Nonaffinity purification of recombinant gp120 for use in AIDS vaccine development.

The gene encoding the major envelope glycoprotein of the HIV-SF2 isolate was engineered for the secretion of recombinant gp120 (rgp120SF2) from permanent Chinese hamster ovary (CHO) cell lines. Cellular production methods were scaled up and a method for purification of the secreted glycoprotein was devised. Mild purification conditions were selected in order to preserve the native structure of the protein. rgp120SF2 exhibits a molecular weight of 120 kDa in reduced or nonreduced SDS gels; thus the polypeptide chain is intact. Deglycosylated rgp120SF2 has the predicted molecular weight of the polypeptide backbone, 54 kDa. Gel-filtration HPLC in a nondenaturing buffer at neutral pH yields a molecular weight estimate of approximately 120 kDa. Purified rgp120 closely resembles authentic viral gp120 by several physical, chemical, and immunochemical tests. rgp120SF2 reacts strongly with human HIV-positive sera, monoclonal antibodies reactive with HIV-SF2 and HIV-MN viral envelope, and a human virus-neutralizing monoclonal antibody that maps to a conserved discontinuous epitope on HIV-1 gp120. Purified rgp120SF2 forms a 1:1 molecular complex with soluble recombinant human CD4 (rCD4) receptor, as demonstrated by gel-filtration HPLC; binding is high affinity (Kd approximately 2 x 10(-9) M).

Importance of hypervariable regions of HIV-1 gp120 in the generation of virus neutralizing antibodies.

Variants of the envelope gene of the HIV-SF2 isolate of HIV-1 with deletions of one or more of the hypervariable domains of gp120 were produced in genetically engineered yeast as nonglycosylated denatured polypeptide analogs of gp120. Purified antigens were used to immunize experimental animals to determine whether the removal of hypervariable regions from this type of gp120 immunogen had any effect on (1) the ability of the antigen to elicit virus neutralizing antibodies; and (2) the isolate specificity of the neutralizing antibodies that were elicited. The results of these studies demonstrate that, in addition to the previously identified V3 domain, at least two other hypervariable regions in gp120 are capable of eliciting neutralizing antibodies in experimental animals. However, when all five of the hypervariable regions were deleted, the resulting antigen was no longer capable of eliciting neutralizing antibodies. Finally, the neutralizing antibodies elicited by all of these nonglycosylated antigens were effective against HIV-SF2, the isolate from which the antigens were derived, but were not able to neutralize two divergent isolates, HIV-BRU or HIV-Zr6.

Complete post-traumatic ptosis: a mechanism for recovery?

Traumatic blepharoptosis, although considered relatively rare, is an entity which demands recognition if one is to achieve optimal results. Reports of levator injury following orbital, ocular, and adnexal surgery, as well as in cataract and blepharoplasty procedures, are well described. In most cases eventuating in complete ptosis, levator disinsertion is the anatomic correlate, the ptosis is usually permanent, and surgical intervention is often indicated. We have observed two cases of transient, complete post-traumatic ptosis which have recovered by 6 weeks with expectant management. We believe this entity to be more pervasive than the current literature seems to reflect and emphasis is placed on nonoperative therapy. This paper reviews the anatomical considerations relevant to the function of the levator complex as well as the possible mechanisms for its injury.

Variants of human tissue-type plasminogen activator substituted at the protease cleavage site and glycosylation sites, and truncated at the N- and C-termini.

Mutations were directed to specific regions of the human tissue-type plasminogen activator (t-PA) gene in an effort to better define structure-function relationships of the enzyme. Three types of modifications were effected by in vitro mutagenesis: elimination of glycosylation sites; substitutions of amino acids at the cleavage site for conversion of single-chain t-PA to two-chain t-PA; and truncations of the N- and C-termini. Thirteen variants were purified from permanent CHO cell lines and analyzed for specific activity, fibrin stimulation, fibrin binding, inhibition by plasminogen activator inhibitor-2 (PAI-2) and half-life. The results of these analyses are: (i) variants with carbohydrate-depleted kringle domains possessed higher specific activities than wild-type t-PA; (ii) a cleavage site variant substituted at Arg275 with Gly had greatly reduced specific activity; (iii) two variants substituted at Lys277 exhibited altered interactions with PAI-2; (iv) the variant with a truncated C-terminus had reduced activity in the absence of fibrin; and (v) no variants had significantly altered half-lives. In order to test the effects of combining mutations, four additional variants were produced. Each combination variant retained at least one of the altered properties observed in the original variants, and in three of the variants the diverse properties were additive.

Rhabdomyosarcoma of the alveolar ridge in an adult.

Rhabdomyosarcoma of the head and neck is rare in adults, and past reports indicate that it is a more aggressive disease than that which is found in children. We report the case of a 63-year-old woman with an alveolar type of rhabdomyosarcoma located on the mandibular alveolar ridge. Rhabdomyosarcoma of the oral cavity appears to carry a particularly poor prognosis, especially when bone is involved. Multimodal treatment with surgery, radiation therapy, and chemotherapy is indicated in all patients.

The effect of ancrod on perfusion of myocutaneous flaps.

Ancrod is a defibrinogenating enzyme that also stimulates production of prostacyclin and, thereby, acts indirectly as a vasodilator and inhibitor of platelet aggregation. The actions of ancrod have provided the rationale for successful clinical use in the treatment of peripheral vascular disease and venous thrombosis. A porcine panniculus carnosus myocutaneous flap model was utilized to study the effects of ancrod on flap perfusion and viability. Ancrod treatment appeared to increase perfusion at one distal flap site, as measured by dermofluorometry. A small but not statistically significant increase in percent of viable flap surface area was also observed in ancrod-treated piglets.

Progressive multifocal leukoencephalopathy and magnetic resonance imaging.

Progressive multifocal leukoencephalopathy developed in a homosexual man with underlying Hodgkin's disease. Computed tomography and magnetic resonance imaging of the brain demonstrated multiple lesions, more in gray than white matter. Brain biopsy established the diagnosis of progressive multifocal leukoencephalopathy. Magnetic resonance imaging was found useful for detecting brain lesions and for localizing an accessible lesion for biopsy.

The effect of alpha-adrenergic stimulation and blockade on perfusion of myocutaneous flaps.

A porcine myocutaneous flap model was utilized to assess the development of denervation adrenergic hypersensitivity and to determine the effects of the alpha-adrenergic blocking agent--phenoxybenzamine--on flap blood perfusion. During intravenous administration of norepinephrine, blood flow to the flaps and control skin was monitored simultaneously, using laser Doppler velocimetry and dermofluorometry. A relative decrease in myocutaneous flap blood flow, as compared to control skin in response to norepinephrine infusion, was observed at between 2 and 7 days following flap elevation. This is the same time period during which norepinephrine content of skin flaps is diminished, and suggests development of an increased sensitivity to adrenergic stimulation. Administration of phenoxybenzamine blunted norepinephrine-induced pressor responses and blocked development of adrenergic hypersensitivity in the porcine myocutaneous flap model. Phenoxybenzamine significantly increased flap blood perfusion (as measured by dermofluorometry).