P. Sriramarao

Neutralizing anti‐vascular endothelial growth factor antibody completely inhibits angiogenesis and growth of human prostate carcinoma micro tumors in vivo

Neovascularization mediated by growth factors produced by tumors is critical for the growth of tumors. Vascular endothelial growth factor (VEGF) is one such growth factor. A neutralizing anti‐VEGF antibody (A4.6.1) was recently shown in vivo to inhibit tumor angiogenesis and growth of the human rhabdomyosarcoma cell line A673. The antibody profoundly changed the growth characteristics of the tumor line from a rapidly growing malignancy to a dormant microcolony.

Integrin expression in human melanoma cells with differing invasive and metastatic properties.

During the process of tumor cell invasion and metastasis, tumor cells are known to interact with extracellular matrix proteins, endothelial cells, platelets and other organ-specific structures. Integrins are cell surface molecules which mediate cell-matrix and cell-cell interactions and are likely to be important for tumor cell survival and dissemination. The purpose of this study was to characterize the integrin and proteolytic enzyme repertoire from low (A375P), medium (A375M) and high metastatic (A375SM) human melanoma cell lines. These cell lines are also invasive through human amniotic membranes in vitro and their invasiveness parallels the reported metastatic phenotype. The types and levels of expression of the various integrin receptors were analysed by quantitative immunoprecipitation using a panel of monoclonal antibodies directed to known integrin subunits. In addition, cDNA probes to the integrin subunits were used in quantitative northern blot analysis. These data show that the integrin αvβ3 increases 50- to 100-fold as these cells progress to a more metastatic phenotype. α4β1 levels also appeared to increase several fold, while other β1 integrins did not differ in their expression levels. The increased αvβ3 expression in the more metastatic cells resulted in an increased adhesion to vitronectin and fibrinogen substrates in cell attachment assays. However, αv- and β3-specific antibodies did not inhibit A375 cell invasion through the amnion. Each cell line was found to release similar quantities of a 72-kDa gelatinase/type IV collagenase and tissue type plasminogen activator. These results suggest that during the progression of these tumor cells from a low to high metastatic phenotype, marked changes in integrin expression occurred which may facilitate interactions with platelets, endothelial cells and specific extracellular matrix proteins to promote metastasis.

A novel tenascin type III repeat is part of a complex of tenascin mRNA alternative splices

Sequence analysis of two human tenascin encoding cDNA clones from a cDNA library of the U251 glioblastoma cell line revealed the presence of a novel 276 bp tenascin type III fibronectin like repeat. This alternatively spliced type III repeat designated AD1 is located between the previously identified repeats 10 and 11 and has sequence homology with human, chicken and mouse tenascin type III repeats. These results show that tenascin has at least 16 consecutive fibronectin like type III repeats. PCR amplification of random primed mRNA with specific type III repeat primers revealed a pattern of multiple alternative splices of AD1 and flanking type III repeats. The alternative splice variants were confirmed by direct sequencing. Differences were observed in the expression of the various alternative splices of tenascin mRNA between tumor and normal cells and may thus indicate differences in tenascin isoform expression and function in normal and tumor cells. PCR and Southern analysis of genomic DNA indicate that AD1 is coded by a single exon present in both human and mouse genome.

Structure-antitubercular activity relationship of phenothiazine-type calmodulin antagonists

Six neuroleptic (antipsyrhotic) phenothiazinc derivatives which are calmodulin antagonists were tested for their activity against Mycobacterium tuberculosis H37R, in order to understand their structure-antitubercular activity relationship. Out of the six derivatives tested (trifluoperazine, chlorpromazine, triflupromazine, thioridazine, acetopromazine and fluphenazine), trifluoperazine appears to he a more potent antitubercular drug than others with a minimum inhibitory concentration (MIC) of 5 γg/ml. Chlorpromazine, triflupromazine and thioridazine are also active but less potent and have a higher MIC of 20 γg/ml. Acetopromazine and fluphenazine could not completely inhibit the growth even at a high concentration of 20 γg/ml. These results indicate that a methylpiperazinylpropyl group attached to the nitrogen (position 10) atom and trifluoromethyl group at the second carbon confer antitubercular activity to the phenothiazine molecule. It is suggested that trifluoperazine or one of its derivatives could be useful as one of the drugs in the multi-drug regimen for the treatment of tuberculosis with psychotic problems or vice versa.

Allergenic cross-reactivity between parthenium and ragweed pollen allergens

Cross-reactivity of allergens from the pollen of the Compositae weeds, Parthenium hysterophorus (American feverfew) and Ambrosia (ragweed), in 2 groups of patients with different geographic distributions was studied. Parthenium-sensitive Indian patients, who were never exposed to ragweed, elicited positive skin reactions with ragweed pollen extracts. A significant correlation in the RAST scores of Parthenium and ragweed-specific IgE was observed with the sera of Parthenium and ragweed-sensitive Indian and US patients, respectively. RAST inhibition experiments demonstrated that the binding of IgE antibodies in the sera of ragweed-sensitive patients to short (W1) and giant (W3) ragweed allergen discs could be inhibited by up to 94% by Parthenium pollen extracts. Similar inhibition (up to 82%) was obtained when the sera of Parthenium rhinitis patients were incubated with ragweed allergen extracts. A dose-dependent proliferation of lymphocytes from a Parthenium-sensitive rhinitis patient with elevated levels of both Parthenium and ragweed-specific IgE was observed when incubated with Parthenium and ragweed pollen extracts. A 1.6-fold higher proliferation, however, was observed with Parthenium pollen extract at a concentration of 100 micrograms/ml. These results suggest that shared epitopes present on Parthenium and ragweed pollen allergens are recognized by both Indian and US patients sensitized by exposure to Parthenium and ragweed pollen, respectively. The high degree of cross-reactivity between Parthenium and ragweed pollen allergens suggests that individuals sensitized to Parthenium may develop type-I hypersensitivity reactions to ragweed and vice versa when they travel to regions infested with the weed to which they had not been previously exposed.

Immediate hypersensitivity to Parthenium hysterophorus. II. Clinical studies on the prevalence of parthenium rhinitis

The airborne pollen of the South American weed, Parthenium hysterophorus (American feverfew), accidentally introduced into India was found to he responsible for severe allergic rhinitis. A random clinical survey conducted on 2035 residents of Bangalore city with the aid of questionnaires and skin tests revealed that 7.1% of the study population was suffering from allergic rhinitis due to exposure to Parthenium pollen. Skin‐prick tests performed on 1294 clinic patients suffering from nasobronchial allergy during the past 4 years have also shown that 42.5% were sensitive to Parthenium pollen. IgE and IgG antibodies specific for Parthenium pollen allergens were demonstrable in the sera of Parthenium‐sensitive rhinitis patients. The specificity of these antibodies in Parthenium allergens was established by ELISA. A 7‐ to 11‐fold higher stimulation was observed when lymphocytes from two Parthenium‐sensitive rhinitis patients were treated in vitro with Parthenium pollen extract. To our knowledge, nowhere in the world has such a high incidence of allergic rhinitis due to a single pollen ever been reported.

Differential regulation of eosinophil adhesion under conditions of flow in vivo

The proinflammatory role of eosinophils in patients with allergic inflammation is now well recognized. However, the molecular mechanisms mediating the sequential events of eosinophil recruitment from the blood stream to sites of allergic inflammation under conditions of shear force have not been clearly established. Using the xenogeneic rabbit model system to study human eosinophil adhesion under conditions of flow in vivo, we have demonstrated that eosinophils like neutrophils roll, adhere, and extravasate across cytokine-stimulated endothelial cells at physiological shear rates in vivo. Eosinophils rolling on venular endothelial cells is mediated by L-selectin and VLA-4. Mediators of cellular activation such as GM-CSF, PAF, or PMA had a differential effect on neutrophil and eosinophil receptor expression and their rolling function. It would thus appear that acting sequentially or in concert a variety of cytokines, including GM-CSF, RANTES, IL-5, and specific cell adhesion molecules (VLA-4/VCAM-1) might play a critical role in the selective sequestration of eosinophils and other proinflammatory leukocytes into the inflamed tissues during episodes of allergic inflammation. Further understanding of the function of these mediators as well as other traffic signals that regulate eosinophil adhesion will help in developing better therapeutic strategies to block the emigration of eosinophils from the blood stream, and also to inhibit the activation of eosinophils once they have reached sites of tissue inflammation.

Genes That Regulate Eosinophilic Inflammation

Eosinophils and neutrophils may be considered close cousins, because of their shared bone-marrow origin, circulation in the blood stream, and egress at tissue sites of inflammation. Under the influence of distinct cytokines, both eosinophils and neutrophils differentiate in the bone marrow and enter the bloodstream. Both cell types interact with the surface of inflamed endothelium, diapedese between endothelial cells, and migrate through the extracellular matrix at tissue sites of inflammation, where they eventually release their stores of inflammatory mediators.

Immediate hypersensitivity to parthenium hysterophorus i. association of hla antigens and parthenium rhinitis

Lymphocytes collected from rhinitis subjects with strong positive skin reactions to the pollen allergens of Parthenium hysterophorus (American feverfew) having moderate to high titres of Parthenium‐specific serum IgE were analysed for association of HLA‐antigens covering 13 specificities of HLA‐A, 17 specificities of HLA‐B and eight specificities of HLA‐DR loci by the NIH two‐stage microlymphocytotoxicity assay. Comparison of the phenotypic frequencies of HLA‐A and B antigens between Parthenium rhinitis subjects (n= 22) and control subjects (n= 137) did not suggest any significant association when tested for these antigen specificities. A significant correlation in the association of HLA‐DR3 antigen with a relative risk of 11·33, however, was observed in Parthenium rhinitis subjects (n= 30) when compared to controls (n= 50).

Demonstration of IgE antibodies to nucleic acid antigens in patients with SLE.

Using a combination of avidin-biotin microELISA and solid phase radioimmunoassay, we examined sera from 23 patients with systemic lupus erythematosus (SLE), two patients with established sensitivity to ingested shrimp, and 15 healthy normal subjects. In addition to IgG antibodies, varying amounts of IgE antibodies specific for native DNA (nDNA), denatured or single-stranded DNA (dnDNA), RNA, and tRNA were demonstrable in the sera of SLE patients, but not in the sera of normal subjects. A comparison of the specificity of nucleic acid-specific IgE antibodies present in the sera of shrimp-sensitive patients with those present in the sera of seven SLE patients revealed that the IgE antibodies in the sera of shrimp-sensitive patients specifically recognized shrimp tRNA but not yeast tRNA, calf thymus RNA, or calf thymus DNA, while those present in the sera of patients with SLE recognized all these nucleic acid antigens. The IgE antibodies directed against nDNA, dnDNA, RNA, and tRNA may mediate mast cell and basophil degranulation and thus contribute both to immediate-type hypersensitivity phenomena including hives seen in patients with SLE and to the localization of IgE-nucleic acid complexes in target tissues.