Exploring the Significance of Gut Microbiota in Diabetes Pathogenesis and Management-A Narrative Review.

Type 2 diabetes is a disease with significant health consequences for the individual. Currently, new mechanisms and therapeutic approaches that may affect this disease are being sought. One of them is the association of type 2 diabetes with microbiota. Through the enteric nervous system and the gut-microbiota axis, the microbiota affects the functioning of the body. It has been proven to have a real impact on influencing glucose and lipid metabolism and insulin sensitivity. With dysbiosis, there is increased bacterial translocation through the disrupted intestinal barrier and increased inflammation in the body. In diabetes, the microbiota's composition is altered with, for example, a more abundant class of Betaproteobacteria. The consequences of these disorders are linked to mechanisms involving short-chain fatty acids, branched-chain amino acids, and bacterial lipopolysaccharide, among others. Interventions focusing on the gut microbiota are gaining traction as a promising approach to diabetes management. Studies are currently being conducted on the effects of the supply of probiotics and prebiotics, as well as fecal microbiota transplantation, on the course of diabetes. Further research will allow us to fully develop our knowledge on the subject and possibly best treat and prevent type 2 diabetes.

mitoBKCa is functionally expressed in murine and human breast cancer cells and potentially contributes to metabolic reprogramming.

Alterations in the function of K+ channels such as the voltage- and Ca2+-activated K+ channel of large conductance (BKCa) reportedly promote breast cancer (BC) development and progression. Underlying molecular mechanisms remain, however, elusive. Here, we provide electrophysiological evidence for a BKCa splice variant localized to the inner mitochondrial membrane of murine and human BC cells (mitoBKCa). Through a combination of genetic knockdown and knockout along with a cell permeable BKCa channel blocker, we show that mitoBKCa modulates overall cellular and mitochondrial energy production, and mediates the metabolic rewiring referred to as the 'Warburg effect', thereby promoting BC cell proliferation in the presence and absence of oxygen. Additionally, we detect mitoBKCa and BKCa transcripts in low or high abundance, respectively, in clinical BC specimens. Together, our results emphasize, that targeting mitoBKCa could represent a treatment strategy for selected BC patients in future.

Recent progress on Pickering emulsion stabilized essential oil added biopolymer-based film for food packaging applications: A review.

Nowadays food safety and protection are a growing concern for food producers and food industry. The stability of food-grade materials is key in food processing and shelf life. Pickering emulsions (PEs) have gained significant attention in food regimes owing to their stability enhancement of food specimens. PE can be developed by high and low-energy methods. The use of PE in the food sector is completely safe as it uses solid biodegradable particles to stabilize the oil in water and it also acts as an excellent carrier of essential oils (EOs). EOs are useful functional ingredients, the inclusion of EOs in the packaging film or coating formulation significantly helps in the improvement of the shelf life of the packed food item. The highly volatile nature, limited solubility and ease of oxidation in light of EOs restricts their direct use in packaging. In this context, the use of PEs of EOs is suitable to overcome most of the challenges, Therefore, recently there have been many papers published on PEs of EOs including active packaging film and coatings and the obtained results are promising. The current review amalgamates these studies to inform about the chemistry of PEs followed by types of stabilizers, factors affecting the stability and different high and low-energy manufacturing methods. Finally, the review summarizes the recent advancement in PEs-added packaging film and their application in the enhancement of shelf life of food.

Lack of seroprotection against diphtheria in the Austrian population, in light of reported diphtheria cases in Europe, 2022.

Following an increase in diphtheria cases in Europe since 2022, we retrospectively estimated the prevalence of seroprotection against diphtheria and tetanus in 10,247 Austrian residents (population: 8,978,929) voluntarily tested between 2018 and 2022. Lack of seroprotection against diphtheria was found in 36% compared with 4% against tetanus. The geometric mean antibody concentration against tetanus was 7.9-fold higher compared with that for diphtheria. Raising awareness of regular booster vaccinations against diphtheria in combination with tetanus and pertussis is urgently needed.

Biological potential of polysaccharides extracted from Nostoc colonies for film production - Physical and biological properties.

Cyanobacteria of the Nostoc genus secrete a number of biologically active compounds, including polysaccharides, which may exhibit antioxidant, antimicrobial, anticancer, and anti-inflammatory properties. The aim of the study was to investigate the biological properties of Nostoc polysaccharides (NPs) (antioxidative and antimicrobial) and the possibility of using NPs addition in the production of biofoils. Our results allow to indicate that NPs were compatible with the used biopolymer matrix (furcellaran and chitosan) and showed antioxidant properties. The tested polysaccharide extracts (0.14%) exhibited the ability to neutralize free radicals - 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) at a level of 4.46% and 10.14%, respectively. NP extracts demonstrated reducing properties of 15.35 and 30.07 mg Trolox equivalents (FRAP and CUPRAC methods, respectively) and 2.64 mg chlorogenic acid equivalents (tested with Folin's reagent). NP extracts showed: a growth-stimulating effect (Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae), no effect (Penicillum sp.), or a slight inhibitory effect (Streptomyces sp.) on the tested microorganisms. The enrichment of the film with NPs influenced the physic-chemical properties of the obtained biofoils. The addition of polysaccharides to furcellaran and chitosan films decreased their water solubility (by approximately 40% and 9%, respectively, compared to the control) and, at the same time increased, their water absorption.

Double-Layered Films Based on Furcellaran, Chitosan, and Gelatin Hydrolysates Enriched with AgNPs in Yerba Mate Extract, Montmorillonite, and Curcumin with Rosemary Essential Oil.

Double-layered active films based on furcellaran (1st layer-FUR), chitosan, and gelatin hydrolysates (2nd layer-CHIT+HGEL) were successfully prepared. Bioactive ingredients were added to the 1st film layer: AgNPs, which were synthesized in situ with yerba mate extract; montmorillonite clay (MMT); and different loads of ethanolic curcumin (CUR) extract enriched with rosemary essential oil (REO). SEM images confirmed the presence of AgNPs with a size distribution of 94.96 ± 3.33 nm throughout the films, and AFM and SEM photos indicated that the higher substance concentrations had rougher and more porous film microstructures. However, the water vapor transmission rate was reduced only at the lowest load of this ingredient. Despite the tensile strength of the films having decreased, the incorporation of the compounds showed a tendency towards reducing the modulus of elasticity, resulting in a lower stiffness of the composites. The addition of CUR and AgNPs improved the UV light barrier properties of the materials. The presented films showed quick reactions to changes in the pH value (from orange to red along with an increase in pH from 2 to 10), which indicates their potential use as indicators for monitoring the freshness of food products. Composite No. 2 showed the highest antimicrobial potential, while none of the presented films showed an antifungal effect. Finally, the antioxidant activities of the films increased dramatically at higher AgNP and CUR loads, suggesting an outstanding potential for active food packaging applications.

The Analysis of Erosive Wear Resistance of WC-Co Carbides Obtained by Spark Plasma Sintering Method.

WC-Co (tungsten carbide-cobalt) composites are widely used in industry, wear-resistant parts, and cutting tools. As successful tool materials, WC-Co carbides are widely applied in metal cutting, wear applications, chipless forming, stoneworking, wood, and plastic working. These materials are exposed to severe solid particle erosion by sand particles, such as in the wood industry. During the production of furniture with HDF (High Density Fibreboard), MDF (Medium Density Fibreboard), or OSB (Oriented Strand Board), there are observed problems with tool erosion. Contamination, mainly of the HDF by sand, is quite often, which is why all tools used for the machining of such materials are exposed to erosion by sand particles. Although many studies have been performed on the erosion of various metals, and erosion models exist to predict their erosion behavior, the issue is still relevant. The aim of the study was to determine the effect of grain size (submicron, ultrafine) and the manufacturing technology (SPS-Spark Plasma Sintering, conventional) used on the erosive properties of WC-Co sintered carbides. Sinters produced by the SPS method with different sizes of WC grains and commercial samples were used for the tests. Ten two-hour cycles were carried out under medium conditions of quartz sand and quartz sand with 10% SiC added. Used samples were characterised using scanning electron microscopy (SEM) and roughness was determined. Furthermore, erosion studies allowed individuating a wear mechanism as well as the possibility to foresee cutting performance in prospective application.

Absent antibody production following COVID19 vaccination with mRNA in patients under immunosuppressive treatments.

Patients undergoing immunosuppressive treatments have a higher need for protection against coronavirus disease (COVID19) that follows infection with the SARS-CoV-2 virus but their ability to respond sufficiently to COVID vaccines is uncertain. We retrospectively evaluated SARS-CoV-2 spike subunit 1 (S1)-specific antibody levels after two mRNA doses in 242 patients with underlying chronic inflammatory, hematooncological or metabolic diseases and in solid organ transplant recipients. S1-specific antibodies were measured 30 days after the second dose. In 15.9% of these patients, no S1-specific antibodies were detectable. Non-responsiveness was linked to administration of B-cell depleting therapies as well as to ongoing therapies that block lymphocyte trafficking (Fingolimod) or inhibit T cell proliferation (Tacrolimus). Thus, it is important to inform immunosuppressed patients about the risk of vaccine non-responsiveness and the necessity to maintain non-pharmaceutical protection measures. In these risk patients antibody testing and cellular analysis are helpful to estimate the benefit/responsiveness to further booster vaccinations.

Neutralising SARS-CoV-2 RBD-specific antibodies persist for at least six months independently of symptoms in adults.

Background: In spring 2020, at the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in Europe, we set up an assay system for large-scale testing of virus-specific and neutralising antibodies including their longevity. Methods: We analysed the sera of 1655 adult employees for SARS-CoV-2-specific antibodies using the S1 subunit of the spike protein of SARS-CoV-2. Sera containing S1-reactive antibodies were further evaluated for receptor-binding domain (RBD)- and nucleocapsid protein (NCP)-specific antibodies in relation to the neutralisation test (NT) results at three time points over six months. Results: We detect immunoglobulin G (IgG) and/or IgA antibodies reactive to the S1 protein in 10.15% (n = 168) of the participants. In total, 0.97% (n = 16) are positive for S1-IgG, 0.91% (n = 15) were S1-IgG- borderline and 8.28% (n = 137) exhibit only S1-IgA antibodies. Of the 168 S1-reactive sera, 8.33% (n = 14) have detectable RBD-specific antibodies and 6.55% (n = 11) NCP-specific antibodies. The latter correlates with NTs (kappa coefficient = 0.8660) but start to decline after 3 months. RBD-specific antibodies correlate most closely with the NT (kappa = 0.9448) and only these antibodies are stable for up to six months. All participants with virus-neutralising antibodies report symptoms, of which anosmia and/or dysgeusia correlate most closely with the detection of virus-neutralising antibodies. Conclusions: RBD-specific antibodies are most reliably detected post-infection, independent of the number/severity of symptoms, and correlate with neutralising antibodies at least for six months. They thus qualify best for large-scale seroepidemiological evaluation of both antibody reactivity and virus neutralisation.

Correction: Invasive pneumococcal diseases in children and adults before and after introduction of the 10-valent pneumococcal conjugate vaccine into the Austrian national immunization program.

[This corrects the article DOI: 10.1371/journal.pone.0210081.].

Invasive pneumococcal diseases in children and adults before and after introduction of the 10-valent pneumococcal conjugate vaccine into the Austrian national immunization program.

BACKGROUND: In February 2012 the ten-valent pneumococcal conjugate vaccine (PCV10) with a 2+1 doses schedule (3, 5, 12 or 14 months of age) without catch-up vaccination was introduced in Austria. We assessed direct and indirect vaccine effects on invasive pneumococcal disease (IPD) by a population-based intervention study. METHODS: The study period was divided into pre- (2009-2011) and post-period (2013-2017, February), regarding 2012 as transition year. Outcomes were defined as PCV10 ST-IPD, the PCV10-related ST 6A and 19A IPD and non-PCV10 excluding ST 6A-/19A-IPD (NVT-IPD). We used national surveillance data and compared average monthly incidence rate (IR) between pre- and post-period among <5, 5-49 and ≥50 years old. Additionally, for the 5-49 and ≥50 years old, and the 50-59 and ≥60 years old, we analyzed monthly incidence data of the pre-, post-period, and estimated trend and level changes by using a segmented time-series regression. RESULTS: The PCV-10 IPD was reduced by 58% (95% CI: 30%; 74%) and 67% (95% CI: 32%; 84%) among <5 and ≥50 years old; the reduction in ≥60 years was 71% (95% CI: 36%; 88%). There were no significant changes in the pre-post-rate or incidence trend of NVT-IPD in the <5 and ≥50 years old. ST-specific analyses revealed no ST 6A- and ST 19A IPD decline in any age-group, and a ST 8 IPD increase among ≥50 years old (IR ratio: 3.5; 95% CI: 1.7; 7.2). We found no vaccine effects among 5-49 years old. CONCLUSIONS: Our study adds to the evidence on direct and indirect protection of a childhood PCV10 vaccine program. Elderlies seem to benefit the most. Findings did not support PCV 10 cross-protection, but indicate replacement at least for ST 8 among the ≥50 years old. Follow-up analyses of IPD surveillance data are needed to fully characterize the magnitude of serotype replacement and further vaccine-attributable IPD reduction with time.

Age-related differences in humoral and cellular immune responses after primary immunisation: indications for stratified vaccination schedules.

Immunosenescence is characterised by reduced B and T cell responses. Evidence shows that booster vaccinations are less effective in elderly people, but data on the efficacy of primary immunisation are sparse. We conducted a monocentric, open label, phase IV trial to compare immune responses to primary vaccinations using the inactivated, adjuvanted Japanese Encephalitis vaccine by 30 elderly people (mean 69, range 61-78 years) and 30 younger people (mean 24, range 18-30 years). Humoral and cellular immune responses were analysed in relation to age and cytomegalovirus (CMV) seropositivity. Vaccine-specific antibody titres were significantly lower in elderly participants and 47% of them were non- or low responders after the two doses of the vaccine neo-antigen. The reduced humoral immune responses in elderly people correlated with reduced cytokine production, such as interferon gamma (IFN-γ) in vitro, as well as higher frequencies of late-differentiated effector and effector memory T cells and T regulatory cells. These cellular changes and lower antibody titres were particularly prominent in CMV-seropositive elderly participants. If primary vaccination before the age of 60 is not possible, elderly patients may require different vaccination strategies to ensure sufficient long-lasting immunity, such as adapted or accelerated schedules and the use of different adjuvants.

Enhanced and long term immunogenicity of a Her-2/neu multi-epitope vaccine conjugated to the carrier CRM197 in conjunction with the adjuvant Montanide.

BACKGROUND: We previously identified three short single peptides (P4, P6 and P7) representing different B-cell epitopes on the extracellular domain of Her-2/neu for a vaccine that was tested in a phase-I clinical trial. Here we describe the improvement of the multi peptide vaccine by fusing the single peptides to a hybrid peptide P467. METHODS: After coupling to either virosomes or to diphtheria toxoid CRM197 (CRM), the hybrid peptide was tested in different concentrations in combination with either Montanide or Aluminium hydroxide (Alum) in preclinical studies. RESULTS: Already low amount (10 µg) of P467 conjugated to CRM led to faster onset of high antibody levels compared to the P467-virosome. The formulation P467-CRM-Montanide induced higher serum IgG antibody titers, compared with P467-CRM-Alum, as examined by ELISA using recombinant Her-2/neu or Her-2/neu natively expressed on the tumor cell line SK-BR-3. Compared to P467-CRM-Alum, higher in vitro production of IL-2 and IFNγ in the Montanide-immunized mice was induced after re-stimulation of splenocytes with CRM but also with P467, indicating a clear Th1-biased response. In contrast to the single B cell peptides, the hybrid peptide led to T cell proliferation and cytokine production as CD4 T cell epitopes were generated in the fusion region of the single peptides P4 and P6 or P6 and P7. Additionally, a significantly higher proportion IFNγ-producing CD8+ T cells was found in the P467-CRM-Montanide immunized mice, probably by Montanide-driven bystander activation. Importantly, anti-P467 IgG antibodies exhibited anti-tumor properties and the combination of anti-P467 specific IgG with Herceptin® was found to inhibit the proliferation of Her-2/neu-overexpressing cell line SK-BR-3 in a significantly higher capacity than Herceptin® alone. CONCLUSIONS: Fusion of the B cell peptides has led to additional generation of CD4 T cell epitopes, and this P467-multi epitope vaccine was found to induce polyclonal antibody responses with anti-proliferative capacity against Her-2/neu. The hybrid vaccine together with Montanide induced higher and long-lasting antibody levels, Th1-biased cellular responses being superior to vaccination with the single B cell peptides. This vaccine formulation is now planned to be evaluated in a phase Ib/II study in Her-2/neu overexpressing cancer patients.

Prevention of birch pollen-related food allergy by mucosal treatment with multi-allergen-chimers in mice.

BACKGROUND: Among birch pollen allergic patients up to 70% develop allergic reactions to Bet v 1-homologue food allergens such as Api g 1 (celery) or Dau c 1 (carrot), termed as birch pollen-related food allergy. In most cases, specific immunotherapy with birch pollen extracts does not reduce allergic symptoms to the homologue food allergens. We therefore genetically engineered a multi-allergen chimer and tested if mucosal treatment with this construct could represent a novel approach for prevention of birch pollen-related food allergy. METHODOLOGY: BALB/c mice were poly-sensitized with a mixture of Bet v 1, Api g 1 and Dau c 1 followed by a sublingual challenge with carrot, celery and birch pollen extracts. For prevention of allergy sensitization an allergen chimer composed of immunodominant T cell epitopes of Api g 1 and Dau c 1 linked to the whole Bet v 1 allergen, was intranasally applied prior to sensitization. RESULTS: Intranasal pretreatment with the allergen chimer led to significantly decreased antigen-specific IgE-dependent ß-hexosaminidase release, but enhanced allergen-specific IgG2a and IgA antibodies. Accordingly, IL-4 levels in spleen cell cultures and IL-5 levels in restimulated spleen and cervical lymph node cell cultures were markedly reduced, while IFN-γ levels were increased. Immunomodulation was associated with increased IL-10, TGF-ß and Foxp3 mRNA levels in NALT and Foxp3 in oral mucosal tissues. Treatment with anti-TGF-ß, anti-IL10R or anti-CD25 antibodies abrogated the suppression of allergic responses induced by the chimer. CONCLUSION: Our results indicate that mucosal application of the allergen chimer led to decreased Th2 immune responses against Bet v 1 and its homologue food allergens Api g 1 and Dau c 1 by regulatory and Th1-biased immune responses. These data suggest that mucosal treatment with a multi-allergen vaccine could be a promising treatment strategy to prevent birch pollen-related food allergy.

Persistence of antibodies in 4-8 year old Austrian children after vaccination with hexavalent DTaP-HBV-IPV/Hib and MMR vaccines.

To determine the proficiency of the Austrian childhood vaccination schedule to induce long lasting seroprotection against vaccine preventable diseases a seroepidemiological study in 348 children between four and eight years of age was conducted. Antibodies against diphtheria, tetanus, pertussis, hepatitis B, measles, mumps and rubella antigens were assessed in children, who had been vaccinated with hexavalent DTaP-HBV-IPV/Hib vaccines at three, four, five months and in the second year of life and/or MMR vaccines in the second year of life at least once, but mostly twice. High seroprotection rates (SPRs) were detected for tetanus (96%) and measles (90%). SPRs regarding diphtheria and mumps were 81% and 72%, respectively. Rubella-SPRs were 68% in females and 58% in males. Hepatitis B-antibody levels ≥10 mIU/mL were present in 52%; antibodies against pertussis were detected in 27% of the children. SPRs for measles and rubella depended on the interval since last vaccination; mumps-antibodies were significantly lower after one MMR-vaccination only. Antibodies against diphtheria, tetanus and pertussis depended on the interval since last vaccination while HBs-antibodies did not. The low levels of antibodies 1-7 years after vaccination against pertussis, rubella and mumps after only one vaccination should be considered when recommending new vaccination schedules.

Delayed tumor onset and reduced tumor growth progression after immunization with a Her-2/neu multi-peptide vaccine and IL-12 in c-neu transgenic mice.

Passive immunotherapy with monoclonal antibodies is a routinely performed but cost intensive treatment against certain cancers. Induction of humoral anti-tumor responses by active peptide immunization has therefore become a favorable treatment concept. We have recently identified three peptides representing B-cell epitopes of the extracellular domain of Her-2/neu each of them inducing Her-2/neu specific immune responses with anti-tumor activity in vitro. The present study was performed to evaluate the in vivo protective capacity of a combined vaccination with these three peptides in FVB/N transgenic mice spontaneously developing c-neu overexpressing breast cancers. The three Her-2/neu peptides coupled to tetanus toxoid were administered with or without addition of recombinant IL-12. At the time all untreated mice had developed tumors about 40% of peptide-immunized mice and nearly 60% of mice immunized with the peptide vaccine co-applied with IL-12 remained tumor free. Moreover, co-administration of IL-12 had a significant impact on the retardation of tumor progression. The enhanced anti-tumor efficacy of the vaccine by IL-12 was associated with a Th1 biased immune response as demonstrated by an increased IFN-gamma production in vitro and elevated Her-2-specific IgG levels. Our findings clearly demonstrate that this multi-peptide vaccine is effective in tumor prevention and support its use against minimal disease, drug-resistant tumors or even for prophylaxis against cancers overexpressing Her-2/neu.

Vaccination with a human high molecular weight melanoma-associated antigen mimotope induces a humoral response inhibiting melanoma cell growth in vitro.

Peptide mimics of a conformational epitope that is recognized by a mAb with antitumor activity are promising candidates for formulations of anticancer vaccines. These mimotope vaccines are able to induce a polyclonal Ab response focused to the determinant of the mAb. Such attempts at cancer immunotherapy are of special interest for malignant melanoma that is highly resistant to chemotherapy and radiotherapy. In this study, we describe for the first time the design and immunogenicity of a vaccine containing a mimotope of the human high m.w. melanoma-associated Ag (HMW-MAA) and the biological potential of the induced Abs. Mimotopes were selected from a pVIII-9mer phage display peptide library with the anti-HMW-MAA mAb 225.28S. The mimotope vaccine was then generated by coupling the most suitable candidate mimotope to tetanus toxoid as an immunogenic carrier. Immunization of rabbits with this vaccine induced a specific humoral immune response directed toward the epitope recognized by the mAb 225.28S on the native HMW-MAA. The induced Abs inhibited the in vitro growth of the melanoma cell line 518A2 up to 62%. In addition, the Abs mediated 26% lysis of 518A2 cells in Ab-dependent cellular cytotoxicity. Our results indicate a possible application of this mimotope vaccine as a novel immunotherapeutic agent for the treatment of malignant melanoma.

Epitope-specific antibody response to Mel-CAM induced by mimotope immunization.

Peptide mimotopes of tumor antigen epitopes have been proposed as components of tumor vaccines. In this study, we determined the immunogenicity of melcam mim1 and melcam mim2, peptide mimics of an epitope of the melanoma cell-adhesion molecule (Mel-CAM). BALB/c mice were vaccinated either with mimotopes or mimotopes coupled to tetanus toxoid (TT). The antibody responses of mice to melcam mim1, melcam mim2, and recombinant Mel-CAM were analyzed by an ELISA and immunoblot analyses. TT-coupled mimotopes led to high titers of IgG mainly of the IgG2a subclass to melcam mim1 and melcam mim2. Immunization with each of the mimotope formulations induced antibodies that cross-reacted with recombinant Mel-CAM. Uncoupled mimotopes induced lymphocyte proliferation and cytokine production in spleen cell cultures indicating that both peptide mimotopes also contained T cell epitopes. TT-coupled mimotopes induced T helper (Th)1 (interleukin (IL)-2, interferon-gamma) and Th2 (IL-4, IL-5) cytokines, whereas uncoupled mimotopes induced a Th1-biased T cell response. Our results suggest that mimotopes potentially represent a novel vaccine approach to induce a tumor antigen-specific humoral and cellular response.

Suppression of human melanoma tumor growth in SCID mice by a human high molecular weight-melanoma associated antigen (HMW-MAA) specific monoclonal antibody.

The lack of efficacy of available therapies for the treatment of malignant melanoma has emphasized the need to develop novel therapeutic strategies to prevent melanoma growth. We have tested whether the anti-HMW-MAA mAb 225.28S is able to inhibit human melanoma tumor growth in SCID mice because in vitro data suggested that this antigen plays a role in spreading, migration and invasion of melanoma cells. Tumors were established by subcutaneous injection of the human melanoma cell line 518A2 into SCID mice. When tumors reached a size of 5 mm, the mAb 225.28S was administered intravenously 4 times in 3 day intervals at 100 microg/injection. Within 14 days after the first administration of the mAb 225.28S, tumor growth was reduced by 52% as compared to control mice. Three hundred and seven genes of >20,000 genes contained on the GeneChip were changed in their expression level at least 2-fold after administration of the mAb 225.28S. The encoded proteins were mostly components or modifiers of the extracellular matrix, tumor suppressors, and melanogenesis associated proteins. Surprisingly, the administration of the control mAb that did not lead to a significant tumor growth inhibition in vivo resulted in the modulation of two-thirds of these genes. This is the first report of suppression of human melanoma tumor growth in SCID mice by the mAb 225.28S. Our results suggest that anti-HMW-MAA mAbs may represent useful reagents to apply passive immunotherapy to patients with malignant melanoma.

Inhibition of tumor cell growth by antibodies induced after vaccination with peptides derived from the extracellular domain of Her-2/neu.

The anti Her-2/neu monoclonal antibody Trastuzumab has strong inhibiting effects on tumor growth in vitro and in vivo and is therefore used for immunotherapy in breast cancer patients. Due to necessity of frequent applications, however, cost intensiveness of Trastuzumab treatment and its limited duration of affectivity, an active immunization inducing a perhaps preventive and long-term immunity to Her-2/neu remains a desirable goal. We attempted to induce anti Her-2/neu antibodies by peptide vaccination and to test their efficacy in inhibiting tumor cell growth in vitro. By computer aided analyses, 7 putative B cell epitopes of Her-2/neu were defined and synthesized. These peptide epitopes were coupled to tetanus toxoid and used for immunization in BALB/c mice. Among these peptides, immunizations with 2 single peptides or a combination of 2 peptides induced anti-peptide antibody levels, primarily of the IgG1 isotype. These antibodies were also directed against the native Her-2/neu antigen, as shown in precipitation assays and ELISA with cell lysates of the Her-2/neu overexpressing breast cancer cell line SK-BR-3. Isolated IgG fractions from immune sera incubated with SK-BR-3 cells led to a moderate inhibition of the tumor cell growth in vitro, as well as to complement dependent cell lyses comparable to that achieved by incubation with Trastuzumab. Moreover, peptide immunization in rabbits generated anti-Her 2-neu IgG that, in contrast to mouse sera, were able to mediate a 31-46% lysis of SK-BR-3 cells in ADCC experiments. We conclude from our data that immunization with Her-2/neu peptides successfully induced humoral immune response with anti-tumor activity in an animal model.