Multidimensional Chromatographic and Hyphenated Techniques for Hydrophilic Copolymers, 1

Summary: The chromatographic analysis of hydrophilic copolymers is complicated due to the fact that in most cases aqueous eluents must be used. In aqueous eluents different polar and ionic effects may disturb the selective interactions between the macromolecules and the stationary phase making it impossible to separate such copolymers with regard to chemical composition. Therefore, 2D chromatography combining a separation according to composition with a separation according to molar mass has been applied mostly to polymers that are soluble in organic solvents. The present contribution describes experimental approaches to analyze such hydrophilic copolymers by 2D‐chromatography. For a model polymer system resulting from the copolymerization of methacrylic acid and a poly(ethylene glycol) macromonomer, it is shown that different analytical techniques including SEC, LC‐CC, MALDI‐TOF MS and 2D chromatography can be used to analyze the different parameters of molecular heterogeneity of such copolymers.

2D separation of poly(MPEG‐MM 2), 1^st dimension: LC‐CC, 2^nd dimension: SEC.     

PG-M1: A New Monoclonal Antibody Directed against a Fixative-Resistant Epitope on the Macrophage-Restricted Form of the CD68 Molecule

A new anti-macrophage monoclonal antibody (PG-M1) was produced by immunizing BALB/c mice with fresh spleen cells from a patient with Gaucher's disease. PG-M1 reacts strongly with a fixative-resistant epitope of an intracytoplasmic molecule, selectively expressed by virtually all macrophages of the human body. Although attempts to immunoprecipitate the molecule recognized by PG-M1 have failed so far, the reactivity of the antibody with COS-1 and WOP cells transfected with a human complementary DNA clone encoding for the CD68 antigen suggests that PG-M1 is a new member of the CD68 cluster. However, unlike other CD68 antibodies (KP1, EBM11, etc.), which react with both macrophages and myeloid cells, PG-M1 detects a fixative-resistant epitope on the macrophage-restricted form of the CD68 antigen. In 957 routinely fixed, paraffin-embedded samples, PG-M1 showed a more restricted reactivity with elements of the monocyte/macrophage lineage than the previously described monoclonal antibodies MAC-387 (anti-calgranulins), KP1 (CD68) and Ki-M1P. Among hematological malignancies, PG-M1 only labels acute leukemias of M4 and M5 type and rare examples of malignant histiocytosis/true histiocytic sarcoma. In contrast, acute leukemias of the M1, M2, M3, M6, M7, and L1-L3 types, non-Hodgkin's lymphomas, and Hodgkin and Reed-Sternberg cells of Hodgkin's disease are consistently PG-M1-negative. In the daily diagnostic practice, PG-M1 seems to be particularly valuable for the diagnosis of myelomonocytic or monocytic leukemia and neoplasms of true histiocytic origin in routine paraffin sections.     

Anti-actin specificity of human smooth muscle antibodies in chronic active hepatitis.

Thirty sera reacting by IFL technique in titres greater than or equal to 100 with smooth muscle fibres of rat stomach, rat renal glomeruli, and with the membrane region of thyroid cells were randomly chosen among sera sent in for routine testing of tissue antibodies. All sera but one were found to be derived from patients with chronic active hepatitis. The smooth muscle and other relevant cell staining were abolished after absorption of sera with actin, prepared from rabbit skeletal muscle and found to be homogeneous by SDS gel-electrophoresis and by electron microscopy. The actin anti-bodies were purified by precipitation of sera with F-actin and elution of the precipitates at acid pH. The purified antibodies stained all tissues in the same way as the original sera. In double immunodiffusion tests all thirty sera gave precipitation with actin. Thus, it was concluded that these broad-reacting SMA are directed against actin. The finding of high-titred SMA is of diagnostic value and supports the clinical diagnosis of active chronic hepatitis. In addition, anti-actin antibodies eluted from human sera are a suitable tool for studying actin-containing cellular structures.     

Somatoform pain disorder in the general population

BACKGROUND: Chronic pain disorder is assumed to represent a frequent and disabling condition. However, data on the prevalence of somatoform pain symptoms and somatoform pain disorder in the community are limited to date. METHODS: German versions of the Composite International Diagnostic Interview were administered to a representative national sample of 4,075 people. Somatoform pain disorder was diagnosed by standardized diagnostic algorithm based on the DSM-III-R criteria (absence of adequate physical findings). One subgroup was identified as also meeting the DSM-IV criterion B for 'significant distress or psychosocial impairment due to the somatoform pain'. RESULTS: A lifetime prevalence rate of somatoform pain disorder according to DSM-III-R of 33.7% and a 6-month rate of 17.3% was found. When applying the DSM-IV B criterion, the prevalence rate dropped to 12.3 and 5.4%, respectively. In both groups more than 95% of the probands had contacted their doctor because of the pain. In 25% of the probands the pain was positively assigned to psychological factors. A female:male ratio of 2:1 was found. CONCLUSIONS: Somatoform pain disorder (DSM-III-R) is a frequent condition. However, only about one third of these subjects is severely distressed or impaired by the pain. A clear operationalized concept of the DSM-IV criterion C 'psychological factors are judged to have an important role in the onset, severity, exacerbation or maintenance of the pain' should be provided in the further development of the diagnosis 'pain disorder' in order to make this diagnosis suitable for general population surveys.     

Antibody response and protection against challenge in mice vaccinated intraperitoneally with a live aroA O4-O9 hybrid Salmonella dublin strain.

An auxotrophic Salmonella dublin (O9,12) strain, SL5631, with a deletion affecting gene aroA, was made into a partial diploid expressing the rfb (O-antigen-repeat-unit-specifying) gene cluster of Salmonella typhimurium (O4,12). By use of O4- and O9-specific antisera in indirect immunofluorescence assays, the resulting hybrid SL7103 was shown to express both the O4- and O9-antigen epitopes in the same bacterium. Qualitative and quantitative sugar analyses by gas-liquid chromatography on peralditol acetates of phenol-water-extracted lipopolysaccharides showed that the S. dublin and S. typhimurium repeating units (estimated on the basis of their tyvelose and abequose contents, respectively) were present in approximately equimolar amounts. The SL7103 hybrid auxotroph was avirulent when given intraperitoneally to NMRI mice in a dose of 10(8) CFU and elicited a protective immunity against intraperitoneal challenge with either virulent S. dublin (50% lethal dose of ca. 1.5 x 10(4) CFU versus < 1 x 10(1) CFU in nonimmunized mice) or virulent S. typhimurium (50% lethal dose of ca. 1 x 10(5) versus < 1 x 10(1) CFU in nonimmunized mice). Compared with the protection elicited in homologous systems (S. dublin SL5631 against S. dublin and S. typhimurium SL1479 against S. typhimurium), the protective efficacy of the hybrid was reduced approximately 70-fold against S. dublin challenge and 100-fold against S. typhimurium challenge. Vaccination with S. typhimurium SL1479 conferred no protection against S. dublin challenge, and vaccination with S. dublin SL5631 conferred no protection against S. typhimurium challenge. The protection elicited by the hybrid strain SL7103 is supposed to be mainly a consequence of serum antibodies directed against the immunodominant O4 and O9 epitopes.     

Salmonella typhimurium infection in calves: protection and survival of virulent challenge bacteria after immunization with live or inactivated vaccines.

Salmonella typhimurium SL1479, an auxotrophic mutant strain having a complete block in the aromatic biosynthetic pathway and therefore requiring p-aminobenzoic acid and 2,3-dihydroxybenzoate not available in mammalian tissues, was given orally in a dose of 10(10) live bacteria to 4- to 5-week-old calves. Only a mild transient fever response was seen. Strain SL1479 was unable to colonize and persist in the calves for more than 2 weeks. In a vaccination experiment, groups of six calves were (i) orally vaccinated with the live S. typhimurium SL1479 strain, (ii) subcutaneously vaccinated with a heat-inactivated S. typhimurium SVA1232 strain with aluminum hydroxide as adjuvant, or (iii) not vaccinated, to serve as controls. Calves were orally challenged with the live, calf-virulent S. typhimurium SVA44 strain: either 10(6) bacteria (equivalent to 100 25% lethal doses [LD25S]) or 10(9) bacteria (100,000 LD25S doses). The live oral vaccine gave significantly better protection than the heat-vaccinated, subcutaneously injected vaccine since (i) only control calves and calves given the killed vaccine developed profuse diarrhea, (ii) clinically, the mild fever responses seen after challenge in calves given the live vaccine were significantly lower (P less than 0.0005), (iii) autopsies performed 21 days after the challenge infection revealed normal findings in calves given the live vaccine, whereas calves given the killed vaccine showed signs of acute enteritis and chronic salmonellosis, (iv) all 12 calves given either 100 X or 100,000 X the LD25 survived the 21-day observation period; the mean survival time in nonvaccinated calves was 8.0 days; in calves given heat-inactivated vaccine and 100 X the LD25 it was 21.0 days, and in calves given 100,000 X the LD25 it was 11.5 days, (v) the fecal bacterial counts of the challenge S. typhimurium SVA44 strain were significantly lower (P less than 0.0005) in both groups given the live vaccine, and (vi) upon autopsy followed by culture, the qualitative recovery of the challenge strain from the alimentary canals and tissues of calves given the live vaccine was significantly lower (P less than 0.005).     

Ultrastructural changes of the liver in spontaneously ketotic cows

The ultrastructure of the liver in normal, mildly ketotic and severely ketotic cows was studied using stereological methods. In the liver of severely ketotic cows there is: (1) a significant increase in the volume fraction of hepatocytes and a decrease in the volume fraction of sinusoids, and (2) an increase in the volume fraction of lipid and smooth endoplasmic reticulum and a decrease in the volume fraction of glycogen and Golgi in parenchyma. A decrease in the profile density of mitochondria per 1 mm2 field and an increase of the volume occupied by mitochondria were not significant nor was the decrease in the volume density of rough endoplasmic reticulum. The degree and duration of negative energy balance obviously affect the morphological changes of the fatty liver. However, additional work is needed to determine the significance of ultrastructural changes in liver function.     

Cytotoxic T cell responses to haptenated cells III. Isolation and specificity analysis of continuously growing clones

Various procedures were used to derive continuously growing cytotoxic T lymphocyte (CTL) clones from a primary culture containing responder cells from immunized mice and 3-(p-sulfophenyldiazo)-4-hydroxylphenyl acetic acid (SP)-or fluorescein isothiocyanate (FL)-coupled stimulator cells. It seems likely that CTL have to undergo some change, possibly genetic, to be able to grow continuously in T cell growth factor conditioned medium in the absence of any stimulator or filler cells. The most convenient and reliable procedure to generate CTL clones with different specificities was to establish from several aliquots of a primary culture cell populations continuously growing in medium conditioned with T cell growth factor(s). Clones with different specificities segregated in the different populations. SP-and FL-specific CTL clones restricted to H-2K^k, and H-2D^d and two FL-specific CTL clones with no apparent H-2 restriction are described.     

The selective processing of briefly presented affective pictures: an ERP analysis

Recent event-related potential (ERP) studies revealed the selective processing of affective pictures. The present study explored whether the same phenomenon can be observed when pictures are presented only briefly. Toward this end, pleasant, neutral, and unpleasant pictures from the International Affective Pictures Series were presented for 120 ms while event related potentials were measured by dense sensor arrays. As observed for longer picture presentations, brief affective pictures were selectively processed. Specifically, pleasant and unpleasant pictures were associated with an early endogenous negative shift over temporo-occipital sensors compared to neutral images. In addition, affective pictures elicited enlarged late positive potentials over centro-parietal sensor sites relative to neutral images. These data suggest that a quick glimpse of emotionally relevant stimuli appears sufficient to tune the brain for selective perceptual processing.