Biological and immunological properties of haemagglutinin and neuraminidase expressed from cloned cDNAs in prokaryotic and eukaryotic cells

To study the biological and immunological properties of influenza virus surface glycoproteins, cDNA copies of the haemagglutinin (HA) and the neuraminidase (NA) genes of A/WSN/33 influenza virus were cloned and expressed in prokaryotic and eukaryotic cells. In Escherichia coli, maximum expression of HA is obtained only as a fusion protein in which the NH₂-terminal portion is provided by a bacterial protein (i.e. βgal or trpLE′). The HA expressed in bacteria (bacterial HA) is recognized by polyclonal anti-WSN antibodies but not by neutralizing monoclonal antibodies. The antibodies made against the bacterial HA bind to the detergent-treated viral HA, intact virus and live influenza infected cells, but fail to show either haemagglutination inhibition (HI) or virus neutralization. These results suggest that the three-dimensional structure as well as the antigenic epitopes of the bacterial HA are different from that of native viral HA. HA, expressed from cDNA in cultured animal cells, is shown to possess the structural features of the native viral HA. It is glycosylated, transported to the apical domain of the plasma membrane of polarized cells, causes haemadsorption and can induce cell to cell fusion at low pH after proteolytic cleavage. An attempt was made to define the structural features of HA required for sorting and directional transport by making chimeras with vesicular stomatitis virus G (VSV G) protein either by switching the amino terminus or the carboxy terminus of HA with that of VSV G. These chimeric proteins were translocated across the rough endoplasmic reticulum (RER) but were blocked in transport between the RER and cell membrane. These preliminary results indicate that the three-dimensional structure, in addition to specific sequences, may play a critical role in the transport process. More precise constructions are in progress to delineate the functions of the different domains of HA molecule. HA has been expressed in the lower eukaryote Saccharomyces cerevisiae (baker's yeast). The HA expressed in yeast (yeast-HA) is glycosylated and membrane-bound. It is recognized by both the polyclonal and neutralizing monoclonal antibodies made against the native HA. These results suggest that the yeast-HA retains the antigenic epitopes present in the native viral HA and therefore may be of significance in the development of a subunit vaccine. NA, the other influenza glycoprotein, has been expressed from the cloned cDNA in cultured monkey kidney cells and the expressed NA, like the native viral NA, is glycosylated and enzymatically active. Furthermore, it has been shown that the NH₂-terminus of NA, in addition to providing the anchor function, also provides the signal function for translocation across the rough endoplasmic reticulum (RER). Like HA, NA expressed from cloned cDNA is preferentially transported to the apical domain of the plasma membrane of polarized epithelial cells.     

Ex vivo elimination of lymphoblastic leukemia cells from human marrow by mafosfamid

Studies were performed to evaluate the anti-tumor activity of mafosfamid, a new synthetic derivative of cyclophosphamide. We tested its ability to eliminate lymphoblastic leukemia cells from autologous bone marrow grafts following a 30 min preincubation in a highly sensitive clonogenic assay. Treatment with 50-100 micrograms mafosfamid/ml eliminated more than 4 logs of contaminating clonogenic tumor cells from a 200-fold excess of normal bone marrow. Flow cytometric studies showed differences in cell cycle kinetics between mafosfamid-resistant and mafosfamid-susceptible tumor cell clones. Compared to drug susceptible clonogenic tumor cells, clones that resisted treatment with 100 micrograms mafosfamid/ml exhibited a smaller percentage of cells in S-phase, indicating that mafosfamid is mostly cytotoxic to rapidly cycling tumor cells. The combination of mafosfamid and a target cell selective immunotoxin containing pokeweed anti-viral protein was superior to mafosfamid alone or immunotoxin alone for purging mafosfamid-resistant leukemic cells from human marrow.     

Anterior chemotherapy in esophageal cancer

Front loading chemotherapy using methotrexate (200 mg/m2) alone or methotrexate (200 mg/m2) with cisplatin (20 mg/m2 daily for 5 days) was used in epidermoid carcinoma of esophagus. Evaluation after two courses showed objective response of 50% or greater in 48% of patients with methotrexate alone. Response rate was increased to 76.2% with addition of cisplatin to methotrexate. Small lesions (less than 10 cm) showed better response as compared to advanced cases. Therapy was generally well tolerated and good palliation was obtained even after the first course. Postchemotherpy treatment either with surgery or radiotherapy was tolerated without any major complications. The data confirm the short-term usefulness of initial chemotherapy with methotrexate and cisplatin in esophageal cancer. Results of prolonged follow-up will help to evaluate the role of front loading chemotherapy on long-term survival.     

STEREOCHEMICAL CRITERIA FOR POLYPEPTIDE AND PROTEIN CHAIN CONFORMATIONS.

As a first step in the study of the effect of the distortions of the angle NC^aC' (τ) at the α-carbon atom and the planarity of the peptide unit (measured by parameter ω) on the steric map of two linked peptide units, these distortions have been analysed from known crystal structure data on amino acids, peptides and the globular proteins, myoglobin, lysozyme, α-chymotrypsin, carboxypeptidase-A, and ribonuclease-S. Corresponding to three different values each of τ and ω around the ideal values (of 110° and 180° respectively) the contact maps are drawn. Most of the observed conformations of non-glycyl residues in the globular proteins are found to be within the allowed regions. The 3 → 1 type of N-H. O = C hydrogen bond between adjacent units is considered and the possibility of the formation of this hydrogen bond around φ= +90° and φ = -90° (leading to a left-handed helix, if continued) is explored in more detail. The conformations at some of the residues in α-chymotrypsin and carboxypeptidase-A are shown to form the right-handed 3 → 1 type of hydrogen bonds.     

Massive gastrointestinal bleeding in a patient with AIDS

Cytomegalovirus enteritis can lead to gastrointestinal bleeding in patients with the acquired immune deficiency syndrome. The commonest site of involvement is the colon, followed by the stomach and terminal ileum. Most of these lesions can be diagnosed by colonoscopy or gastroscopy. We present our experience of a patient with cytomegalovirus infection involving only the proximal jejunum causing massive lower gastrointestinal bleeding. Conventional endoscopy and imaging had failed to locate the source of bleeding. Enteroscopy performed at the time of laparotomy showed an ulcerated lesion in the jejunum. Resection followed by histological examination of the resected area confirmed the diagnosis of cytomegalovirus infection. In addition to highly active antiretroviral therapy, ganciclovir was given for 14 days in a dose of 5 mg/kg twice a day and tapered over a period of 3 months. There has been no further episode of gastrointestinal bleeding over a follow up of 9 months.     

Anthropometric indicators of body composition in young adults: relation to size at birth and serial measurements of body mass index in childhood in the New Delhi birth cohort

BACKGROUND: South Asians have a muscle-thin but adipose body phenotype and high rates of obesity-related disease. Adult body composition may be predictable in early life. OBJECTIVE: Anthropometric indexes of adult body composition were examined in relation to birth size and body mass index (BMI) during childhood. DESIGN: A population-based cohort of 1526 men and women aged 26-32 y in Delhi, India, who were measured sequentially from birth until 21 y of age were followed up. Adult weight, height, skinfold thicknesses, and waist and hip circumferences were measured. BMI and indexes of adiposity (sum of skinfold thicknesses), central adiposity (waist-hip ratio), and lean mass (residual values after adjustment of BMI for skinfold thicknesses and height) were derived. RESULTS: Mean birth weight was 2851 g. As children, many subjects were underweight-for-age (>2 SDs below the National Center for Health Statistics mean; 53% at 2 y), but as adults, 47% were overweight, 11% were obese, and 51% were centrally obese (according to World Health Organization criteria). Birth weight was positively related to adult lean mass (P < 0.001) and, in women only, to adiposity (P = 0.006) but was unrelated to central adiposity. BMI from birth to age 21 y was increasingly strongly positively correlated with all outcomes. BMI and BMI gain in infancy and early childhood were correlated more strongly with adult lean mass than with adiposity or central adiposity. Higher BMI and greater BMI gain in late childhood and adolescence were associated with increased adult adiposity and central adiposity. CONCLUSIONS: Birth weight and BMI gain during infancy and early childhood predict adult lean mass more strongly than adult adiposity. Greater BMI gain in late childhood and adolescence predicts increased adult adiposity.     

Effect of gender on the manifestations of celiac disease: evidence for greater malabsorption in men

OBJECTIVE: Because celiac disease is a female-predominant disease we investigated the influence of gender on clinical manifestations of the disease in the United States. MATERIAL AND METHODS: Data were obtained on biopsy-proven adult patients with celiac disease from a database of patients seen between 1981 and 2001 in a University-based referral center. Z scores were calculated to adjust for age, ethnicity and gender using the National Health and Nutrition Examination Survey database as controls. RESULTS: The cohort consisted of 323 patients (211 F, 112 M). Men had a shorter duration of symptoms than women (p=0.006). There was no gender difference in the age at diagnosis or mode of presentation. Body mass index (BMI), mean hemoglobin and ferritin values were lower in women than in men, but the Z scores for these values were not significantly different, indicating that the differences are physiological. All lipid values were low (negative Z scores). Men had lower total cholesterol (162.0+/-46.5mg/dl) compared to women (181.0+/-40.0mg/dl), p=0.02 and lower Z scores (-1.10+/-1.1) compared to women (-0.71+/-0.9), p=0.04. Men had lower bone density T scores at the radius (p=0.07). Autoimmune diseases were present in 30.7% with a female to male ratio of 1:1, compared to the general population in which 3.2% have autoimmune diseases with a female predominance. CONCLUSIONS: Most gender differences in celiac disease are physiological. However, men have indirect evidence of greater malabsorption than females and have female-predominant associated diseases when they present with celiac disease.