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.     

Bone metastases from thyroid carcinoma: a histopathologic study with clinical correlates

CONTEXT: Only limited information exists on the pathologic aspects of thyroid carcinomas with bone metastases, most large studies having concentrated mainly on their clinical features. OBJECTIVE: To study in detail the morphologic features of thyroid carcinomas with skeletal metastases. DESIGN: Seventy-nine cases of thyroid carcinoma with bone metastases treated at Memorial Sloan-Kettering Cancer Center, New York, NY, between 1964 and 1998 were investigated, with emphasis on the pathology of the primary and/or metastatic tumors and comparison of the morphologic features of the tumors at both the sites, wherever possible. The tumors were also compared for various clinical parameters. RESULTS: The cohort consisted of 22 papillary, 17 follicular, 16 insular, 10 anaplastic, 9 Hürthle cell, and 5 medullary carcinomas. Of these cases, 68% had poorly differentiated or undifferentiated features in the primary and/or metastatic tumors. The metastatic tumors were better differentiated than the primary in one third of the cases (6 of 18). Only one case showed a less differentiated metastasis. The overall 5- and 10-year survival probabilities after the bone metastases were 29% and 13%, respectively (Kaplan-Meier method). Although both the tumor type and differentiation seemed to affect survivals after bone metastasis (P =.007 and.012, respectively) (log-rank test), this was primarily due to the much worse prognosis in the cases of anaplastic and medullary carcinoma. Cases of Hürthle cell carcinoma showed the longest median survival. There was no significant difference in survival among patients up to or older than 45 years at the time of metastases (P =.31). CONCLUSIONS: Most thyroid carcinomas with bone metastases are of papillary type, and most have poorly differentiated or undifferentiated features. The influence of the microscopic tumor type and tumor differentiation on survival after bone metastasis primarily appears to be due to the much worse prognosis among anaplastic and medullary carcinomas. Age at diagnosis of bone metastases does not influence survivals.     

Low molecular weight proteinuria exacerbates experimental ischemic renal injury

To determine whether tubular reabsorption of low molecular weight proteins (LMWPs) alters ischemic tubular injury, rats were infused with 25 mg of lysozyme (isoelectric point (pI) 11.3), cytochrome C (pI 10.6), ribonuclease (pI 8.7), or myoglobin (pI 7.0), and during this time 25 minutes of bilateral renal artery occlusion (RAO) was induced. RAO control rats received either saline or 25 mg of albumin. Renal injury was assessed 24 hours later by blood urea nitrogen, creatinine, and histology. Lysozyme, ribonuclease, and myoglobin each exacerbated ischemic damage (increased tubular necrosis, cast formation, azotemia), but to comparable degrees (e.g., blood urea nitrogen range 75 +/- 8 to 100 +/- 5 mg/dl versus controls, 29 +/- 2 to 36 +/- 7; p less than 0.01). Rendering lysozyme anionic (pI 4.5) by succinylation did not diminish its acute renal failure-potentiating effect. Cytochrome C which is freely filtered but poorly reabsorbed had a minimal impact on the ischemic process. Infusion of LMWPs did not alter blood pressure, renal blood flow, or induce renal injury in the absence of RAO. During a sublethal ischemic event (10 minutes of RAO) LMWP infusion exacerbated proximal tubular luminal membrane damage before an adverse effect on other critical determinants of cell integrity were apparent (adenine nucleotide pools, oxidant stress). We conclude that endocytic LMWP reabsorption by proximal tubules can exacerbate superimposed ischemic tubular necrosis independent of any direct nephrotoxic protein effect. This action is not influenced by protein isoelectric point and appears to be mediated by a primary intensification of ischemic luminal membrane damage.     

Gender paradox in cardiac calcium deposits in middle-aged and elderly patients: mitral annular and coronary calcifications interrelationship

OBJECTIVES: mitral annular calcification (MAC) occurs mainly in middle-aged and elderly patients and can lead to serious clinical consequences. Male predominance in the prevalence of coronary disease is well-established. Paradoxically, the prevalence of MAC, which is theoretically based on the same etiological mechanisms as coronary atherosclerosis, seems to be predominant in postmenopausal women. The goal of this work was to investigate gender influences on interrelationship between MAC and coronary calcifications (CC) in the same population of middle-aged and elderly patients with increased cardiovascular risk. METHODS: the study comprised 522 patients (284 men and 238 postmenopausal women, aged 52-80 years, mean 65+/-6), who were recruited to the International Nifedipine GITS Study of Intervention as a Goal in Hypertension Treatment (INSIGHT) study in our region. They underwent both fast spiral computed tomography of the heart and echo-Doppler. MAC was defined as advanced when its thickness was > or =5mm; otherwise it was defined as trivial. RESULTS: there were 37 (16%) women and 25 (9%) men with advanced MAC (AMAC), 97 (41%) women and 118 (42%) men with trivial MAC and 104 (44%) women and 141 (50%) men without MAC. The prevalence of any type of CC was significantly higher among men (P=0. 001). In sharp contrast to the distinct male predominance in coronary disease, AMAC was more prevalent among women. In patients without CC prevalence was 9 and 4%, increasing to 16 and 8% in those with nonsevere CC and to 38 and 14% in patients with severe CC, respectively (P=0.001). Multivariate analysis showed that AMAC can predict the presence of severe CC in women and men, with OR of 4.1 and 2.6 (CI 1.2-14.8 and 1.0-10.6) and coronary disease with OR of 2. 5 and 2.5 (CI 0.6-10.6 and 1.0-6.4), respectively. CONCLUSIONS: AMAC signifies a high probability of coronary atherosclerosis in patients of both genders. The inverted gender predominance in the prevalence of annular calcification and CC could be explained by additional etiological (likely osteoporotic) mechanisms of MAC development among postmenopausal women.     

Analysis of the RNA species isolated from defective particles of vesicular stomatitis virus.

Serial high multiplicity passage of a cloned stock of vesicular stomatitis virus was found to generate defective interfering particles containing three size classes of RNA, with sedimentaiton coefficients of 31 S, 23 S and 19 S. The 31 S and 23 S RNA species were found to be complementary to both the 12 to 18 S and 31 S size classes of VSV mRNAs. The 19 S class of RNA was found to be partially base-paired. All three RNA species were found to contain ppAp at their 5' termini.     

Phorbol myristate acetate and calcium ionophore A23187-stimulated human T cells do not express high-affinity IL-2 receptors

Phorbol myristate acetage (PMA) and Ca2+ ionophore A23187 mimic early signal transduction pathways and activate purified human T cells to secrete large quantities of interleukin-2 (IL-2) and to proliferate. Despite producing 50-100-fold more IL-2 than phytohaemagglutinin (PHA)-activated peripheral blood lymphocytes (PBL), PMA/A23187-stimulated human T cells proliferate less than cells activated by PHA. Washing the cells to remove PMA/A23187 was found to increase cellular proliferation two to five-fold. High-affinity IL-2R (HA-IL-2R) were found to be expressed by human T cells that had been washed 24 hr after PMA/A23187 stimulation and recultured without stimulus for an additional 48 hr, but not by T cells constantly exposed to PMA/A23187 for 72 hr. Radioligand binding studies with [125I]IL-2 demonstrated that while the alpha (p55) and beta (p70-75) subunits of HA-IL-2R were both present on the constant PMA/A23187-stimulated T cells, they did not appear to associate to form functional HA-IL-2R. This defect in the expression of bio-active HA-IL-2R on constant PMA/A23187-stimulated human T cells seems to account for their low proliferative response.     

Utilization of the 2017 diagnostic criteria for hEDS by the Toronto GoodHope Ehlers–Danlos syndrome clinic: A retrospective review

The new 2017 diagnostic criteria for hypermobile Ehlers–Danlos Syndrome (hEDS) provide a framework for diagnosing hEDS but are more stringent than the previous Villefranche criteria. Our clinical experience at the GoodHope EDS clinic was that the 2017 criteria left many highly symptomatic patients without a diagnosis of hEDS. We conducted a retrospective cohort study to confirm our clinic experience and assess the accuracy of the 2017 diagnostic criteria for hEDS in patients who had a previous hEDS diagnosis based on the Villefranche criteria. Our study found that 15% (n = 20 of 131) of patients with a prior diagnosis of hEDS met the 2017 diagnostic criteria, and many of the traits used to distinguish hEDS were not significantly more frequent in patients who met 2017 criteria versus those who did not. In both groups objective systemic manifestations were found less frequently than subjective systemic manifestations. Beighton score (BS) as assessed by primary care practitioner was found to be higher than assessment by EDS practitioner in 81% (n = 74 of 91) of cases. Generalized joint hypermobility was confirmed in only 46% (n = 51 of 111) of patients who had a previous diagnosis of hEDS. Higher BS did not correlate with increased number of systemic manifestations in our cohort. Common comorbidities of hEDS were found with similar frequency in those who met 2017 criteria and those who did not. Based on our cohort, the 2017 hEDS diagnostic criteria require refinement to improve its diagnostic accuracy.     

Hepatitis C virus and its genotypes in patients suffering from chronic hepatitis C with or without a cryoglobulinemia-related syndrome

Recently, evidence has been presented for a possible association between hepatitis C virus (HCV) infection and essential mixed cryoglobulinemia (EMC). Eleven consecutive patients with EMC and two with cryoglobulinemia type I were examined for the presence of markers of HCV infection. Eleven of 13 patients (10 with EMC and 1 with type I cryoglobulinemia) had anti-HCV antibodies (as determined by a second generation anti-HCV assay) and HCV-RNA in plasma or serum. HCV-RNA was also detected in liver biopsies of five patients. Genotyping showed that HCV genotype 1 was found in 10 of 11 patients with HCV-RNA (9 genotype 1b and 1 genotype 1a) and only one patient had HCV genotype 2. However, a similar high prevalence of genotype 1b (100%) was found in a group of 14 consecutive patients with chronic hepatitis C, who had no clinical evidence of cryoglobulinemia. Concomitant infection was present in three patients with genotypes 2, 3 and 4, respectively. These findings stress the high prevalence of HCV infection in patients with EMC and further study shows that a difference in genotype prevalence was not found between HCV-related EMC and chronic hepatitis C without clinical manifestations of EMC. © 1994 Wiley-Liss, Inc.     

Malignancy may adversely influence the quality and behaviour of oocytes

A case series of eight cycles of in-vitro fertilization (IVF) in five women diagnosed with malignant disorders is presented. These patients chose to defer definitive treatment for a chance for preservation of potential fertility. The response of these patients to ovarian stimulation, and the outcome, was compared with 17 IVF cycles in 12 age- matched patients with isolated tubal infertility. An apparent adverse influence of malignant disease on the quality and behaviour of oocytes was observed. Despite a comparable total number of oocytes per cycle in the two groups, a significantly reduced percentage of mature oocytes was retrieved per cycle from patients with malignant diseases. The oocytes from patients with malignant disorders were of a poorer quality and exhibited a significantly impaired fertilization rate compared to the controls. We propose that neoplastic processes, irrespective of the site or cell of origin, may have a detrimental impact on the biology of oocytes, an effect akin to that seen on spermatozoa in men with certain malignancies.      

Cellular basis of an auto-anti-allotypic mechanism for the maintenance of chronic allotype suppression in the rabbit.

Immunocytochemical identification of antibody-forming cells (AFCs) in situ was used to test the hypothesis that the maintenance of chronic allotype suppression in heterozygous rabbits is the result of an autoimmune B-cell-mediated response. Appreciable numbers of B cells with antibody activity directed against the suppressed allotypic determinant were found in spleen and bone marrow sections of all chronically suppressed rabbits examined. Appropriate double-staining was used to determine that such cells were of the non-suppressed allotype. These cells were indistinguishable from anti-allotypic AFCs found in larger numbers in spleens of normal heterozygous rabbits that had been immunized against a heterologous allotypic determinant. Auto-anti-allotypic AFCs were not found in suppressed rabbits less than 8 week old, nor were they found in normal (non-suppressed) heterozygous rabbits or chimeric rabbits formed by the injection of histocompatible but allotype-mismatched lymphoid cells at birth. The findings reported here support the hypothesis that the long-term maintenance of allotype suppression in the rabbit may result from the suppressive activities of autoimmune B cells. It is suggested that the suppression of an allotype during the first few weeks of life could result in a loss of tolerance to a self-determinant. The kinetics of auto-anti-AFC production support this idea in showing that such cells are generated following the decline of the antibody used to induce suppression. The triggering event may be the emergence of B cells expressing the previously suppressed gene product.