Vaccination against pandemic H1N1 (2009) in patients after allogeneic hematopoietic stem cell transplantation: a retrospective analysis.

PURPOSE: Limited data are available on immunologic responses to primary pandemic H1N1 (2009) vaccination in recipients of allogeneic hematopoietic stem cell transplantation (HSCT) recipients. In 2009 serologic responses to either pandemic H1N1 (2009) vaccine (n = 36) or pandemic H1N1 (2009) infection (n = 2) were studied in 38 HSCT recipients. METHODS: Responses were measured with a standard hemagglutination-inhibition assay. Fourteen patients had active chronic graft-versus-host disease (cGvHD) at the time of vaccination/infection and seven patients had cGvHD in remission; 11 patients had no immunosuppressive therapy, and 27 patients were on immunosuppressive therapy. Nineteen patients (53%) responded to pandemic H1N1 (2009) vaccination. Two patients had pandemic H1N1 (2009) infection without prior vaccination, and one patient had severe pandemic H1N1 (2009) infection with acute respiratory distress syndrome despite prior single vaccination. RESULTS: Non-responders to pandemic H1N1 (2009) vaccination more often had cGvHD (65 vs. 53%) and received second- or third-line therapy (53 vs. 11%), while responders mostly had first-line therapy for cGvHD. While vaccine responders had no or single agent immunosuppressive therapy, non-responders frequently received moderate or intense immunosuppressive therapy. All vaccine recipients previously treated with rituximab were non-responders. CONCLUSIONS: In summary, the overall response to pandemic H1N1 (2009) vaccination in HSCT recipients was modest. Patients receiving combined immunosuppressive therapy for steroid-refractory cGvHD barely responded to pandemic H1N1 (2009) vaccination.

Toward genome editing in X-linked RP-development of a mouse model with specific treatment relevant features.

Genome editing represents a powerful tool to treat inherited disorders. Highly specific endonucleases induce a DNA double strand break near the mutant site, which is subsequently repaired by cellular DNA repair mechanisms that involve the presence of a wild type template DNA. In vivo applications of this strategy are still rare, in part due to the absence of appropriate animal models carrying human disease mutations and knowledge of the efficient targeting of endonucleases. Here we report the generation and characterization of a new mouse model for X-linked retinitis pigmentosa (XLRP) carrying a point mutation in the mutational hotspot exon ORF15 of the RPGR gene as well as a recognition site for the homing endonuclease I-SceI. Presence of the genomic modifications was verified at the RNA and protein levels. The mutant protein was observed at low levels. Optical coherence tomography studies revealed a slowly progressive retinal degeneration with photoreceptor loss starting at 9 months of age, paralleling the onset of functional deficits as seen in the electroretinogram. Early changes to the outer retinal bands can be used as biomarker during treatment applications. We further show for the first time efficient targeting using the I-SceI enzyme at the genomic locus in a proof of concept in photoreceptors following adeno-associated virus mediated gene transfer in vivo. Taken together, our studies not only provide a human-XLRP disease model but also act as a platform to design genome editing technology for retinal degenerative diseases using the currently available endonucleases.

Effect of growth conditions on the content of the major groups of carbohydrates in chick embryo fibroblasts.

The levels of glycogen, hyaluronic acid, chondroitin sulfates, N-acetylneuraminic acid, all of the monosaccharide components of the glycoprotein and glycolipid fractions, and the monosaccharide pools were measured in cultured chick embryo fibroblasts. Under all growth conditions, the glycogen plus the glucose phosphate pool contained approximately 50% of total monosaccharide content of the cells. However, marked qualitative and quantitative alterations were found in the glycoprotein, glycolipid, and mucopolysaccharide fractions when growing cells reached confluence, when the growth temperature was shifted from 36 to 41 degrees, or when the cells were transformed with Rous sarcoma virus. From 65 to 95% of the total monosaccharide residues in these complex carbohydrates were found in the glycoprotein fraction, while the glycolipids contained only 5 to 10% of the residues, and the mucopolysaccharides contained 5 to 25%. Changes in the complex carbohydrates in normal cells following changes in cell density or growth temperature were so great that they obscured any transformation-dependent changes that might have occurred consistently in the virus-infected cells under different growth conditions.

Purification and characterization of a nuclear DNA-binding phosphoprotein in fetal and tumor tissues.

The basic nonhistone phosphoprotein 110/8.4 (M.W. X 10(-3)/pI) was found in 0.35 M NaCl nuclear extracts of four tumor tissues, i.e., fast-growing Novikoff hepatoma, Morris hepatoma 3924A, HeLa cells, and Namalwa cells; it was also found in fetal rat liver. This protein was not detected in normal or regenerating liver and thus may represent an "oncofetal" protein of potential interest as a cancer "marker." Protein 110/8.4 was purified approximately 4000- to 5000-fold under nondenaturing condition from 0.35 M NaCl nuclear extracts of Novikoff hepatoma cells or Namalwa cells by ammonium sulfate fractionation, calcium phosphate gel treatment, and phosphocellulose chromatography. Sodium dodecyl sulfate:polyacrylamide gel electrophoresis of the purified native protein revealed a single polypeptide chain with a molecular weight of approximately 110,000. The pI of the protein was estimated to be 8.4 by nonequilibrium pH gradient electrophoresis in 9 M urea; accordingly, this protein was designated 110/8.4. Amino acid analysis showed that Protein 110/8.4 had an acidic:basic amino acid ratio of 1.25 and a high lysine and serine content; approximately 20% of the serine residues were found to be phosphorylated. Hydrazinolysis indicated that the carboxyl-terminal amino acid was serine; the amino terminus appeared to be blocked. Binding of Protein 110/8.4 to DNA was studied by the nitrocellulose filter assay. High-affinity binding occurred at ionic strength equal to or below 0.15 M.

Turnover of cellular carbohydrates in normal and Rous sarcoma virus-transformed cells.

We have analyzed the distribution of glucosamine-labeled polymers on the cell surface, in the growth medium, and inside the cell and the net turnover of these polymers during the process of malignant transformation of chick embryo fibroblasts by Rous sarcoma virus. The distribution of label and the turnover kinetics for hyaluronic acid, total glycoproteins, and chondroitins were found to be identical in both normal and transforming cultures. We conclude that nonspecific degradative processes are probably not involved in causing transformation-related alterations in cell surface carbohydrates, althougn degradation of specific macromolecules is not excluded.

Integrative rehabilitation of residents chronic post-stroke in skilled nursing facilities: the design and evaluation of the BrightArm Duo.

PURPOSE: To describe the novel BrightArm Duo bimanual upper extremity (UE) rehabilitation system; to determine its technology acceptance and clinical benefit for older hemiplegic participants. METHODS: The system table tilted to adjust arm gravity loading. Participants wore arm supports that sensed grasp strength and wrist position on the table. Wrist weights further increased shoulder exertion. Games were designed to improve UE strength, motor function, cognition and emotive state and adapted automatically to each participant. The system underwent feasibility trials spanning 8 weeks in two skilled nursing facilities (SNFs). Participants were evaluated pre-therapy and post-therapy using standardized clinical measures. Computerized measures of supported arm reach, table tilt and number of arm repetitions were stored on a remote server. OUTCOMES: Seven participants had significant improvements in their active range of shoulder movement, supported arm reach, shoulder strength, grasp strength and their ability to focus. The group demonstrated higher arm function measured with FMA (p = 0.01) and CAHAI (p = 0.05), and had an improvement in depression (Becks Depression Inventory, II). BrightArm Duo technology was well accepted by participants with a rating of 4.4 out of 5 points. CONCLUSIONS: Given these findings, it will be beneficial to evaluate the BrightArm Duo application in SNF maintenance programs. Implications for Rehabilitation Integrative rehabilitation that addresses both physical and cognitive domains is promising for post-stroke maintenance in skilled nursing facilities. Simultaneous bilateral arm exercise may improve arm function in older hemiplegic patients several years after stroke. Virtual reality games that adapt to the patient can increase attention and working memory while decreasing depression in elderly.

Comparison of the carbohydrate and amino acid composition of normal and S-variant alpha-1-antitrypsin.

alpha-1-Antitrypsin has been isolated and purified from the serum of an individual with the Pi S phenotype whose serum contains only 50--60% as much alpha-1-antitrypsin as normal M-type serum. The preparation was homogeneous by the criteria of sodium dodecyl sulfate polyacrylamide gel electrophoresis and sedimentation equilibrium ultracentrigufation. When analyzed in the ultracentrifuge, the S-type alpha-1-antitrypsin exhibited a molecular weight of 47,500 which was essentially the same as that of the M-type (47,300) and the Z-type (47,500) alpha-1-antitrypsin. The S-type alpha-1-antitrypsin contains 15.2% carbohydrate consisting of 16.4 residues/mol of N-acetylglucosamine, 7.8 residues/mol of mannose. 6.7 residues/mol of galactose and 7.1 residues/mol of sialic acid which is essentially the same as the carbohydrate composition of the M-type alpha-1-antitrypsin. In addition, M- and S-type alpha-1-antitrypsin have very similar amino acid compositions.

Identification of consensus epitope structures expressed in recombinant DNA libraries.

In the course of screening cDNA expression libraries with a monospecific polyclonal antibody to topoisomerase I, we isolated three different immunopositive cDNA clones. By comparing their derived amino acid sequences, a consensus region of similarity in otherwise completely dissimilar sequences was identified as an epitope. The approach described here should identify both continuous and discontinuous topological epitopes. The probability of occurrence of "spurious" immunopositive clones is shown to depend on the number of codons of each critical amino acid within the antigenic determinant.

Selective reduction of adrenal medulla response to angiotensin induced by suppression of renin-angiotensin.

Angiotensin II (AII) induced an increase of adrenaline (A) release from rat adrenal glands in vitro. The response of the adrenal glands was completely abolished 22--24 h after bilateral nephrectomy. Adrenal glands from DOCA-salt-treated rats did not respond to AII as well, whereas adrenal glands from rats treated with furosemide and a low salt diet retained this response. High potassium in the medium increased significantly the release of A from in vitro incubated adrenal glands of nephrectomized rats from 0.43 +/- 0.04 to 0.63 +/- 0.06 microgram/gland and salbutamol (in calcium-free medium) increased the release from 0.34 +/- 0.03 to 0.43 +/- 0.03 microgram/gland. These results indicate that the adrenal medulla develops subsensitivity to AII in vitro, following a reduction in levels of this agonist in vivo.

Single-case experimental designs in medical education: an innovative research method.

This paper presents an argument for more extensive use of single-case experimental research designs in medical education research. Single-case experimental designs consist of a group of experimental techniques that are widely used in the social sciences but are just beginning to be utilized by medical researchers. The method emphasizes reliable observations of behavior, repeated measurements of outcome, and individualized tailoring of objectives for each subject; all of these occur within a system that allows an experimental analysis to be conducted. Single-case designs are particularly useful when only small numbers of participants are available for a relatively long period of time. Trends in medical education toward individualized instruction, adult-centered learning, and fine-grained analyses of medical skills and knowledge make this field especially amenable to single-case experimental designs. Issues of internal and external validity, generality, practicality, and ethics are discussed, and several typical designs are illustrated. While the emergence of qualitative research methods in medical education may prove useful, single-case designs can maintain experimental science's emphasis on methodologic rigor, while allowing the flexibility often needed to conduct research in applied settings.

Synthesis and PMR and mass spectra of potential metabolites and other derivatives of bis(2-ethylhexyl) phthalate.

The 3- and 4-hydroxylated derivatives of the plasticizer bis(2-ethylhexyl) phthalate and the monoesters derived from these hydroxylated derivatives were synthesized. The structures of the two 4-hydroxy monoesters were confirmed by lithium borohydride reduction to their corresponding hydroxymethylbenzoic acids, followed by cyclization of the reduced compounds to known phthalides. All compounds were characterized by PMR and mass spectra. The PMR data reiterate the difficulty in predicting chemical shifts in multisubstituted benzene rings.

Hongoquercins A and B, new sesquiterpenoid antibiotics: isolation, structure elucidation, and antibacterial activity.

Two new sesquiterpenoid antibiotics, hongoquercins A and B, were isolated from the extracts of an unidentified fungus. The structures of both metabolites were determined by spectroscopic analysis. They are related to a class of compounds commonly found in brown algae and dictyoceratid sponges. Hongoquercin A exhibited moderate activity against gram-positive bacteria.

[A comparison of different methods of calculating clearances using 99mTc-mercaptoacetyltriglycine and 123I- or 131I-ortho-iodohippuric acid]. / Vergleich verschiedener Clearance-Berechnungs-methoden bei Verwendung von 99mTc-Mercaptoacetyltriglycin sowie 123J- oder 131J-Orthojod-hippursäure.

Using labelled MAG3 (n = 172) or OIH (n = 102) the results of three different techniques of calculating renal clearance were compared. The following techniques were used: Tauxe (single plasma concentration determination), Oberhausen (modified, combined partial body and plasma disappearance clearance) and Winkler (partial body clearance). For OIH, linear regression analysis revealed a positive correlation of Tauxe and Oberhausen clearances with a correlation coefficient (r) of 0.90 and a mean ratio (mean) Oberhausen/Tauxe clearances of 1.03. For MAG3, the Oberhausen values were found to be significantly lower than the Tauxe values with an equally good correlation. For this radiopharmaceutical the Winkler values were significantly higher than the Tauxe and Oberhausen values. For OIH, no such difference was noted. However, the correlation coefficient was significantly lower. Whereas with the Tauxe method, a mean ratio MAG3-/OIH clearances of 0.62 was observed in an earlier investigation, from the present data a ratio of 0.53 is calculated for the Oberhausen clearance. The difference between the Tauxe and Oberhausen clearances with MAG3 is of importance when comparing clearance values obtained with clearance calculation techniques and for establishing normal values for the MAG3 clearance.

Polycystic ovary syndrome. An autoimmune disease?

OBJECTIVE: To determine whether there is an autoimmune cause of polycystic ovary disease (PCOD) implicating antiovarian antibodies in the pathophysiology of the syndrome. STUDY DESIGN: This study examined 31 women diagnosed as having PCOD according to internationally recognized criteria and who attended our infertility clinic during 1994-1995. RESULTS: We could not confirm the findings of previous studies. Only one patient in our group with type II diabetes mellitus had antiovarian antibodies present in her serum. CONCLUSION: It seems that an autoimmune mechanism probably is not of prime importance in this multifaceted syndrome.

Modification of nasal resonance in cleft-palate children by informative feedback.

Vibration of the walls of the nasal cavities during production of an English vowel or diphthong defined a hypernasal response. A crystal transducer on one side of the nose activated a voice-operated relay when vibration exceeded an arbitrary limit to provide an apparatus definition of the response. During training sessions, responses without nasal vibration turned on a white light in the sound-treated chamber. Subject 1 had a repaired cleft palate and mild hypernasal speech. Introduction of the differential feedback, reversal, and reinstatement of the feedback conditions resulted in a rapid decrease, increase, and decrease in percentage of nasalized productions of the /eI/ sound Subject 2 had a cleft of the soft palate and severe hypernasal speech. Introduction of feedback produced a gradual decline in the percentage of hypernasal productions of the /##/ sound over 22 sessions. Removal and reinstatement of feedback resulted in a rapid increase and decrease respectively in the percentage of nasalized responses.

Behavioral engineering: postural control by a portable operant apparatus.

Recent studies suggested a general behavioral engineering approach to behavioral disorders by portable operant treatment instruments. The approach was applied to the problem of poor posture, specifically rounding of the back or slouching. An apparatus was developed that provided a warning stimulus followed by an aversive tone for the duration of slouching. Slouching was thereby punished by onset of the tone, and non-slouching was reinforced by tone termination and postponement. Twenty-five adults wore the apparatus during their normal working day during alternate periods in which the aversive tone was connected and disconnected experimentally. A miniature time-meter recorded the duration of slouching. The results showed that slouching decreased for each subject during each period in which slouching produced the aversive tone. For two subjects, a second control procedure was applied in which slouching terminated the tone. The result was an increase of slouching, demonstrating that the postural changes were controlled by the scheduled relation between the aversive tone and the response, and not by other factors such as simple response feedback. The substantial changes in posture indicate that the present procedure may prove to be an effective treatment alternative and suggests the general value of the behavioral engineering approach.