Retinol binding protein and vitamin D associations with serum antibody isotypes, serum influenza virus-specific neutralizing activities and airway cytokine profiles.

Vitamin A supports the induction of immunoglobulin (Ig)A responses at mucosal surfaces in mice, but much less is known about the influence of vitamins on antibody isotype expression in humans. To address this knowledge gap, we examined 46 residual blood samples from adults and children, some of whom were experiencing influenza virus infections of the respiratory tract. Assays were performed for retinol binding protein (RBP, a surrogate for vitamin A), vitamin D (a related vitamin) and antibody isotypes. Results showed that all but two tested samples exhibited RBP and/or vitamin D insufficiencies or deficiencies. Vitamin D correlated with blood IgM and IgG3, while RBP correlated with IgG4 and IgA. RBP also correlated positively with age and with influenza virus-specific antibody neutralization titres. Individuals with low blood RBP levels exhibited the highest frequencies of over-expressed cytokines and growth factors in nasal wash samples, an indication of inflamed mucosal tissues. While cause-effect relationships were not discerned, results support a hypothesis that vitamins directly influence B cell isotype expression in humans, and by so doing may help protect mucosal surfaces from respiratory viral disease.

A two-millimetre free margin from invasive tumour minimises residual disease in breast-conserving surgery.

AIMS: In breast-conserving surgery, the width of free margin around a tumour to ensure adequate excision is controversial. The aim of this study was first to evaluate the frequency of residual disease in wider excision specimens in patients who undergo further surgery because of close margins of < 5 mm. Secondly, the ability of demographic and tumour-related factors to predict the close margins was appraised. PATIENTS AND METHODS: Three-hundred-and-three patients were included in the study. Patients undergoing wider excision were assessed for the presence of residual disease, and this was tested for association with the width of the initial free margin. Various factors were studied for association with close or involved margins by univariate analysis. RESULTS: Fifty-three per cent of patients were eligible for re-excision based on the need for a 5-mm clearance. With a free margin of 2 mm or more from invasive tumour, the probability of finding residual disease was 2.4%. The probability of residual disease was higher for ductal carcinoma in situ (DCIS) and did not decline with increasing the free margin width. Tumour size, lobular cancer type, vascular invasion and nodal involvement were associated with close margins. CONCLUSIONS: We suggest that a free margin of 2 mm from invasive tumour is adequate to minimise residual disease, whereas the equivalent free margin for DCIS remains unclear. Patients with large tumours and lobular cancer type should be counselled at the time of first surgery concerning the higher risk of further excision and mastectomy.

Gastrointestinal effects following acupuncture at Pericardium-6 and Stomach-36 in healthy dogs: a pilot study.

OBJECTIVES: To quantify changes in gastric and intestinal emptying times in the conscious dog following gastrointestinal acupoint stimulation. MATERIALS AND METHODS: In a randomised, blinded crossover study, six dogs were fed 30×1.5 mm barium-impregnated polyethylene spheres and underwent: (1) no acupuncture (Control); (2) stimulation of target points PC6 and ST36 (Target) and (3) stimulation of non-target points LU7 and BL55 (Sham). Abdominal radiographs were assessed immediately after feeding the spheres and every hour for 12 hours and their number in the stomach and large intestines was counted. RESULTS: The number of barium-impregnated polyethylene spheres found distal to the stomach was less in the Target group compared to the Control and Sham groups between hours 2 and 4, but no differences between groups were seen for the remainder of the treatment period. The number of spheres found within the colon/rectum was less in the Target group compared to the Control and Sham groups between hours 4 and 6, and compared to the Sham group only at hour 7 but no differences between groups were seen after hour 8. CLINICAL SIGNIFICANCE: Acupuncture targeted at the gastrointestinal tract of dogs was associated briefly with slowed gastric emptying and gastrointestinal transit time. This foundational study lays the groundwork for additional studies of acupuncture effects associated with altered physiologic states.

Antisense-mediated reduction in insulin-like growth factor-I receptor expression suppresses the malignant phenotype of a human alveolar rhabdomyosarcoma.

The expression of the insulin-like growth factors (IGFs) and their receptors has been linked to cellular proliferation and tumorigenicity in a number of model systems. Since rhabdomyosarcoma cells express IGF-I receptors, an autocrine or paracrine loop involving this receptor and its ligands could be responsible in part for the growth characteristics of this tumor. To assess directly the role of the IGF-I receptor in rhabdomyosarcoma cell growth and tumorigenicity, a human alveolar rhabdomyosarcoma cell line with high IGF-I receptor expression was transfected with an amplifiable IGF-I receptor antisense expression vector. Four unique, transfected clones were analyzed and found to have reduced IGF-I receptor expression relative to the parental line. Integration of the antisense sequence was demonstrated by Southern blot analysis, and expression of antisense message in these clones was shown by S1 nuclease protection assay. Reduced IGF-I receptor surface expression in the transfectants was shown by decreased immunofluorescence with an IGF-I receptor monoclonal antibody and by decreased IGF-I binding as measured by Scatchard analysis. These clones had markedly reduced growth rates in vitro, impaired colony formation in soft agar, and failed to form tumors in immunodeficient mice when compared with vector-transfected clones. These results demonstrate that reduction of IGF-I receptor expression can inhibit both the in vitro and in vivo growth of a human rhabdomyosarcoma cell line and suggest a role for the IGF-I receptor in mediating neoplastic growth in this mesenchymally derived tumor.

DNA-binding specificity and trans-activating potential of the leukemia-associated E2A-hepatic leukemia factor fusion protein.

Hybrid transcription factors, resulting from gene fusions in the wake of chromosomal translocations, have been implicated in leukemogenesis, but their precise contributions to oncogenic conversion remain unclear. The E2A-HLF fusion gene, formed by a t(17;19)(q22;p13) in childhood pro-B-cell acute lymphoid leukemia, encodes a hybrid protein that contains the trans-activation domain of E2A (E12/E47) linked to the bZIP DNA-binding and dimerization domain of hepatic leukemia factor (HLF). Here we report that both HLF and E2A-HLF bind to a 10-bp consensus sequence, 5'-GTTACGTAAT-3', with a core dyad-symmetric motif characteristic of the bZIP scissors-grip model of DNA binding. A probe containing this sequence bound chimeric E2A-HLF proteins in nuclear extracts of a leukemic cell line (UOC-B1) containing the t(17;19), as demonstrated by complexes supershifted with antibodies specific for amino-terminal epitopes of E2A or carboxyl-terminal eptiopes of HLF. E2A-HLF functioned as a potent trans activator of reporter gene expression from a plasmid that contained the consensus DNA-binding sequence. Interestingly, wild-type HLF was restricted in its capacity to act as a trans activator, functioning in human fetal kidney cells but not HepG2 hepatocarcinoma cells or NIH 3T3 mouse fibroblasts. The ability of the E2A-HLF hybrid protein to bind DNA in a sequence-specific manner and trans activate the expression of artificial reporter genes suggests that it could subvert transcriptional programs that normally control the growth, differentiation, and survival of lymphoid progenitor cells.

Anatomical study of the placement of proximal oblique locking screws in intramedullary tibial nailing.

Intramedullary tibial nailing was performed in ten paired cadavers and the insertion of a medial-to-lateral proximal oblique locking screw was simulated in each specimen. Anatomical dissection was undertaken to determine the relationship of the common peroneal nerve to the cross-screw. The common peroneal nerve was contacted directly in four tibiae and the cross-screw was a mean of 2.6 mm (1.0 to 10.7) away from the nerve in the remaining 16. Iatrogenic injury to the common peroneal nerve by medial-to-lateral proximal oblique locking screws is therefore a significant risk during tibial nailing.

Vitamin Supplementation at the Time of Immunization with a Cold-Adapted Influenza Virus Vaccine Corrects Poor Mucosal Antibody Responses in Mice Deficient for Vitamins A and D.

Vitamin A and D deficiencies and insufficiencies are prevalent worldwide in developed and developing countries. Vitamin metabolites are functionally intertwined in that they are high-affinity ligands for related receptors of the nuclear receptor superfamily. The effects of vitamin A deficiencies (VAD) on antibody responses to respiratory virus vaccines have already been demonstrated. Of particular concern was the reduction in IgA, a first line of defense against pathogens in the respiratory tract. Here, we describe the individual and combined effects of vitamin A and D deficiencies in mice immunized with an attenuated influenza virus vaccine. Relative to VAD, vitamin D deficiency (VDD) had a limited effect, but double deficiencies for vitamins A and D (VAD+VDD) further reduced antibody responses in the respiratory tract. The administration of supplemental vitamins A and D to VAD+VDD mice at the time of vaccination restored responses in a dose-dependent manner. Results suggest that vitamin supplementation programs may be beneficial in a clinical setting to promote healthy immune responses to respiratory virus vaccines in vitamin-deficient individuals.

Lack of homozygously inactivated p73 in single-copy MYCN primary neuroblastomas and neuroblastoma cell lines.

We examined 18 neuroblastoma cell lines and 32 primary single-copy MYCN tumor specimens to determine whether mutations of p73, a novel p53-related gene located in chromosome band 1p36.33, contribute to the genesis or progression of childhood neuroblastoma. By fluorescence in situ hybridization, 16 of the 18 cell lines, but only 3 of the 32 primary tumors, had evidence of a deleted p73 allele. Sequence analysis of the p73 coding region in the mRNAs expressed by these cell lines and tumors did not reveal inactivating mutations, suggesting that p73 is not homozygously inactivated in neuroblastoma. However, several novel splice forms of p73 mRNAs were identified, including one without exon 11 that predominated in multiple MYCN-amplified cell lines. Its encoded p73 protein differed from other splice forms in that the C-terminus was derived from an alternative reading frame. Further study of the functional properties of the protein encoded by this splice form of p73 will be needed to determine whether it contributes to the pathogenesis of childhood neuroblastoma with MYCN gene amplification.

Cloning and chromosomal localization of the gene encoding human cyclin D-binding Myb-like protein (hDMP1).

The murine transcription factor murine cyclin D-binding Myb-like protein (mDmp1) arrests the cell cycle in G1 phase, through an activity that can be overridden by direct interaction with the D-type cyclins. Here, we describe the identification, sequence, chromosomal localization, and expression of the human cognate, hDMP1. The hDMP1 cDNA contains a 2280bp open reading frame that shares a high degree of identity with the mDmp1 coding region. The 4.4kb hDMP1 messenger RNA is ubiquitously expressed in normal human tissues, with highest levels in testis and substructures within the brain. By use of fluorescence in situ hybridization with a human genomic P1 probe, we assigned hDMP1 to chromosome 7, band q21. This chromosomal region is frequently deleted as part of the 7q-minus and monosomy 7 abnormalities of human acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We analyzed hDMP1 copy number by fluorescence in situ hybridization in leukemic blasts from nine patients with abnormalities of the long arm of chromosome 7, and in each case one allele of the hDMP1 gene was deleted. Functional analysis of the mDmp1 protein has shown that it negatively regulates cell proliferation, which suggests that this gene is a candidate suppressor of malignant transformation. Further study will be needed to determine whether gene-specific mutations implicate hDMP1 as a tumor suppressor in acute leukemias with deletions of the long arm of chromosome 7 or in other types of human malignancy.

Lipid peroxidation and homocysteine induced toxicity.

Homocysteine induced toxicity has been examined in cultures of human umbilical vein endothelial cells. The toxic effects of the amino acid alone and the amino acid plus Cu2+ could be prevented by catalase and decreased by desferal, when either was present in the culture medium. When desferal was allowed to accumulate intracellularly, no significant protection from homocysteine induced toxicity was observed. Even though lipid peroxidation accompanied the toxicity induced by homocysteine and homocysteine plus Cu2+, inhibition of lipid peroxidation in either case had no effect on cell viability. The significance of these results is discussed.

Homocysteine mediated endothelial cell toxicity and its amelioration.

In response to homocysteine induced toxicity in human umbilical vein endothelial cells, minimal changes in the concentration of cellular protein thiols but substantial changes in the concentration of intracellular soluble thiols were observed. The latter correlated closely with changes in cellular glutathione levels. No correlation existed between cellular glutathione levels and cell viability, whereas a close correlation between NAD+ levels and cell viability was demonstrated. Large decreases in cellular NAD+ occurred in response to homocysteine induced toxicity which were accompanied by the production of single stranded DNA. 3-Aminobenzamide, an inhibitor of poly (ADP-ribose) polymerase preserved cell viability and cellular NAD+ levels. Evidence that DNA synthesis was also compromised was revealed by the decreased capacity of homocysteine treated cells to incorporate deoxyuridine. Radical scavengers were also effective in preventing homocysteine induced toxicity. It is likely that the major threat to cells derives from radicals generated intracellularly. Eicosanoid metabolism and the xanthine oxidase system have been identified as two potential sources of radicals.

Microcalorimetry does not predict the cellular phagocytosis of latex microspheres.

Current literature highlights the potential suitability of microcalorimetry for the investigation of cell-drug interactions. Previous work using bacteria or antigens derived from infectious organisms yielded conclusions that heat production is a quantitative means of measuring phagocytosis. In this study we evaluated the potential of flow-through microcalorimetry as a method of quantifying the phagocytosis of microsphere particulates. The technique avoids the need to incorporate radioactive or fluorescent markers into the particulate formulation, and would be widely applicable in biopharmaceutical research. Using the monocyte cell line Mono Mac 6 a power output of 9.00 microW per million cells was increased significantly on addition of zymosan, lipopolysaccaride (LPS) and phorbol myristate acetate but not following exposure to FITC labelled latex microspheres (LM). TNFalpha production increased on exposure to zymosan, LPS and LPS-phorbol myristate acetate, though not on exposure to LB. An assay was developed which allowed the quantification of internalised particulates in phagocytic cells using fluorescent activated cell sorting (FACS). In contrast to the microcalorimetric and TNFalpha data FACS revealed that 20% of the MM6 population phagocytosed a mean of 1.35 LM. Microcalorimetry and measurements of TNFalpha production are assays of cellular activation a phenomenon not necessarily associated with phagocytosis. FACS, however, serves as a specific and quantitative measure of phagocytosis. Microcalorimetry may not be a suitable technique for the quantitative assessment of the phagocytosis of drug delivery particulates.

The inhibition of phagocytosis of respirable microspheres by alveolar and peritoneal macrophages.

Respirable poly(lactic co-glycolic acid) (PLGA) microspheres (2-3 microm diameter), were fabricated as a model drug delivery system whose uptake by macrophages could be quantified by fluorescent activated cell sorting. The microspheres exhibited minimal release of the entrapped flourophore (rhodamine B) and thus avoided possible fluid phase uptake of the flourophore. Externally bound microspheres were removed from the cell membrane by acid washing. The fluorescent intensity associated with the cells arose, therefore, from the internalised microspheres. NR8383 continuous culture alveolar macrophages were verified against primary cultures as a good model of alveolar phagocytosis. Peritoneal macrophages were also isolated and systemic and alveolar phagocytosis compared. Poloxamer 338 adsorbed at the microsphere surface did not reduce phagocytosis by NR8383 macrophages. It did, however, reduce the number of microspheres contained in primary alveolar macrophages but did not reduce the percentage of phagocytic cells. Poloxamer coatings did not reduce phagocytosis by peritoneal macrophages once the ratio of five microspheres per cell was exceeded. Dipalmitoylphosphatidylcholine (DPPC), the major component of lung surfactant, was added to cultures to model the alveolar environment where it was observed to reduce phagocytosis. In light of this finding, microspheres were coated in DPPC, which reduced their uptake by all cell types at all microsphere to cell ratios.

Retarding effects of DNA on the autoxidation of liposomal suspensions.

Deoxyribonucleic acid (DNA) is associated with the cell membrane of prokaryotes and the inner nuclear membrane of eukaryotes. The unsaturated fatty acids of phospholipids, which constitute the bilaminar structure of membranes, undergo autoxidation in the presence of O2. Calf thymus DNA was incuabted with methyl archidonate-enriched phosphatidyl choline liposomes in order to study the effect of DNA upon the oxidation of phospholipids while present in their natural in vivo bilayer configuration. DNA retarded the rate of lipid oxidation as monitored by both diene conjugation and the TBA test, but it did not alter the induction period. These results suggest that DNA is scavenging free radicals produced within the phospholipid bilayer.

Lung surfactant phospholipids inhibit the uptake of respirable microspheres by the alveolar macrophage NR8383.

Fluorescent poly(lactic-co-glycolic acid) microspheres of a respirable size were fabricated for use in a fluorescent activated cell sorting assay utilizing the continuous alveolar macrophage NR8383. This is a suitable model of alveolar phagocytosis, which permitted an investigation of the influence of phospholipid structure on the inhibition of phagocytosis of microspheres. Phospholipid inhibition was found to be independent of phosphatidylcholine alkyl chain length. Head group effects were investigated by studies employing phosphatidyl-choline, -serine, and -ethanolamine, and inhibition was shown to be independent of head group. Closer modelling of the lung environment by co-culturing NR8383 on A549 alveolar epithelium showed type II secretions to also down-regulate phagocytosis. In addition, pre-incubation with microspheres coated with dipalmitoylphosphatidylcholine reduced the uptake of a second microsphere (fluorescein isothiocyanate-labelled latex).

Identification of historical lead sources in roof dusts and recent lake sediments from an industrialized area: indications from lead isotopes.

X-ray fluorescence and stable lead (Pb) isotopic analyses have been undertaken on dusts, known from microscopic investigation to contain significant quantities of industrially- and urban-derived particulate matter, present in the roof cavities of houses in the Illawarra region (N.S.W., Australia), with the objective of examining the historic record of Pb pollution. All investigated houses contained in excess of 250 micrograms g-1 Pb, with dwellings close to a copper smelter, in a large industrial complex including a major steelworks, containing higher (> 2500 micrograms g-1) Pb concentrations. The isotopic composition in the dusts, expressed here as 206Pb/204Pb, is relatively constant at 17.0, irrespective of dwelling age or distance from the industrial complex. Contamination of the dusts by Pb sourced from paint cannot explain the isotopic uniformity of the dust samples. Isotopic modelling indicates that the dusts contain Pb derived from the copper smelter, gasoline-air Pb and a minor contribution from coal-utilising sources. Lead loading was also investigated in the adjacent lagoon, which acts as a natural sink for particulate matter in the Illawarra region. Isotopic data and modelling indicate that one natural and four anthropogenic sources contribute to the Pb burden of this lagoon. The natural source consists of Permian rocks cropping out in the catchment area which have a 206Pb/204Pb of approximately 18.7. The suggested anthropogenic sources are an old disbanded base-metal (Pb) smelter (206Pb/204Pb approximately 16.2-16.3), the copper smelter (206Pb/204Pb approximately 17.9), gasoline-air derived Pb (206Pb/204Pb approximately 16.4-16.5) and industries utilising coal, for example the recently closed thermal coal-fired power station (206Pb/204Pb approximately 18.9). The relative contributions of the base-metal (mainly lead) smelter and gasoline-air Pb in the sediment can only be partly assessed due to the isotopic similarity of these sources. Likewise the natural background and coal source (e.g. power station) contributions can only be estimated from historical data. Age estimations for sediment cores, using 137Cs, provide some control on these assessments. Near surface sediments in the lagoon have a relatively constant 206Pb/204Pb of 17.6-17.7, irrespective of sample location. Isotopic calculations, together with records of particulate matter pollution emissions, indicate a link between the Pb in roof dusts (206Pb/204Pb approximately 17.0) and Pb contamination of the near surface (upper 20 cm) lagoonal sediments via a homogeneous, non-unique source of lead whose isotopic composition closely matches that of the dusts. Over the last 5 decades, atmospheric fallout of Pb-bearing particulate matter appears to have been the dominant pathway for addition of Pb to the lagoon and dwellings in the Illawarra region.

The effectiveness of a nutrition education program for family practice residents conducted by a family practice resident-dietitian.

BACKGROUND AND OBJECTIVES: Despite current interest in the role of diet in disease prevention, physicians frequently lack adequate nutrition knowledge. This project sought to test family practice residents' nutrition knowledge, heighten their understanding of nutrition concepts, and implement a method of teaching nutrition in a residency program. METHODS: Following a pretest, family practice residents participated in four teaching sessions during a 5-month period conducted by a fellow resident who was also a dietitian. The residents were asked to keep a 3-day diet diary, which was analyzed with nutrition analysis software. A posttest measure changes in nutrition knowledge and interest. Pretests and posttests were also given to a control group of residents at another campus of the same university-administered program. RESULTS: The intervention group's mean scores increased from 54.7% to 70% (P<.001). Residents who kept a diet diary increased their scores by 23.5%, compared with a 7.4% increase for those who did not. Scores for control-group residents who took both pretests and posttests fell from 43.4% to 42.1%. CONCLUSIONS: Residents who attended lectures conducted by a family practice resident- dietitian and examined their diets showed remarkable improvement in posttest scores of nutrition knowledge. Factors most influential in increasing residents' nutrition knowledge include increasing residents' interest in nutrition, involving them in a longitudinal series of lectures, and providing support for their knowledge and its application.

Applications of centrographic techniques to the study of urban phenomena: Atlanta, Georgia 1940-1975.

"A limited number of centrographic techniques are applied to data for total population, black population, households by income quartiles, and housing characteristics for the five-county metropolitan region of Atlanta over the period from 1940 to 1975. Results of the analysis are compared to expectations from urban economic theory, and descrepancies are noted."

Influence of low back pain on total knee arthroplasty outcome.

BACKGROUND: Preoperative pain and functional status are strong determinants of postsurgical success in total knee arthroplasty. Patients suffering chronic pain from other coexistent musculoskeletal problems may respond differently postoperatively, with potentially poorer outcomes after surgery. The aim of the study was to determine the influence of low back pain on the outcome of total knee replacement surgery. METHODS: All patients completed Oxford Knee Scores (OKS), American Knee Society Scores (AKSS) and SF-12 (both physical and mental components). Patients were divided into those with (n=40) and without a documented history of low back pain (n=305). RESULTS: OKS, AKSS and SF-12 physical scores were significantly worse for patients with low back pain at 24 months following surgery. The mental component of the SF-12 measure demonstrated a significant improvement in median mental health post-operatively for patients with no current history of low back pain. In contrast the group with low back pain showed no improvement in mental health scores post-operatively. CONCLUSION: This study demonstrates that symptomatic low back pain influences functional outcome after total knee arthroplasty surgery and that patients with low back pain show limited or no improvement in mental health post-operatively. Level of evidence II.

Oral retinyl palmitate or retinoic acid corrects mucosal IgA responses toward an intranasal influenza virus vaccine in vitamin A deficient mice.

Vitamin A deficiency (VAD) is a leading cause of pediatric morbidity and mortality due to infectious diseases. Recent pre-clinical studies have revealed that VAD impairs mucosal IgA-producing antibody forming cell (AFC) responses toward a paramyxovirus vaccine in the upper respiratory tract (URT), thus impeding a first line of defense at the pathogen's point-of-entry. The studies described here tested the hypothesis that VAD may also impair immune responses after FluMist vaccinations. Results show that (i) IgA-producing antibody forming cells (AFCs) are significantly reduced following FluMist vaccination in VAD mice, and (ii) oral doses of either retinyl palmitate or retinoic acid administered on days 0, 3, and 7 relative to vaccination rescue the response. Data encourage the conduct of clinical studies to determine if there are FluMist vaccine weaknesses in human VAD populations and to test corrective supplementation strategies. Improvements in vaccine efficacy may ultimately reduce the morbidity and mortality caused by influenza virus worldwide.