Rationally engineered nanoparticles target multiple myeloma cells, overcome cell-adhesion-mediated drug resistance, and show enhanced efficacy in vivo.

In the continuing search for effective cancer treatments, we report the rational engineering of a multifunctional nanoparticle that combines traditional chemotherapy with cell targeting and anti-adhesion functionalities. Very late antigen-4 (VLA-4) mediated adhesion of multiple myeloma (MM) cells to bone marrow stroma confers MM cells with cell-adhesion-mediated drug resistance (CAM-DR). In our design, we used micellar nanoparticles as dynamic self-assembling scaffolds to present VLA-4-antagonist peptides and doxorubicin (Dox) conjugates, simultaneously, to selectively target MM cells and to overcome CAM-DR. Dox was conjugated to the nanoparticles through an acid-sensitive hydrazone bond. VLA-4-antagonist peptides were conjugated via a multifaceted synthetic procedure for generating precisely controlled number of targeting functionalities. The nanoparticles were efficiently internalized by MM cells and induced cytotoxicity. Mechanistic studies revealed that nanoparticles induced DNA double-strand breaks and apoptosis in MM cells. Importantly, multifunctional nanoparticles overcame CAM-DR, and were more efficacious than Dox when MM cells were cultured on fibronectin-coated plates. Finally, in a MM xenograft model, nanoparticles preferentially homed to MM tumors with ∼10 fold more drug accumulation and demonstrated dramatic tumor growth inhibition with a reduced overall systemic toxicity. Altogether, we demonstrate the disease driven engineering of a nanoparticle-based drug delivery system, enabling the model of an integrative approach in the treatment of MM.

Repair of experimental Achilles tenotomy with porcine renal capsule material in a rat model.

Porcine small intestinal submucosa (SIS) is a collagenous acellular matrix which has found substantial utility as a tissue growth scaffold. In the present study, the utility of porcine renal capsule matrix (RCM) was compared to SIS in a rat Achilles tenotomy repair model. Groups of rats underwent surgical tenotomy followed by either no repair, repair with a SIS graft, or repair with a RCM graft. The weight-bearing ability of the manipulated limb was evaluated for 10 days following surgery using a subjective scale. Tenotomy sites sampled 28 days after surgery were numerically graded for degree of histologic change. There were no statistically significant differences between groups with respect to return to weight-bearing ability (p >or= 0.05) or degree of histologic change (p >or= 0.001); however, a non-significant trend suggested that rats treated with SIS or RCM experienced a faster return to limb function than untreated rats, and RCM-treated rats had slightly higher scores for degree of histologic change, suggesting a more rapid repair of the tenotomy site than in SIS-treated or untreated rats. The harvested tenotomy sites in all treatment groups were characterized by marked fibroplasia and presence of macrophages. Remnants of SIS surrounded by macrophages and multi-nucleated giant cells were still present in some rats, however remnants of RCM were not observed, suggesting more rapid incorporation of RCM. The results show that RCM is equivalent to SIS as a material for repair of Achilles tendon injury and merits further study in other tendon injury models.

Use of porcine renal capsule matrix as a full-thickness dermal wound-healing material in rats.

OBJECTIVE: To compare the utility of porcine renal capsule matrix (RCM) with porcine small intestinal mucosa (SIS) in a rat full-thickness skin wound model. METHOD: Groups of rats had surgically-created wounds filled with either SIS or RCM. On each rat a contralateral wound was left unfilled (RCM-U or SIS-U). Wound diameter was measured 3, 7, 12, 17, 26 and 30 days after creation. Wound sites sampled 3, 7, 14, 28, 42 and 56 days after wound creation were numerically graded for degree of histologic change and for collagen content, based on intensity of trichrome staining. RESULTS: Wounds in all groups rapidly contracted to less than 50% of the original diameter within 12 days. There were no differences in wound diameter between RCM- and SIS-treated wounds at any time point, but these wounds had significantly greater (p < 0.001) diameters than their unfilled counterparts on days 7, 12 and 17. There were no differences in histologic scores or trichrome-staining scores between RCM- and SIS-treated wounds and their unfilled counterparts at any time point, except for a greater (p < 0.05) histologic score in SIS-treated wounds compared with unfilled controls on day 14. In both treatment groups an acute inflammatory response at the wound site was soon replaced by an influx of macrophages and fibroblasts. CONCLUSION: The results show that RCM is equivalent to SIS for the treatment of full-thickness wounds and that these materials may enhance wound healing in terms of wound-tissue collagenisation and maturation. These materials therefore merit further study in other wound-care models.

In utero transplantation of wild-type fetal liver cells rescues factor X-deficient mice from fatal neonatal bleeding diatheses.

Factor X (FX)-deficient embryos suffer partial embryonic lethality with approximately 30% of the embryos arresting at midgestation. The remaining animals survive to term but die perinatally mainly from abdominal or intracranial hemorrhage. We have rescued FX-deficient mice by transplanting fetal liver cells from FX+/+, Rosa26 fetuses into midgestation embryos derived from FX+/- heterozygous crosses. FX-/- embryos were born at the expected frequency and approximately 50% of the FX-/- neonates survived longer than 4 months. FX-/- embryos receiving saline injections that survived to term died perinatally similar to untreated FX-deficient mice. The plasma levels of FX in the rescued 16-week-old FX-/- mice were approximately 1-6% of wild-type levels. beta-Galactosidase-staining cells derived from the donor Rosa26 fetal liver cells were detected in 47% of the livers of adult mice. In addition, donor-derived cells were also recovered in the bone marrow, spleen, lung, and occasionally in the brain and testis. These results suggest that in utero cell transplantation could be an effective therapeutic strategy to treat pathologies resulting from the deficiency of hepatic-expressed factors.

Sebaceous adenocarcinoma of the external auditory canal in a New Zealand white rabbit.

A rare sebaceous gland carcinoma of the external auditory canal in a rabbit is described. The tumour was characterized histologically by foci and cords of markedly pleomorphic cells with abundant cytoplasm and variable numbers of vacuoles. A single pulmonary mass had similar histological characteristics. This is the first such tumour reported in a rabbit.

Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha.

We have investigated the methylotrophic yeast Hansenula polymorpha as a host for the co-integration and expression of multiple heterologous genes using an rDNA integration approach. The ribosomal DNA (rDNA) of H. polymorpha was found to consist of a single rDNA cluster of about 50-60 repeats of an 8-kb unit located on chromosome II. A 2.4-kb segment of H. polymorpha rDNA encompassing parts of the 25S, the complete 5S and the non-transcribed spacer region between 25S and 18S rDNA was isolated and inserted into conventional integrative H. polymorpha plasmids harboring the Saccharomyces- cerevisiae-derived URA3 gene for selection. These rDNA plasmids integrated homologously into the rDNA repeats of a H. polymorpha (odc1) host as several independent clusters. Anticipating that this mode of multiple-cluster integration could be used for the simultaneous integration of several distinct rDNA plasmids, the host strain was co-transformed with a mixture of up to three different plasmids, all bearing the same URA3 selection marker. Transformations indeed resulted in mitotically stable strains harboring one, two, or all three plasmids integrated into the rDNA. The overall copy number of the plasmids integrated did not exceed the number of rDNA repeats present in the untransformed host strain, irrespective of the number of different plasmids involved. Strains harboring different plasmids co-expressed the introduced genes, resulting in functional proteins. Thus, this approach provides a new and attractive tool for the rapid generation of recombinant strains that simultaneously co-produce several proteins in desired stoichiometric ratios.

Intranasal vaccination of rabbits with Pasteurella multocida A:3 outer membranes that express iron-regulated proteins.

OBJECTIVE: To determine efficacy of intranasal vaccination of rabbits with Pasteurella multocida A:3 outer membrane proteins (OMP) expressing iron-regulated OMP (IROMP) in conferring protection against experimental challenge exposure. ANIMALS: 52 male New Zealand White rabbits. PROCEDURE: Rabbits were vaccinated intranasally on days 0, 7, and 14; some vaccines included cholera toxin (CT) as an adjuvant. Concentrations of intranasal IgA and serum IgG antibodies against P multocida OMP were determined. In experiment A, rabbits were vaccinated with either phospate-buffered saline solution (PBSS), PBSS-CT, OMP-CT, or IROMP-CT, challenge-exposed intranasally on day 16, and euthanatized and necropsied on day 28. Rabbits were also vaccinated with OMP or IROMP without CT and were not challenge-exposed. In experiment B, rabbits were vaccinated with PBSS, PBSS-CT, IROMP, or IROMP-CT. On day 17, rabbits were challenge-exposed intranasally. Nasal bacteria and antibodies were determined on day 24. RESULTS: In experiment A, OMP-CT vaccination stimulated mucosal and systemic antibody responses to the bacterium and enhanced resistance against challenge exposure. Intranasal bacterial counts were not significantly reduced. Vaccination with IROMP-CT stimulated mucosal and systemic antibodies, enhanced resistance to challenge exposure, and significantly reduced nasal bacterial counts. In experiment B, natural infection was detected in several rabbits at challenge exposure; however, IROMP-CT-vaccinated rabbits had significantly higher serum and nasal antibody responses, compared with other rabbits IROMP-CT-vaccinated rabbits had significantly lower nasal bacterial counts compared to control rabbits. CONCLUSIONS AND CLINICAL RELEVANCE: Intranasal vaccination of rabbits with P multocida outer membranes containing IROMP and CT stimulated immunity against experimental pneumonic pasteurellosis.

The characterization of mice with a targeted combined deficiency of protein c and factor XI.

Activated protein C functions directly as an anticoagulant and indirectly as a profibrinolytic enzyme. To determine whether the fibrin deposition previously observed in PC(-/-) murine embryos and neonates was mediated through the FXI pathway, PC(+/-)/FXI(-/-) mice were generated and crossbred to produce double-deficient progeny (PC(-/-)/FXI(-/-)). PC(-/-)/FXI(-/-) mice survived the early lethality observed in the PC(-/-)/FXI(+/+) neonates, with the oldest PC(-/-)/FXI(-/-) animal living to 3 months of age. However, the majority of these animals was sedentary and significantly growth-retarded. On sacrifice or natural death, all of these PC(-/-)/FXI(-/-) mice demonstrated massive systemic fibrin deposition with concomitant hemorrhage and fibrosis, as confirmed through histological analyses. Several of these animals also presented with enlarged lymph nodes and extensive lymphatic fluid in the thoracic cavity. Thus, although a number of the PC(-/-)/FXI(-/-) mice survived the lethal perinatal coagulopathy seen in the PC(-/-) neonates, they nonetheless succumbed to overwhelming thrombotic disease in later life. This combined deficiency state provided the first clear indication that the course of a severe thrombotic disorder could be manipulated by blocking the intrinsic pathway and provided the first opportunity to study a total protein C deficiency in an adult animal.

Immunization of rabbits against Pasteurella multocida using a commercial swine vaccine.

Elaboration of heat-labile toxin (PMT) is an important virulence factor in some isolates of Pasteurella multocida from rabbits. Previously, we reported that immunization with inactivated PMT (IPMT) stimulated protective immunity to challenges from PMT. To test the hypothesis that immunization with a commercial swine vaccine containing IPMT stimulates similar protective immunity, groups of five rabbits were inoculated twice intramuscularly (i.m.), 10 days apart, with 0.5 ml of sterile saline or a commercial swine P. multocida bacterin-toxoid (BT). In addition, a group was inoculated intranasally with 5 microg of IPMT. Serum and nasal lavage samples were taken on days 0, 7, 14 and 21 after initial immunization and assayed by ELISA for anti-PMT antibody. Serum IgG and nasal lavage IgA were detectable by day 14 in BT and IPMT-immunized rabbits, but not in the saline controls. Groups of similarly inoculated rabbits were then challenged intranasally with 28 microg of PMT 21 days after initial immunization, and necropsied 7 days later, along with control challenged and non-challenged rabbits. Histological lesion severity was graded on a numerical scale. Non-immunized and saline, challenged controls developed more severe pneumonia, pleuritis, nasal turbinate atrophy and testicular atrophy than IPMT and BT-immunized rabbits. The results confirm the hypothesis that immunization with a commercial swine P. multocida BT confers protective immunity in rabbits against challenges from PMT.

Intranasal vaccination of New Zealand white rabbits against pasteurellosis, using alginate-encapsulated Pasteurella multocida toxin and potassium thiocyanate extract.

OBJECTIVE: We evaluated the efficacy of intranasal administration of Pasteurella multocida toxin (PMT) and a potassium thiocyanate extract of P. multocida (CN) encapsulated in alginate microspheres, compared with unencapsulated PMT and CN antigens, in protection of rabbits against pasteurellosis. METHODS: New Zealand male rabbits (n=24) were allotted randomly into four intranasally administered vaccine groups: 1, PMT/CN; 2, microencapsulated PMT/CN with or; 3, without subcutaneous priming; and 4, empty microspheres (control). Blood samples and nasal wash specimens were collected before vaccination and one week after each vaccination (days 7, 21, 35, and 49). Rabbits were primed subcutaneously with either unencapsulated PMT/CN or aluminum hydroxide (control) (day 0), vaccinated intranasally (days 14, 28, and 42), challenged intranasally with live P. multocida (day 56), and necropsied (day 60). RESULTS: Compared with controls, PMT/CN-immunized rabbits had significantly higher concentrations of serum IgG and IgM, nasal IgG, and bronchoalveolar lavage fluid IgA and IgG against CN. Immunized rabbits had 100% survival rate and low numbers of bacteria in liver and lungs; the control group had 50% survival rate and higher numbers of bacteria (> 4x) per gram of tissue in liver and lungs. CONCLUSION: The PMT/CN microspheres stimulated systemic and mucosal immune responses similar in effectiveness (protection) to those in response to unencapsulated PMT/CN administration.

Immunogenicity of antigens in boiled alginate microspheres.

Vaccine efficacy can be enhanced by delivery of antigens in synthetic microspheres. The process of antigen incorporation into microspheres can expose fragile antigens to damaging conditions, such as high temperatures, and to bacterial contamination. Maintenance of immunogenicity of several antigens and reduction of bacterial load in alginate microspheres following boiling was evaluated. Mice were immunized subcutaneously, initially and again 21 days later, with either non-boiled or boiled microspheres containing ovalbumin (OVA), a culture supernatant vaccine of Pasteurella haemolytica (PHV), or a potassium thiocyanate extract of P. multocida (PTE). Serum samples were obtained prior to immunization and at the time of euthanasia 28 days later. Culture of microspheres showed that boiling completely eliminated aerobic bacterial growth for OVA-containing microspheres, and reduced growth by a factor of 10(4) for PTE microspheres. More bacteria were cultured after boiling than before for PHV microspheres. ELISA performed on serum and intestinal lamina propria explant supernatants showed that immunogenicity of PHV microspheres was not altered by boiling. Boiled OVA microspheres were still able to stimulate a significant serum IgG anti-OVA titer in mice, but boiled PTE microspheres completely lacked immunogenicity. Elispot assays of spleens showed that only PHV microspheres were able to retain immunogenicity after boiling. Results indicate that boiling is not an effective means for reducing the bacterial load of alginate microspheres and that the process is associated with a diminution of vaccine immunogenicity.

[Therapy of lymphomatoid papulosis with balneo-PUVA-photochemotherapy]]. / Therapie der lymphomatoiden Papulose mit Bade-PUVA-(Balneo-) Photochemotherapie.

Lymphomatoid papulosis is a rare lymphoproliferative disorder of the skin. The standard therapeutic regimen is systemic (oral) photochemotherapy (PUVA). We examined the efficacy of PUVA-bath photochemotherapy in a patient requiring heart transplantation because of idiopathic dilatated cardiomyopathy and a relative contraindication against systemic 8-methoxypsoralen. The 42-years old male patient had suffered for 15 years with itching papules and plaques which clinically, immunohistochemically and molecular biologically were diagnosed as lymphomatoid papulosis. PUVA-bath photochemotherapy with 8-MOP was initiated. After 29 treatments the plaques disappeared completely. After 44 sessions (cumulative UV-A dose 206 J/cm2) the patient's skin almost was clear. PUVA-bath photochemotherapy proved to be a therapeutic alternative to systemic PUVA-treatment in this case of lymphomatoid papulosis.

[Pruritus of dark skin in hookworm infection]. / Pruritus auf dunkler Haut bei Hakenwurminfektion.

Using the example of a young man from Senegal we would like to point out that in cases of pruritus without an underlying skin disease an intestinal parasitosis might be the cause of the pruritus should the patient come from the tropics. However, it is important to use concentration methods when examining stool specimens so as to be able to diagnose the parasitosis. The causal connection between a pruritus and an intestinal parasitosis needs to be confirmed by a successful treatment.

Enhanced bone regeneration using porcine small intestinal submucosa.

Small intestinal submucosa (SIS) is an easily produced material that has been used experimentally for tissue engineering. To evaluate the ability of SIS to facilitate bone growth within a long-bone defect, a segment of the radius was surgically removed in adult, female Sprague-Dawley rats. The defect was either left unfilled or implanted with SIS, demineralized cortical bone (DMCB), or ovalbumin. The defect was evaluated radiographically and histologically after 3, 6, 12, and 24 weeks. Tissue remodeling within the defect was evident by week 3 in SIS- and DMCB-treated rats. Filling was characterized initially by infiltration of mononuclear cells and extracellular material in SIS-implanted rats and multifocal remodeling bone particles and cartilage formation in DMCB-implanted rats. Cartilage was observed as early as 3 weeks and bone as early as 6 weeks in SIS-implanted rats. Filling of the defect arose from multiple foci in DMCB-implanted rats, but was contiguous with and parallel to the ulnar shaft in SIS-implanted rats, suggesting that defect repair by SIS may be conductive rather than inductive. Rats in which the defect was left unfilled demonstrated slow but progressive filling of the defect, characterized by mononuclear cell infiltrates and fibrous extracellular material. In summary, SIS facilitated rapid filling of a long-bone defect. These results suggest that SIS may be useful as a bone repair material.

Biodegradable alginate microspheres as a delivery system for naked DNA.

Sodium alginate is a naturally occurring polysaccharide that can easily be polymerized into a solid matrix to form microspheres. These biodegradable microspheres were used to encapsulate plasmid DNA containing the bacterial beta-galactosidase (LacZ) gene under the control of either the cytomegalovirus (CMV) immediate-early promoter or the Rous sarcoma virus (RSV) early promoter. Mice inoculated orally with microspheres containing plasmid DNA expressed LacZ in the intestine, spleen and liver. Inoculation of mice with microspheres containing both the plasmid DNA and bovine adenovirus type 3 (BAd3) resulted in a significant increase in LacZ expression compared to those inoculated with microspheres containing only the plasmid DNA. Our results suggest that adenoviruses are capable of augumenting transgene expression by plasmid DNA both in vitro and in vivo.

Evaluation of hypothermia-induced analgesia and influence of opioid antagonists in leopard frogs (Rana pipiens).

Hypothermia results in diminished voluntary muscle activity, and is frequently used as a means of providing deep anesthesia to ectotherms and some mammals. In ectotherms, however, it is unclear if hypothermia produces true pain insensation. A needle-probe thermometer was used to demonstrate in frogs (Rana pipiens) that local hypothermia (9 degrees C) could be induced by placement of a tourniqueted leg into ice water (6 degrees C) for 10 min in contrast to the contralateral nontourniqueted leg (21.8 degrees C) kept out of ice water. Analgesia was tested by placement of dilutions of acetic acid on the rear leg. Further tests using groups of 10 frogs demonstrated that frogs with local hypothermia tolerated greater concentrations of acetic acid (mean acetic acid test score = 11) than morphine (10 mg/kg)-treated (9.6) or nontreated (5.8) frogs. Additional studies showed that morphine analgesia was blocked with naloxone doses as low as 0.01 mg/kg and hypothermia-induced analgesia at 10 mg/kg. Naltrexone blocked morphine analgesia at dosages as low as 0.01 mg/kg and hypothermia-induced analgesia at 0.10 mg/kg. In summary, this study demonstrates that hypothermia induces significant analgesia in an amphibian, and that this analgesia is partially blocked by naloxone and naltrexone, suggesting that the effect is mediated at least partially by opioid receptors.

Oral vaccination of animals with antigens encapsulated in alginate microspheres.

Most infectious diseases begin at a mucosal surface. Prevention of infection must therefore consider ways to enhance local immunity to prevent the attachment and invasion of microbes. Despite this understanding, most vaccines depend on parenterally administered vaccines that induce a circulating immune response that often does not cross to mucosal sites. Administration of vaccines to mucosal sites induces local immunity. To be effective requires that antigen be administered often. This is not always practical depending on the site where protection is needed, nor comfortable to the patient. Not all mucosal sites have inductive lymphoid tissue present as well. Oral administration is easy to do, is well accepted by humans and animals and targets the largest inductive lymphoid tissue in the body in the intestine. Oral administration of antigen requires protection of antigen from the enzymes and pH of the stomach. Polymeric delivery systems are under investigation to deliver vaccines to the intestine while protecting them from adverse conditions that could adversely affect the antigens. They also can enhance delivery of antigen specifically to the inductive lymphoid tissue. Sodium alginate is a readily available, inexpensive polymer that can be used to encapsulate a wide variety of antigens under mild conditions. Orally administered alginate microspheres containing antigen have successfully induced immunity in mice to enteric (rotavirus) pathogens and in the respiratory tract in cattle with a model antigen (ovalbumin). This delivery system offers a safe, effective means of orally vaccinating large numbers of animals (and perhaps humans) to a variety of infectious agents.

A novel feature of DNA recognition: a mutant Gcn4p bZip peptide with dual DNA binding specificities dependent of half-site spacing.

Homodimeric DNA-binding proteins with relaxed half-site spacing requirements for their DNA targets have been described. As an example, the yeast transcriptional activator Gcn4p binds in vitro equally well to the AP1 site (5'A4T3G2A1C0T1'C2'A3'T4'3') and the ATF/CREB site (5'A4T3G2A1C0G0'T1'C2'A3'T4'3'), which have identical but differently spaced half-site blocks. We describe a novel feature for the bZip class of DNA-binding proteins. The N-14 mutant of a Gcn4p-derived bZip peptide shows a diametrically opposed base-pair recognition specificity depending on the half-site spacing of its DNA target: on pseudo-palindromic, AP1 site-like binding sites, guanine is required in position 2 for proper binding; in contrast, on palindromic, ATF/CREB site-like targets, position 2 must be cytosine to prevent a loss of binding. Modeling studies suggest that the different base-pair requirements on differently spaced DNA targets are due to minimal alterations of the distances between the relevant atoms of the N-14 side-chain and the corresponding target groups on the DNA. Although the N-14 peptide does not have a natural counterpart, its behavior hints at the possibility that dual binding modi dependent on half-site spacing may occur also for natural homodimeric DNA-binding proteins.

Induction of protective immunity in rabbits by coadministration of inactivated Pasteurella multocida toxin and potassium thiocyanate extract.

Pasteurella multocida is a bacterial pathogen that causes rhinitis (snuffles), pneumonia, otitis media, septicemia, metritis, and death in domestic rabbits. Currently, there are no effective vaccines to prevent infection by this organism. Subcutaneous (s.c.) immunization with either exotoxin or thiocyanate extracts of P. multocida induces partial protection in rabbits. Since disease begins at mucosal sites, induction of local immunity may be important in preventing systemic disease. Little is known concerning the efficacy of intranasal (i.n. ) administration of these antigens in inducing protective mucosal immunity to P. multocida in rabbits. The purpose of this study was twofold: (i) to investigate the effectiveness of vaccination with purified P. multocida toxin (PMT) and a potassium thiocyanate extract of P. multocida (CN) in combination and (ii) to evaluate the efficacy of administration of these antigens i.n. versus s.c. Forty-eight rabbits were randomly divided into eight different treatment groups. Rabbits received either one or both antigens by either s.c. or i.n. administration. Following vaccination, each group received an i.n. challenge of P. multocida. Rabbits vaccinated with both antigens i.n. or s.c. had a 100% survival rate, few or no bacteria in the liver and lungs, high serum immunoglobulin G (IgG) and IgM antibody titers, and significant numbers of IgG antibody-secreting cells (ASC) in the spleen and tracheobronchial lymph node. Rabbits vaccinated i.n. had significant nasal and bronchoalveolar lavage IgA antibody levels. Rabbits vaccinated with only one antigen, either PMT or CN, had lower antibody titers, moderate to severe liver and lung infections, and fewer ASC compared to rabbits receiving both antigens. Rabbits in the control groups had moderate to severe liver and lung infections. This study indicates that i.n. immunization with both PMT and CN induces an effective response against homologous P. multocida challenge.