively analysed seminal plasma HIV-1 shedding by 362 treated HIV-infected men attending a medically assisted reproduction centre (1998–2013) in order to determine its frequency, the impact of the antiretroviral regimen on HIV shedding, and to identify shedding patterns. The HIV-1 virus loads in 1396 synchronized blood and semen samples were measured, and antiretroviral treatment, biological and epidemiological data were recorded.We detected isolated HIV-1 shedding into the seminal plasma in 5.3% of patients on efficient antiretroviral treatment, but there was no association with the HIV antiretroviral drug regimen or the CD4 cell count. These men had undergone more regimen changes since treatment initiation and had been on the ongoing drug regimen longer than the non-shedding men. The patterns of HIV seminal shedding among patients with undetectable HIV blood virus load varied greatly. HIV seminal shedding can occur as long as 5 years after starting antiretroviral treatment.

Conclusions

The seminal HIV load was used to monitor risk for infertile HIV-infected patients on an assisted reproductive technology program. This can still be recommended for patients who recently (6 months) started ART, or those with a poor history of adherence to ART but may also be usefull for some patients during counselling. Residual HIV seminal shedding is probably linked to breaks in adherence to antiretroviral treatment but local genital factors cannot be ruled out.

     

Harmonization of the Volume of Interest Delineation among All Eleven Radiotherapy Centers in the North of France

Background

Inter-observer delineation variation has been detailed for many years in almost every tumor location. Inadequate delineation can impair the chance of cure and/or increase toxicity. The aim of our original work was to prospectively improve the homogeneity of delineation among all of the senior radiation oncologists in the Nord-Pas de Calais region, irrespective of the conditions of practice.

Methods

All 11 centers were involved. The first studied cancer was prostate cancer. Three clinical cases were studied: a low-risk prostate cancer case (case 1), a high-risk prostate cancer case (pelvic nodes, case 2) and a case of post-operative biochemical elevated PSA (case 3). All of the involved physicians delineated characteristically the clinical target volume (CTV) and organs at risk. The volumes were compared using validated indexes: the volume ratio (VR), common and additional volumes (CV and AV), volume overlap (VO) and Dice similarity coefficient (DSC). A second delineation of the same three cases was performed after discussion of the slice results and the choice of shared guidelines to evaluate homogenization. A comparative analysis of the indexes before and after discussion was conducted using the Wilcoxon test for paired samples. A p-value less than 0.05 was considered to indicate statistical significance.

Results

The indexes were not improved in case 1, for which the inter-observer agreement was considered good after the first comparison (DSC = 0.83±0.06). In case 2, the second comparison showed homogenization of the CTV delineation with a significant improvement in CV (81.4±11.7 vs. 88.6±10.26, respectively, p = 0.048), VO (0.41±0.09 vs. 0.47±0.07, respectively; p = 0.009) and DSC (0.58±0.09 vs. 0.63±0.07, respectively; p = 0.0098). In case 3, VR and AV were significantly improved: VR: 1.71(±0.6) vs. 1.34(±0.46), respectively, p = 0.0034; AV: 46.58(±14.50) vs. 38.08(±15.10), respectively, p = 0.0024. DSC was not improved, but it was already superior to 0.6 in the first comparison.

Conclusion

Our prospective work showed that a collaborative discussion about clinical cases and the choice of shared guidelines within an established framework improved the homogeneity of CTV delineation among the senior radiation oncologists in our region.     

Impaired Humoral Immunity and Tolerance in K14-VEGFR-3-Ig Mice That Lack Dermal Lymphatic Drainage

Lymphatic vessels transport interstitial fluid, soluble Ag, and immune cells from peripheral tissues to lymph nodes (LNs), yet the contribution of peripheral lymphatic drainage to adaptive immunity remains poorly understood. We examined immune responses to dermal vaccination and contact hypersensitivity (CHS) challenge in K14-VEGFR-3-Ig mice, which lack dermal lymphatic capillaries and experience markedly depressed transport of solutes and dendritic cells from the skin to draining LNs. In response to dermal immunization, K14-VEGFR-3-Ig mice produced lower Ab titers. In contrast, although delayed, T cell responses were robust after 21 d, including high levels of Ag-specific CD8(+) T cells and production of IFN-γ, IL-4, and IL-10 upon restimulation. T cell-mediated CHS responses were strong in K14-VEGFR-3-Ig mice, but importantly, their ability to induce CHS tolerance in the skin was impaired. In addition, 1-y-old mice displayed multiple signs of autoimmunity. These data suggest that lymphatic drainage plays more important roles in regulating humoral immunity and peripheral tolerance than in effector T cell immunity.     

Determination of sodium cromoglycate in human plasma by liquid chromatography–mass spectrometry in the turbo ion spray mode

A highly sensitivity liquid chromatography–tandem mass spectrometry method has been developed for the quantitation of sodium cromoglycate (SCG) in human plasma. The method was validated over a linear range of 0.100–50.0 ng/ml, using ¹³C₄ sodium cromoglycate as the internal standard. Compounds were extracted from 1.0 ml of lithium heparin plasma by methanol elution of C₁₈ solid-phase extraction cartridges. The dried residue was reconstituted with 100 μl of 0.01 N HCl, and 30 μl was injected onto the LC–MS–MS system. Chromatographic separation was achieved on a C8 (3.5 μm) column with an isocratic mobile phase of methanol–water–0.5 M ammonium acetate (35:64.8:0.2, v/v/v). The analytes were detected with a PE Sciex API 3000 mass spectrometer using turbo ion spray with positive ionization. Ions monitored in the multiple reaction monitoring (MRM) mode were m/z 469.2 (precursor ion) to m/z 245.1 (product ion) for SCG and m/z 473.2 (precursor ion) to m/z 247.1 (product ion) for ¹³C₄ SCG (I.S.). The average recoveries of SCG and the I.S. from human plasma were 91 and 87%, respectively. The low limit of quantitation was 0.100 ng/ml. Results from a 4-day validation study demonstrated excellent precision (C.V.% values were between 1.9 and 6.5%) and accuracy (−5.4 to −1.2%) across the calibration range of 0.100–50.0 ng/ml.