Acceleration of idiopathic pneumonia syndrome (IPS) in the absence of donor MIP-1 alpha (CCL3) after allogeneic BMT in mice

Idiopathic pneumonia syndrome (IPS) is a significant cause of morbidity and mortality after bone marrow transplantation (BMT) in humans. We developed a murine IPS model in which lethal pre-BMT conditioning and allogeneic T cells results in the recruitment of host monocytes and then donor T cells into the lung by day 7 after BMT, concomitant with development of severe lung dysfunction. We reported the T cell-dependent production of the T cell-attracting chemokine macrophage inflammatory protein-1 alpha (MIP-1 alpha) in the lungs of such recipient mice. We reasoned that MIP-1 alpha might be a critical mediator of IPS. Lethally conditioned mice received transplants of major histocompatibility complex-disparate marrow and either wild-type (MIP-1 alpha(+/+)) or knockout (MIP-1 alpha(-/-)) spleen cells. Recipients of MIP-1 alpha(-/-) cells exhibited accelerated mortality and a decrease in specific compliance that appeared earlier than in recipients of MIP-1 alpha(+/+) cells. Donor CD4(+) and CD8(+) T cell expansion was increased in the spleens of recipients of MIP-1 alpha(-/-) cells. Lungs of recipients of MIP-1 alpha(-/-) cells had earlier recruitment of both T-cell subsets by day 3 after BMT, concomitant with the influx of cells expressing the cytolysins granzymes A and B. Monocyte recruitment was not altered. Levels of inflammatory cytokines were not increased and levels of T cell-attracting chemokines were decreased. The level of the anti-inflammatory cytokine interleukin 13 (IL-13) was lower in the serum and lungs of recipients of MIP-1 alpha(-/-) cells, indicating a skewing toward a more inflammatory T helper cell type 1 (Th1) cytokine milieu. Donor-derived MIP-1 alpha may play a role in allogeneic-induced IPS by limiting aggressive expansion of CD4(+) and CD8(+) T cells.     

An inhibitory monoclonal antibody to factor X that blocks prothrombin activation but not prothrombinase enzyme assembly

A monoclonal antibody (designated alpha BFX-2b) prepared against bovine factor X inhibited factor X activity in human, bovine, porcine, rabbit, and canine plasma. In assays using purified prothrombinase components, factor Xa, factor Va, phospholipid vesicles, and calcium ion with the fluorescent active site thrombin inhibitor dansylarginyl-N-(3-ethyl-1,5- pentanediyl)amide, the antibody inhibited the conversion of prothrombin to thrombin. Antibody alpha BFX-2b also blocked prothrombinase cleavage of the macromolecular substrates prethrombin 1 and prethrombin 2 but did not inhibit factor Xa hydrolysis of the synthetic substrate benzoyl- Ile-Glu-Gly-Arg-p-nitroanilide. The antibody also prevented the inactivation of factor Xa by antithrombin III but did not prevent the inactivation by soybean trypsin inhibitor. Antibody alpha BFX-2b bound factor Xa with a stoichiometry of 1:1 and an apparent dissociation constant of 9.0 x 10(-11) mol/L as estimated from its inhibition of prothrombinase activity. Antibody alpha BFX-2b did not prevent binding of factor Xa to factor Va-phospholipid as measured by using fluorescence polarization or high-pressure liquid gel chromatography with the fluorescent Factor Xa analogue dansyl-glutamyl-glycyl-arginyl- Xa. Immunoblotting of factor X following electrophoresis on sodium dodecyl sulphate-polyacrylamide gels and transfer to nitrocellulose indicated that the antigenic determinant recognized by antibody alpha BFX-2b was found on the heavy chain of factors X and Xa. From these observations it can be concluded that antibody alpha BFX-2b recognizes a highly conserved epitope on the factor X heavy chain that is remote from the topographic sites required for prothrombinase complex assembly and substrate hydrolysis but may be located at or near a portion of the macromolecular substrate binding site.     

Cystic fibrosis: An update for clinicians. Part 2: Hepatobiliary and pancreatic manifestations

This paper, the second in the series, will build on the first and explore the importance of liver and pancreatic manifestations of cystic fibrosis (CF) and the effect on morbidity and mortality of this multifaceted genetic condition. It will also further develop the critical role of the gastroenterologist as part of the multidisciplinary group of clinicians and allied health staff in the effective management of patients with CF.     

Further delineation of putative ACTB loss‐of‐function variants: A 4‐patient series

ACTB encodes β‐cytoplasmic actin, an essential component of the cytoskeleton. Based on chromosome 7p22.1 deletions that include the ACTB locus and on rare truncating ACTB variants, a phenotype resulting from ACTB haploinsufficiency was recently proposed. We report putative ACTB loss‐of‐function variants in four patients. To the best of our knowledge, we report the first 7p22.1 microdeletion confined to ACTB and the second ACTB frameshifting mutation that predicts mRNA decay. A de‐novo ACTB p.(Gly302Ala) mutation affects β‐cytoplasmic actin distribution. All four patients share a facial gestalt that is distinct from that of individuals with dominant‐negative ACTB variants in Baraitser‐Winter cerebrofrontofacial syndrome. Two of our patients had strikingly thin and sparse scalp hair. One patient had sagittal craniosynostosis and hypospadias. All three affected male children have attention deficits and mild global developmental delay. Mild intellectual disability was present in only one patient. Heterozygous ACTB deletion can allow for normal psychomotor function.     

Ex vivo expansion with stem cell factor and interleukin-11 augments both short-term recovery posttransplant and the ability to serially transplant marrow

The characterization of many cytokines involved in the control of hematopoiesis has led to intense investigation into their potential use in ex vivo culture to expand progenitor numbers. We have established the optimum ex vivo culture conditions that allow substantial amplification of transient engrafting murine stem cells and which, simultaneously, augment the ability to sustain serial bone marrow transplantation (BMT). Short-term incubation of unfractionated BM cells in liquid culture with stem cell factor (SCF) and interleukin-11 (IL- 11) produced a 50-fold amplification of clonogenic multipotential progenitors (CFU-A). Following such ex vivo expansion, substantially fewer cells were required to rescue lethally irradiated mice. When transplanted in cell doses above threshold for engraftment, BM cells expanded ex vivo resulted in significantly more rapid hematopoietic recovery. In a serial transplantation model, unmanipulated BM was only able to consistently sustain secondary BMT recipients, but BM expanded ex vivo has sustained quaternary BMT recipients that remain alive and well more than 140 days after 4th degree BMT. These results show augmentation of both short-term recovery posttransplant and the ability to serially transplant marrow by preincubation in culture with SCF and IL-11.     

Chaperonin-mediated assembly of wild-type and mutant subunits of human propionyl-CoA carboxylase expressed in Escherichia coli

We developed a bacterial expression system for the human alpha and beta cDNAs of propionyl-CoA carboxylase (PCC). These cDNAs (less the putative mitochondrial matrix targeting presequences) were co-expressed in Escherichia coli on one plasmid vector with each cDNA having its own IPTG-inducible promoter. Only negligible amounts of active PCC were measured despite the presence of both alpha and beta subunits as indicated by Western blot analysis and the almost complete biotinylation of the alpha subunit. Co-expression of this plasmid with a second plasmid vector over-expressing the E. coli chaperonin proteins, groES and groEL, resulted in a several hundred-fold increase in PCC specific activity, to a level comparable with that found in crude human liver extracts. PCC was partially purified on monomeric avidin affinity resin and the presence of both alpha and beta subunits was demonstrated, thereby confirming the assembly of both subunits into an active enzyme. Deficiency of either alpha PCC or beta PCC results in propionic acidemia, an autosomal recessive disorder. We used this expression system to characterize one missense mutation previously described in five Japanese alleles, namely C1283T (Thr428lle) in beta PCC. This mutation, when expressed in E.coli under the same conditions as that of wild-type PCC, had null activity, despite the presence of assembled alpha PCC and beta PCC subunits. This bacterial expression system can be useful for analysis of either alpha PCC or beta PCC mutations. Our findings indicated that the groES and groEL chaperonin proteins were essential for folding and assembly of the human PCC heteromeric subunits.      

Overview and update on molecular testing in non-small cell lung carcinoma utilizing endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) samples

Lung carcinoma remains one of the most frequent and aggressive human neoplasms. Fortunately, in the last decades, the increasing knowledge of the molecular mechanisms leading to cancer development has allowed the use of targeted therapies with improvement of prognosis in many patients. Clinical management has also changed after the introduction of endobronchialultrasonographic bronchoscopy that allows a conservative staging of lung tumors, avoiding the need of mediastinoscopy for lymph node staging. Lung pathologists and cytopathologists are facing the challenge of giving the more comprehensive prognostic and predictive information with ever smaller tissue or cytological samples. The aim of this review is to summarize the molecular testing for non-small cell lung carcinoma and how pathologists can contribute to the patient's outcome with a conscious management of biological samples.     

Adult heart disease prevention in childhood: a national survey of pediatricians' practices and attitudes

There is controversy about the role of pediatric care givers in reducing presumed risk factors for cardiovascular diseases in children. A national mail survey of 2,000 pediatricians was designed to determine the attitudes, current practices, and knowledge among primary care pediatricians regarding these risks. The response rate was 60% (779 primary care pediatricians). Responses indicated that a majority of pediatricians take a family history of cardiovascular diseases, assess BP, recommend exercise to school-aged children, and advise patients and parents against smoking. Few pediatricians felt confident in their ability to affect change in patient life-styles. There was a relatively low level of provision of dietary advice, and most pediatricians do not measure serum cholesterol levels, except in high-risk older children. A substantial minority do not discuss smoking, even with adolescents. Practices and attitudes varied with the age of the patient. Obesity was the topic most frequently chosen for continuing medical education. Older pediatricians were most likely to advocate and practice risk reduction in children. Attitudes and reported practices also varied by gender and geographic location of the pediatrician as well as the ethnic composition of the practice. Implications for continuing medical education are discussed.     

Comparative investigations on the retrieval capabilities of various baskets and graspers in four ex vivo models

OBJECTIVES: The increasing application of ureterorenoscopy for the treatment of urolithiasis has produced a myriad of different help-tools for stone retrieval. In this study, we compared the retrieval capabilities of different baskets and graspers in ex vivo models and attempted to find the most appropriate tool for stone extraction considering the location and size of stone, number of stones and potential harm to urinary tract tissue. METHODS: We created four different ex vivo models with porcine kidneys and ureters and natural human stones: (1) single ureteral stone (4mm); (2) single impacted ureteral stone (6mm); (3) Steinstrasse; (4) single lower-pole stone (5mm). With the aid of four baskets of different design (flat-wire basket Segura and Nitinol basket Zerotip, helical Gemini, and Parachute) and three graspers (two-prong, three-prong Tricep, and Nitinol grasper Graspit) we performed repeated stone extraction 10 times in each model. The time for complete stone removal was calculated. Macroscopical evaluation of tissue damage was performed after each series. In the Steinstrasse model the frequency of instrument reinsertion into the ureter was also calculated. RESULTS: In the single ureteral stone model, the fastest stone removal was achieved with the two-prong graspers, three-prong graspers and helical basket (20, 26 and 31s, respectively). Segura and Parachute needed more time (55 and 86s, respectively). Impacted stones were removed fastest with the two- and three-prong graspers (38 and 52s, respectively), Segura and Gemini were slower (89 and 114s, respectively). The Steinstrasse was cleared fastest by the helical basket with the lowest frequency of endoscope reinsertion (66s, 1.4 reinsertion). The Segura, 149s with 3.2 reinsertion was needed for the same procedure. With the Parachute and two-prong graspers 163s, 1.8 reinsertion and 261s, 4.6 reinsertion were needed, respectively. During postprocedural macroscopical evaluation of uretral tissue, the Parachute basket and three-prong graspers demonstrated the highest risk of mucosal and muscle damage. In the lower-pole kidney model, the poor deflectability of the ureterorenoscope prevented access to the lower-pole with the two- and three-prong graspers. There was no significant difference in stone retrieval time between the remaining Nitinol basket Zerotip and Nitinol grasper Graspit (87 and 61s, respectively ). The number of failures was 4/10 for Zerotip and 5/10 for Graspit. No significant mucosal damage was noted with these tools. CONCLUSIONS: Our ex vivo models demonstrated that the design of graspers and baskets affects the time of stone retrieval in different situations and causes the various grades of tissue damage.