Metastatic hibernomas in transgenic mice expressing an alpha-amylase-SV40 T antigen hybrid gene.

Mice transgenic for a hybrid gene containing the liver promoter of the mouse amylase gene (Amy-1a) fused to the SV40 tumor antigen coding region unexpected developed malignant brown adipose tissue tumors (malignant hibernomas). Expression of the alpha-amylase gene had previously been thought to be confined to the liver parotid, and pancreas; however, analysis of white and brown adipose tissue from nontransgenic mice revealed expression of the endogenous Amy-1a gene in these tissues. Gene constructs driven by the Amy-1a liver promoter thus provide a means of targeting gene expression to the adipocyte cell lineage in transgenic mice. Moreover the high frequency of metastases in the liver, lungs, spleen, heart, and adrenals of these mice provides an experimental system in which to study the development of disseminated malignancy.

Budding yeast Cdc20: a target of the spindle checkpoint.

The spindle checkpoint regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. In the two-hybrid system, three proteins that are components of the checkpoint, Mad1, Mad2, and Mad3, were shown to interact with Cdc20, a protein required for exit from mitosis. Mad2 and Mad3 coprecipitated with Cdc20 at all stages of the cell cycle. The binding of Mad2 depended on Mad1 and that of Mad3 on Mad1 and Mad2. Overexpression of Cdc20 allowed cells with a depolymerized spindle or damaged DNA to leave mitosis but did not overcome the arrest caused by unreplicated DNA. Mutants in Cdc20 that were resistant to the spindle checkpoint no longer bound Mad proteins, suggesting that Cdc20 is the target of the spindle checkpoint.

Acute exacerbations of chronic type B hepatitis are accompanied by increased T cell responses to hepatitis B core and e antigens. Implications for hepatitis B e antigen seroconversion.

T cell proliferative responses to hepatitis B virus-encoded envelope antigen (S + preS2 + preS1), recombinant core antigen (HBcAg), and natural hepatitis B e antigen (HBeAg) were examined in 22 HBeAg-positive patients with chronic type B hepatitis and 17 healthy hepatitis B surface antigen (HBsAg) carriers. The results showed that HBeAg-positive patients had (a) higher levels of T cell responses to HBcAg/HBeAg than those of healthy HBsAg carriers (P less than 0.001 and P less than 0.01, respectively); (b) a further increase in these T cell responses during acute exacerbations (P less than 0.05 and P less than 0.05, respectively); (c) subsidence in the T cell responses to HBcAg/HBeAg after recovery from acute exacerbations and HBeAg seroconversion, whereas the responses would persist at high levels if the patients did not enter a clinical remission; and (d) low levels of T cell responses to S + preS2 + preS1 either before or after HBeAg seroconversion. The appearance of increasing T cell responses to HBcAg/HBeAg usually occurred in the early phase of acute exacerbations. These findings imply that HBcAg/HBeAg-specific T cells play an important role in the exacerbations of chronic hepatitis B and in HBeAg seroconversion. HBcAg/HBeAg-specific precursor T cell frequencies were serially studied in selected cases by limiting dilution assay. Elevation (two- to fourfold) of HBcAg/HBeAg-specific precursor T cell frequencies contributed to the increase of HBcAg/HBeAg-specific T cell proliferation during acute exacerbations.

A novel yeast screen for mitotic arrest mutants identifies DOC1, a new gene involved in cyclin proteolysis.

B-type cyclins are rapidly degraded at the transition between metaphase and anaphase and their ubiquitin-mediated proteolysis is required for cells to exit mitosis. We used a novel enrichment to isolate new budding mutants that arrest the cell cycle in mitosis. Most of these mutants lie in the CDC16, CDC23, and CDC27 genes, which have already been shown to play a role in cyclin proteolysis and encode components of a 20S complex (called the cyclosome or anaphase promoting complex) that ubiquitinates mitotic cyclins. We show that mutations in CDC26 and a novel gene, DOC1, also prevent mitotic cyclin proteolysis. Mutants in either gene arrest as large budded cells with high levels of the major mitotic cyclin (Clb2) protein at 37 degrees C and cannot degrade Clb2 in G1-arrested cells. Cdc26 associates in vivo with Doc1, Cdc16, Cdc23, and Cdc27. In addition, the majority of Doc1 cosediments at 20S with Cdc27 in a sucrose gradient, indicating that Cdc26 and Doc1 are components of the anaphase promoting complex.

Induction of antitumor immunity with combination of HER2/neu DNA vaccine and interleukin 2 gene-modified tumor vaccine.

The therapeutic effects of both cytokine-secreting tumor vaccine and DNA vaccine were studied using mouse MBT-2 bladder cancer cells as a model. Cytokine-secreting MBT-2 cells were obtained by infecting cells with retroviral particles containing interleukin (IL) 2-, IL-4-, or granulocyte-macrophage colony-stimulating factor (GM-CSF)-expression vector. The MBT-2-IL-2 cells were not tumorigenic in syngenic C3H mice at all. Tumor formation decreased significantly for the MBT-2-GM-CSF cells. MBT-2-IL-2, -IL-4, and -GM-CSF cells were killed by irradiation and tested as tumor vaccines. The irradiated MBT2-IL-2 cells could complete protect mice from the growth of the preexisting tumor cells, and the immune memory lasted for 8 months. On the other hand, irradiated MBT-2-IL-4 and MBT-2-GM-CSF cells were less effective. When the loading tumor mass increased, all tumor vaccines lost protective effects. DNA vaccine encoding the tumor antigen neu was additionally tested to improve the therapeutic efficacy. Coinjection of 60 microg pSV-neu DNA was effective in enhancing the antitumor effects of MBT2-IL-2; however, DNA vaccine alone cannot prevent the progression of the preexisting tumor. Immunohistochemical analysis of tumor infiltrate revealed massive increase of CD4+ lymphoid cells in the group of mice treated with both DNA vaccine and IL-2-secreted tumor vaccine. Western blotting demonstrated the presence of anti-neu antibody in the serum from immunized mice. In contrast, combination of DNA vaccine and MBT-2-GM-CSF has no additive effect. The results indicate the combination of DNA vaccine and IL-2-secreting tumor vaccine can additionally improve therapeutic efficacy, and the efficacy is correlated with the increase of CD4+ T lymphocytes and anti-neu antibody.

Tumor-induced immunosuppression: a barrier to immunotherapy of large tumors by cytokine-secreting tumor vaccine.

An active immunotherapy strategy with cytokine-assisted tumor vaccine, although often effective for small tumor burdens, is much less so for large tumor burdens. This study examines how large tumors might suppress the T cell functions and escape from the immune responses elicited by a granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor vaccine. According to our results, the T cells isolated from the tumor-bearing mice treated late with the vaccine failed to confer protective activity on naive mice against a wild-type tumor challenge, unlike those isolated from the early-treated group. Nevertheless, the antitumor activity of the inactive T cells could be restored on in vitro stimulation. Expression of transforming growth factor beta (TGF-beta) and interleukin 10 (IL-10), the potent immunosuppressive factors, was detected in the parental tumor cell line RLmale 1 (a murine T leukemia cell line), as well as in the tumor region, the levels of which correlated with tumor progression. An in vitro assay of T cell functions revealed that the TGF-beta in the conditioned medium of RLmale 1 cells mainly affected the activation, whereas the IL-1male affected the activation to a lesser extent, but significantly affected the cytolytic activity, of tumor-specific T cells. The immunosuppressive activity of IL-10 was also signified by the findings that administration of the conditioned medium of RLmale 1 cultured in a serum-free medium, in which the TGF-beta activity was then lost while the IL-10 activity still remained, or of recombinant IL-10 to the early-treated group of mice abrogated the known efficacy of tumor vaccine on the small tumors. These data suggested that the efficacy of cytokine-secreting tumor vaccine was blocked by the immunosuppressive factors secreted from the large tumors. The results have important implications for the clinical design of immunotherapeutic strategies for advanced cancer patients.

Regression of established mouse leukemia by GM-CSF-transduced tumor vaccine: implications for cytotoxic T lymphocyte responses and tumor burdens.

This study investigated the therapeutic effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on a mouse leukemia model. By using a retroviral vector, mouse GM-CSF cDNA was transduced into a highly tumorigenic T leukemia cell line, RL male 1. Injection of GM-CSF-secreting RL male 1 cells into syngeneic BALB/c mice elicited protective immunity in the animals, which could regress preestablished tumors introduced either by a subcutaneous or in an intravenous route. However, the therapeutic effects were less prominent in the mice inoculated with a large tumor load or in mice treated later. Winn tests further demonstrated that the splenocytes from the late-treated group conferred poorer protective effects in terms of reducing the growth of parental RL male 1 cells in naive mice than the splenocytes from the early-treated group. Nonetheless, upon stimulation in vitro, the activity of tumor-specific cytotoxic T lymphocytes (CTL) was comparable in the splenocytes of both groups of mice. Histological analysis also indicated that the CD8+ T cells appeared as early as 3 days following vaccination at the vaccine sites and at the tumor sites in both groups of mice. Above observations implied that the T cells in the animals bearing large tumors appeared to be in a state of suppression or anergy. Systematic histological analyses for 2 weeks provided further insight into various infiltrates at the vaccine sites and at the tumor sites in response to the inoculation of GM-CSF-secreting tumor vaccine.

Long-term expression of the biologically active growth hormone in genetically modified fibroblasts after implantation into a hypophysectomized rat.

We employed the hypophysectomized rats as an animal model to explore the feasibility of using genetically engineered fibroblast cells for growth hormone gene therapy. An internal ribosome entry site (IRES)-directed bicistronic retroviral vector, PSN, which contained a porcine growth hormone (pGH) cDNA at the first cistron and a Neo(r) gene at the second cistron was used to infect primary rat embryo fibroblast (REF) cells. The infected cells (5 x 10(6) cells/rat) were injected directly into the peritoneum of syngeneic hypophysectomized rats. We demonstrate that the implanted PSN-infected REF cells could secrete biologically active pGH in vivo, leading to significant growth of the tibia at day 15 and day 57 post-implantation. We also treated the PSN-infected REF cells with collagen to form a tissue-like structure. The skin-like discs were grafted underneath the skin on the back of rats and cells were retrieved at different times. Using two criteria, semiquantitative reverse transcription-polymerase chain reaction on the pGH RNA extracted from the explants and G418 resistance conferred from the explanted cells, we demonstrate that pGH was expressed in the implanted fibroblasts up to 70 days. Despite the fact that the total pGH RNA level was reduced in the explants of long-time post-implantation, which was probably due to the reduction of transduced cells retained in the explants, the specific efficiencies of pGH RNA expression from these explants were maintained as high as the primary PSN-infected REF prior implantation. These results suggest that fibroblast cells are capable of expressing the foreign genes persistently in vivo.

Regression of orthotopic brain tumors by cytokine-assisted tumor vaccines primed in the brain.

This study investigated the therapeutic effects of a rat glioma cell line, C6, that was engineered to secrete mouse GM-CSF (mGM-CSF) on intracerebral (i.c.) brain tumors. Significant antitumor immunity was induced in rats when the live or irradiated mGM-CSF-secreting tumor vaccine was implanted i.c. The antitumor activity was effective on small tumors and, to a lesser extent, on large tumors or tumors existing in vivo for a longer duration. Immunohistochemical analysis revealed cellular infiltrates (granulocytes, macrophages, and CD4+ and CD8+ T cells) at both the vaccine site and the tumor site, indicating that immune responses were similarly activated when tumor vaccine was inoculated in the brain, as at the subcutis. Additional studies demonstrated that the therapeutic effects of tumor vaccines on the large tumors or the long-existing tumors were enhanced by strategies such as increasing the dosage of tumor vaccines, using combined vaccines consisting of mGM-CSF and human interleukin-2, or combining tumor vaccine with herpes simplex virus thymidine kinase/ganciclovir treatment. All of the modified strategies yielded synergistic therapeutic effects on the large tumor burdens. The data presented herein suggest that cytokine gene therapy is highly promising for the treatment of i.c. gliomas.

Long term clinical and virologic outcome of primary hepatitis C virus infection in children: a prospective study.

To investigate the long term natural course of primary hepatitis C virus infection in children from the beginning, we prospectively followed up 88 children at risk because of frequent blood transfusions or of hepatitis C virus infection from the mother. Ten of the 88 children contracted primary infection during follow-up. In the acute stage of infection acute hepatitis with elevation of aminotransferases and a positive IgM antibody was found in both children infected during open heart surgery, 3 of the 5 multiply transfused children with congenital hemolytic anemia and none of the 3 infants infected by their mothers. Four of the 10 children later lost hepatitis C virus RNA, whereas 6 had a chronic course. Three of the latter 6 children had abnormal aminotransferase activities in the chronic phase. Our study suggests that the very young age of primary infection and the underlying status of the host may affect the clinical course of hepatitis C virus infection in children.

ATP-binding domain of NTPase/helicase as a target for hepatitis C antiviral therapy.

To enhance the inhibitory potential of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) vs hepatitis C virus (HCV) NTPase/helicase, ribavirin-5'-triphosphate (ribavirin-TP) was synthesized and investigated. Ribavirin-TP was prepared with the use of modified Yoshikawa-Ludwig-Mishra-Broom procedure (cf. Mishra & Broom, 1991, J. Chem. Soc., Chem. Commun, 1276-1277) involving phosphorylation of unprotected nucleoside. Kinetic analysis revealed enhanced inhibitory potential of ribavirin-TP (IC50=40 microM) as compared to ribavirin (IC50 > 500 microM). Analysis of the inhibition type by means of graphical methods showed a competitive type of inhibition with respect to ATP. In view of the relatively low specificity towards nucleoside-5'-triphosphates (NTP) of the viral NTPase/helicases, it could not be ruled out that the investigated enzyme hydrolyzed the ribavirin-TP to less potent products. Investigations on non- hydrolysable analogs of ribavirin-TP or ribavirin-5'-diphosphate (ribavirin-DP) are currently under way.

Sonoporation-mediated anti-angiogenic gene transfer into muscle effectively regresses distant orthotopic tumors.

Ultrasound (US) is an effective tool for local delivery of genes into target tumors or organs. In combination with microbubbles, US can temporarily change the permeability of cell membranes by cavitation and facilitate entry of plasmid DNA into cells. Here, we demonstrate that repeated US-mediated delivery of anti-angiogenic genes, endostatin or calreticulin, into muscle significantly inhibits the growth of orthotopic tumors in the liver, brain or lung. US-mediated anti-angiogenic gene therapy also seems to function as an adjuvant therapy that significantly enhances the antitumor effects of the chemotherapeutic drug doxorubicin and adenovirus-mediated cytokine gene therapy. Significantly higher levels of tumor apoptosis or tumor-infiltrating lymphocytes were observed after combined therapy consisting of either anti-angiogenic therapy and chemotherapy, or anti-angiogenic therapy and immunotherapy. Taken together, our experiments demonstrate that intramuscular delivery of anti-angiogenic genes by US exposure can effectively treat distant orthotopic tumors, and thus has great therapeutic potential in terms of clinical treatment.

Expression of genes introduced into cells by retroviral infection is more efficient than that of genes introduced into cells by DNA transfection.

Calcium phosphate-mediated DNA transfection and retroviral infection are two alternative gene transfer techniques designed to introduce specific DNA fragments into the chromosomes of recipient cells. To compare the efficiency of expression of genes introduced into cells by either of these two techniques, a retrovirus-derived vector was constructed from the genome of Moloney murine leukemia virus by replacing the coding sequences of the envelope gene with the bacterial Neor gene derived from Tn5, termed rEnv-Neor. Expression of the hybrid Neor gene in NIH 3T3 cells after DNA transfection or retroviral infection was determined by measuring the steady-state levels of the corresponding cytoplasmic polyadeylated RNA species. Cells containing one copy of the integrated rEnv-Neor DNA introduced into cells by retroviral infection expressed 10- to 50-fold-higher levels of vector-specific RNA compared with cells harboring one copy of the same DNA derived by DNA transfection. Analysis of the integrated rEnv-Neor DNA with the methylation-sensitive restriction enzyme SmaI has shown that DNA integrated after DNA transfection but not after viral infection is partially methylated, predominantly in the 5' long terminal repeat, the region involved in initiation of transcription.

Metabolites of Bacillus sp. isolated from flax-suppressive soil.

A culture of Bacillus sp. was isolated from the suppressive flax soil. It could inhibit the growth of many plant pathogens that were found in suppressive flax soil, and an acidic ethyl acetate extract of culture broth of the isolated Bacillus showed significant inhibition of the growth of some plant pathogens. The antimicrobial activity of culture extracts (15 times concentrated) was higher than that of 400 micrograms/ml actidione or 200 micrograms/ml mycostatin (nystatin). The antimicrobial substances were relatively stable. After heating at 100 degrees C for 10 min. and 30 min., the activities were still 100% and 72%, respectively. Properties of the compounds were investigated by UV, TLC, GC and GCMS. The component with the retention time of 13.4 min in a gas chromatography was biologically active and the parent peak was at m/z 142.

Induction of antitumor immunity by intracerebrally implanted rat C6 glioma cells genetically engineered to secrete cytokines.

To test whether cytokine gene therapy can be applied to an immunologically privileged site, such as the brain, we investigated antitumor immunity in the brain induced by cytokine-secreting glioma cells. Three cytokine genes, interleukin-2 (IL-2), interleukin-4 (IL-4), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were transduced into a rat C6 glioma cell line via a retroviral vector, S2. Rats intracerebrally (IC) implanted with the C6 cells genetically engineered to secrete the cytokines, especially GM-CSF, manifested significantly higher survival rates than those with C6 cells or with C6 cells bearing the control vector (p < 0.002). In vivo, C6 tumors bearing the cytokine genes grew more slowly than wild-type tumors at any time point, and eventually diminished within 6 weeks after tumor cell implantation. Histopathological and immunohistochemical studies revealed that different cytokines induced diverse immune reactions. In the IL-2 group, CD4+ and CD8+ T cells dominated from day 3 to week 4, but disappeared at week 6. Some granulocytes were noted between weeks 2 and 4. In the IL-4 group, eosinophils were noted from day 3 to week 4, and CD4+ and CD8+ T cells, as well as macrophages at week 2. At week 6, only residual levels of macrophages and CD8+ T cells remained. In the GM-CSF group, granulocytes appeared as early as day 1 post-IC tumor implantation, and macrophages at day 2. CD4+ and CD8+ T cells were found from day 3 to week 4. At week 6, only residual CD4+ T cells and macrophages remained. Long-lasting antitumor immunity was confirmed in all groups by rechallenging surviving rats with wild-type C6 cells in the brain 100 days after implanting cytokine gene-bearing C6 cells. In vivo depletion of GM-CSF by anti-GM-CSF antibody further confirmed that the immune reaction induced by GM-CSF-secreting tumor cells were mainly from the action of GM-CSF, rather than the immunogenicity of C6 cells.

High level expression of porcine growth hormone in Escherichia coli from an expression vector containing bacteriophage lambda PL and N gene untranslated region.

An Escherichia coli expression vector, pG408N containing a PL promoter and the upstream untranslated region of the N gene of bacteriophage lambda has been constructed. We have designed a PvuII site immediately behind the untranslated region. A DNA fragment starting with an initiation codon ATG could be inserted into this site for expression. This vector also contains 7 additional cloning sites downstream from the PvuII site. A gene could be cloned into one of these sites and the 5' sequence of this gene could be modified with synthetic oligonucleotides and ligated to the PvuII for the purpose of increasing gene expression. We have also cloned the lambda cl gene into a p15A plasmid. Cotransformation of this plasmid with the expression vector allows the cloning vector pG408N to be used in any E. coli strain. Using this system, we were able to express porcine growth hormone to approximately 35% of total proteins in E. coli DH5 alpha.

Characterization of a bicistronic retroviral vector composed of the swine vesicular disease virus internal ribosome entry site.

We cloned the 5' nontranslated region (NTR) from the genome of swine vesicular disease virus (SVDV), a member of the family Picornaviridae, and used it to construct a bicistronic retroviral vector. The vector is characterized by coexpression of two genes from a single transcript. We found that inclusion of the 5' NTR of SVDV did not negate the viral vector titer. Protein analysis indicated that the 5' NTR could efficiently direct internal initiation, thus allowing the downstream gene to be translated. Translation of the internally initiated porcine growth hormone gene was about 30-fold less than that when the porcine growth hormone gene was at the upstream position in NIH 3T3 cells but was about equivalent to that in HeLa cells, implying that some cellular factors that stimulated internal initiation of the SVDV 5' NTR are present in HeLa cells. However, in G418-selected clones, the Neor-encoding gene was expressed with equivalent efficiency either at a downstream position or at an upstream position in either NIH 3T3 or HeLa cells. Compared with the conventional double-gene vector or the U3-based vector, the bicistronic vector coexpressed two genes much more efficiently, owing to elimination of promoter interference. Furthermore, this type of vector infected and expressed the target genes efficiently in two primary cell lines, rat embryo and human skin fibroblast cells, which we tested. These experimental data suggest a better design for the retroviral vector and provide evidence that internal initiation of the SVDV 5' NTR was stimulated cell specifically.

The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3).

To assess the RNA helicase activity of hepatitis C virus (HCV) nonstructural protein 3 (NS3), a polypeptide encompassing amino acids 1175 to 1657, which cover only the putative helicase domain, was expressed in Escherichia coli by a pET expression vector. The protein was purified to near homogeneity and assayed for RNA helicase activity in vitro with double-stranded RNA substrates prepared from a multiple cloning sequence and an HCV 5' nontranslated region (5'-NTR) or 3'-NTR. The enzyme acted successfully on substrates containing both 5' and 3' single-stranded regions (standard) or on substrates containing only the 3' single-stranded regions (3'/3') but failed to act on substrates containing only the 5' single-stranded regions (5'/5') or on substrates lacking the single-stranded regions (blunt). These results thus suggest 3' to 5' directionality for HCV RNA helicase activity. However, a 5'/5' substrate derived from the HCV 5'-NTR was also partially unwound by the enzyme, possibly because of unique properties inherent in the 5' single-stranded regions. Gel mobility shift analyses demonstrated that the HCV NS3 helicase could bind to either 5'- or 3'-tailed substrates but not to substrates lacking a single-stranded region, indicating that the polarity of the RNA strand to which the helicase bound was a more important enzymatic activity determinant. In addition to double-stranded RNA substrates, HCV NS3 helicase activity could displace both RNA and DNA oligonucleotides on a DNA template, suggesting that HCV NS3 too was disposed to DNA helicase activity. This study also demonstrated that RNA helicase activity was dramatically inhibited by the single-stranded polynucleotides. Taken altogether, our results indicate that the HCV NS3 helicase is unique among the RNA helicases characterized so far.

Protease-like sequence in hepatitis B virus core antigen is not involved in the cleavage processes of core protein in Escherichia coli.

A DNA fragment, coding for hepatitis core antigen (HBcAg), was amplified by polymerase chain reaction and inserted into a lambda PL promoter-derived expression vector. The recombinant plasmid was transformed into Escherichia coli and proteins produced after heat induction were analyzed. In addition to the 21 kDa HBcAg protein, several smaller related polypeptides, particularly one of 17 kDa in size, were also detected with rabbit anti-HBcAg antiserum. Whether the protease-like sequence of core protein involved in the self-cleavage process to form the 17 kDa polypeptide was investigated by a deletion experiment. Our results with a mutant in which 7 amino acids of the conserved protease-like region in the core protein have been deleted suggest that the cleavage does not depend on the presence of these protease-like sequence. In addition, the core protein synthesized from in vitro translation reaction was not cleaved. Core particles from E. coli lysate were purified by sucrose and cesium chloride density gradient centrifugations and subsequently treated with 0.2% of SDS and 0.2% of beta-mecaptoethanol. Immunoblotting analysis, however, did not reveal any conversion of the 21 kDa protein to smaller ones. In conclusion, our results suggest that the protease-like domain at the N-terminus of the core protein does not contain intrinsic autocleavage activity, nor could the HBcAg be converted to smaller antigens by detergent treatment.

Controlled delivery systems for proteins based on poly(lactic/glycolic acid) microspheres.

This paper describes an investigation of the use of poly(lactic/glycolic acid) polymers for long-term delivery of high molecular weight, water-soluble proteins. Poly(lactic/glycolic acid) (PLGA) microspheres, containing (fluorescein isothiocyanate)-labeled bovine serum albumin and (fluorescein isothiocyanate)-labeled horseradish peroxidase, were prepared by a modified solvent evaporation method using a double emulsion. The microspheres were spherical with diameters of 55-95 microns and encapsulated more than 90% of the protein. The preparation method was gentle and maintained enzyme activity and protein solubility. Stability studies showed that the encapsulation of an enzyme inside PLGA microspheres can protect them from activity loss. When not placed inside PLGA microspheres, (fluorescein isothiocyanate)-labeled horseradish peroxidase lost 80% of its activity in solution at 37 degrees C in a few days, whereas inside the PLGA microspheres it retained more than 55% of its activity after 21 days of incubation at 37 degrees C. In vitro release studies revealed that different release profiles (i.e., near-constant or biphasic) and release rates can be achieved by simply modifying factors in the preparation procedure such as mixing rate and volume of inner water and organic phases. Degradation studies by scanning electron microscopy and gel-permeation chromatography suggested that the mechanism responsible for protein release is mainly through matrix erosion.