Problemas bioéticos en la investigación de nuevas vacunas: ¿obedecen a razones de salud pública? / Bioethical problems in researching new vaccines: Do they respond to public health reasons?

Los problemas éticos de las investigaciones sobre vacunas han crecido en las últimas décadas en frecuencia y magnitud debido a la posición dominante de la industria farmacéutica en el desarrollo de esos estudios. Las tradicionales cuestiones de seguridad y eficacia se han visto agravadas por el conflicto de intereses introducido por la competencia comercial en un mercado a escala global de miles de millones de dólares. La integridad profesional de los investigadores, la responsabilidad moral de los patrocinadores, y la regulación y control por parte de los Estados nacionales, se muestra cuestionada en varios ejemplos. Los resultados de estos cambios son las amenazas a la protección de los derechos de las personas incluidas en estas investigaciones y el discutible progreso que resulta para la salud pública.

The ethical problems in vaccine research have grown in frequency and magnitude in last decades, due to the dominant place of the pharmaceutical industry in the development of such studies. Traditional issues of security and efficacy have been aggravated by the conflicts of interests introduced by commercial competition in a global market worth billions of dollars. We present here a few examples in which the professional integrity of researchers, the moral responsibility of sponsors, and the public regulation and control by national States are put into question. The consequences of these changes represent serious threats to the rights of people included in these studies as well as disputable progress for public health.

Role of novel cardiac biomarkers in prediction, diagnosis, and severity assessment of atherosclerotic coronary artery disease

Atherosclerotic cardiovascular disease is a major health problem around the world. Insulin resistance assessed by homeostatic model assessment of insulin resistance [HOMA-IR], triglyceride/ HDL, serum high sensitive C-reactive protein [hs-CRP], and HbA1c are probably associated with atherosclerosis. The aim of this study was to find if there was an association between HOMA-IR, triglyceride/ HDL ratio and their product as well as hs-CRP and HbA1c with the presence and the severity of coronary artery disease and to find the best cut off value for clinical practice. The study involved 170 consecutive patients with suspected or known CAD referred for coronary angiography divided into two groups; CAD group [Group I] and non CAD group [Group II] based on coronary angiography. The blood samples including HbA1c, hs-c-reactive protein, fasting blood glucose creatinine, urea, insulin and lipid spectrum were obtained after overnight fasting. Patients with CAD had higher HOMA-IR than those no CAD [4.47 +/- 4.26 vs. 2.49 +/- 1.99, p=0.002]. Stepwise multiple logistic regression analysis demonstrated that HOMA-IR > 2.66 was independently associated with CAD [odds ratio: 3.057 ; 95% confidence interval: 1.211-7.717; p=0.018] after adjustment for age, male sex, diabetes and waist circumference. HOMA-IR correlates positively with BMI [p=0.008 and r=0.227], waist circumference [p=0.002 and r=0.267] and HbA1c [p=0.015 and r=0.213]. Also HOMA-IRxTG/HDL was higher in patients with CAD compared to those no CAD [18.35 +/- 22.72 vs, 9.50 +/- 10.50, p=0.018]. However there were insignificant differences in levels of triglyceride/HDL, HbA1c, and hs-CRP among both groups [p= 0.124, 0.523, 0.250 and 0.764 respectively]. HOMI-IR was significantly higher in patients with DVD [Double-vessel Disease] and MVD [Milti-vessel Disease] compared to patients with no CAD [p=0.002 and 0.000 respectively]. Moreover HOMA-IR was significantly higher in patients with DVD and MVD compared to patients with SVD [Single-vessel Disease] [P=0.035, 0.001 respectively]. HOMA-IR x TG/HDL was significantly increased in DVD and MVD patients [P=0.012 and 0.001 respectively] compared to patients with no CAD and also when compared to patients with SVD [P=0.005 and 0.000 respectively]. HOMA-IR is considered as a marker for prediction of CAD and severity assessment

Bioinformatics of mouse uteroglobin binding protein and its polyclonal antibody preparation / 生理学报

To prepare anti-mouse uteroglobin binding protein (mUGBP) polyclonal antibody, two polypeptides were synthesized based on the bioinformatics analysis of mUGBP, and New Zealand white rabbits were immunized separately with each peptide coupled with keyhole limpet hemocyanin (KLH). The data indicate that a 13-amino acid polypeptide (positions 221st-233rd) was able to generate anti-peptide antibodies. The titer of the antisera detected with ELISA was 1:10(8). The antisera were then purified with immuno-affinity chromatography to obtain antibodies. Western blot analysis of mUGBP expressed as a fusion protein with a green fluorescent protein (GFP) was performed on the cell lysates of COS-1 cells with the purified antisera, suggesting that the antisera specifically recognized UGBP. By immunohistochemistry and indirect immunofluorescence analysis, we examined the expression of UGBP in the lung tissues from a patient undergoing surgical lung resection for a tumor and from normal mouse lung tissue, and found for the first time that UGBP protein was widely expressed in both mouse and human lung tissue with the most abundant expression in bronchial epithelial cells. These results suggest that the antigen epitopes of mUGBP are well predicted by using bioinformatics analysis. We have obtained anti-mUGBP polyclonal antibody, which will be useful for further investigation.

Hemocianinas, una herramienta inmunológica de la biomedicina actual / Hemocyanins as immunostimulants

Hemocyanins, the giant oxygen transporter glycoproteins of diverse mollusks, are xenogenic to the mammalian immune system and they display a remarkable immuno-genicity. Therefore they are ideal non-specific immunostimulants to treat some types of cancer. They are used as an alternative therapy for superficial urinary bladder cancer (SBC), that has been traditionally treated with Bacillus Calmette-Guerin (BCG). In contrast to BCG, hemocyanins do not cause side-effects, making them ideal for long-term repetitive treatments. Hemocyanins have also been exploited as carriers to develop antibodies against hapten molecules and peptides, as carrier-adjuvants for cutting-edge vaccines against cancer, drug addiction, and infectious diseases and in the diagnosis of parasitic diseases, such as Schistosomiasis. The hemocyanin from Megathura crenulata, also known as keyhole limpet hemocyanin (KLH), has been used for over thirty years for the purposes described above. More recently, hemoc yanin from the Chilean mollusk Concholepas concholepas (CCH) has proved to be a reliable alternative to KLH, either as carrier protein, and as a likely alternative for the immunotherapy of SBC. Despite KLH and CCH differ significantly in their origin and structure, we have demonstrated that both hemocyanins stimulate the immune system of mammals in a similar way by inducing a potent Thl-polarized cellular and humoral response.

Purificação e caracterização de peptídeos antimicrobianos presentes na hemolinfa de Acanthoscurria rondoniae (Mygalomorphae, Theraphosidae) / Purification and characterization of antimicrobial peptides in the Haemilymph of the Acanthoscurria rondoniae spider( Mygalomorphae, Theraphosidae

Todos os organismos vivos, abrangendo desde microrganismos até plantas e animais, evoluíram de forma a desenvolver mecanismos para ativamente defender-se contra o ataque de patógenos. Peptídeos antimicrobianos são importantes componentes do sistema imune dos vertebrados e invertebrados podendo ser agrupados de acordo com suas propriedades químicas e estruturais. Podem agir na membrana plasmática de microorganismos formando poros na bicamada de fosfolipídeos ou serem internalizados e atuar sobre alvos intracelulares. Estudos sobre a biodiversidade de moléculas antimicrobianas nos vários grupos de artrópodes podem ser importantes para se entender o processo imunológico de uma maneira mais ampla, possibilitando compreender as relações existentes entre os vários sistemas, bem como sua origem, além de poder contribuir para o desenvolvimento e utilização de novas drogas para o uso na medicina e na agricultura. O objetivo deste trabalho foi verificar a produção de substâncias antimicrobianas, como observada em outros artrópodes, utilizando a aranha Acanthoscurria rondoniae como exemplo, através da purificação e caracterização de moléculas presentes na hemolinfa e caracterizando-as para um entendimento mais amplo dos processos envolvidos no sistema imune de aracnídeos e dos artrópodes em geral. Neste trabalho foram encontradas três frações com atividade antimicrobiana nos hemócitos que apresentaram massa molecular de 2.270,3 Da, 418,2 Da e 10.111,8 Da, respectivamente. Essas massas quando comparadas as encontradas em A. gomesiana, mostram similaridade com a gomesina, mygalina e acanthoscurrina, podendo indicar que as duas espécies apresentam as mesmas moléculas antimicrobianas em seus hemócitos. No plasma, foram encontradas seis frações, mais somente uma foi caracterizada, que apresentou massa molecular de 1.236,776 Da, recebeu o nome de rondonina em homenagem a espécie estudada. Pela primeira vez foi observado...

The primary evaluation of its safety and immune effect of NogoA vaccination for spinal cord injury / 中华外科杂志

<p><b>OBJECTIVES</b>To prepare and purify NogoA vaccination for treatment of spinal cord injury. To study the safety and immune effect of this vaccination.</p><p><b>METHODS</b>Artificial NogoA-13 polypeptide was coupled with KLH to improve the immunogenicity of vaccination. Sixty three-week-old Wistar female rats were divided into 3 groups randomly. Group A was immunized with NogoA vaccination, group B with incomplete freund's adjuvant + complete freund's adjuvant; group C with KLH. Rats received abdominal cavity immunization. The level of antibody and the binding capability were detected with ELISA. The safety of vaccination was evaluated by the incidence and severity of experimental autoimmune encephalomyelitis (EAE).</p><p><b>RESULTS</b>The IgG antibody against the NogoA-13 polypeptide had been detected with ELISA in group A. A value of serum presented regular gradient during multiple proportion dilution. In group B and C, no antibodies were detected. The statistical significant difference in A value was revealed between group A and B, C group. No statistical significant difference was found in A value between group B and group C and non-immunized negative control serum. The features of EAE were not found in the immunized rats.</p><p><b>CONCLUSIONS</b>NogoA polypeptide vaccination can stimulate the antibody against the polypeptide. The immune effect of this vaccination is confirmed by binding reaction revealed in the ex vivo experiment. The good safety of vaccination is revealed by no features of EAE found in the immunized rats.</p>

Preparation of anti-mouse PGRP-L single-epitope polyclonal antibody / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare a single-epitope polyclonal antibody against mouse long peptidoglycan recognition protein (mPGRP-L).</p><p><b>METHODS</b>B cell dominant epitopes of mPGRP-L predicted by bioinformatics were synthesized, and the immunogen was prepared by conjugation of the synthetic peptide and the carrier protein key-hole limpet hemocyanin (KLH) by MBS method. The single-epitope polyclonal antibody was obtained by immunizing rabbits with the KLH-peptide conjugate, purified by SPG affinity columns or antigenic peptide affinity columns, and identified by ELISA and Western blotting.</p><p><b>RESULTS AND CONCLUSION</b>A dominant epitope in N-terminal region of mPGRP-L, with amino acid sequence of NH(2)-(C)DPHSLSPELQALISEVAQHD-COOH, was chosen and synthesized. The titer of anti-serum of the rabbits immunized with the KLH-peptide conjugate was 1:256,000. The polyclonal antibody purified with SPG affinity columns and antigenic peptide affinity columns were named as mPGRP-Ln1 and mPGRP-Ln2, respectively. Western blotting demonstrated that the antibody mPGRP-Ln1 could recognize the recombined N-terminal fragment of mPGRP-Ln with a clear band at relative molecular mass of about 29,000, suggesting the successful preparation of the single-epitope polyclonal antibody against the N-terminal region of mPGRP-L.</p>

Evaluation of NR2B peptide as subunit vaccines against experimental neuropathic pain / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>NR2B containing N-methyl-D-aspartate (NMDA) receptor plays an important role in the facilitation and maintenance of neuropathic pain. The discrete distribution of NR2B subunit in the central nervous system (CNS) may support reduced side effects of agents that act selectively at this site. Therefore, we investigated the hypothesis that a humoral autoimmune response targeting the NR2B subunit of NMDA receptor relieves pain like behaviours produced by peripheral injury.</p><p><b>METHODS</b>Rats were immunized subcutaneously with NR2B-Keyhole Limpet Hemocyanin (NR2B-KLH) three times at two-week intervals. NR2B specific IgG titres in sera and cerebrospinal fluid were determined by indirect ELISA. Seven days after the third immunization, 2 of the 3 terminal branches of the sciatic nerve (tibial and common peroneal nerves) were tightly ligated. Behavioural testing was carried out on every other day after surgery, until 7 days after surgery. The lumbar spinal cord (L4-6) was removed on day 7 after ligation. The expression of NR2B protein in the lumbar spinal cord was determined using Western blotting.</p><p><b>RESULTS</b>After the second vaccination, NR2B specific IgG in sera was detected to be > 0.5 microg/ml in six of nine rats. After the third vaccination, all the immunized rats had > 2.2 microg/ml. Titres of NR2B specific IgG in sera peaked 42 days post initial immunization and persisted for over 70 days. No NR2B specific IgG was detected in sera from PBS or KLH group. The behavioural thresholds in NR2B group were significantly higher than those in PBS and KLH groups on day 7 after ligation. NR2B specific IgG in CSF in NR2B group could not be detected on day 1 before spinal dissection; but could be detected on day 7 after surgery. The expression of NR2B protein in group NR2B was significantly lower than in PBS and KLH groups on day 7 after surgery.</p><p><b>CONCLUSION</b>The NR2B peptide could be used as a vaccine against neuropathic pain, which could be associated with reduction of NR2B protein in the lumbar spinal cord.</p>

Primitive research on the effect of FSH autoantibody on the spermatogenic capability of the rat testis / 中华男科学杂志

<p><b>OBJECTIVE</b>To establish rat models of FSH autoantibody and to investigate the effect of FSH autoantibody on the spermatogenic capability of rat testis.</p><p><b>METHODS</b>Thirsty 21-day old SD rats were randomly divided into an experimental and a control group of equal number. A specific polypeptide corresponding to the rat FSHbeta subunit was synthesized and coupled to (keyhole limpet hemocyanin) KLH. The rats in the experimental group were immunized with polypeptide-KLH and these in the control group with KLH. Further immunization was performed every 2 weeks for 7 times. On the 77th, 91st and 105th day of the immunization, 5 rats from the experimental group and another 5 from the control group were killed. Then the structures of the seminiferous tubule and epididymal sperm were observed by light and electron microscope, respectively. Meanwhile, the counts of sperms and the percentage of swelled sperm were calculated. And the level of serum testosterone was detected by enzyme-linked immunospecific assay (ELISA).</p><p><b>RESULTS</b>The titer of the anti-polypeptide antibody was 1:200 on the 49th day of the immunization, and reached 1:400 on the 63rd. Compared with the control group, the percentage of swelled sperm significantly decreased on the 91st day (60.4 +/- 6.23 vs 50.60 +/- 3.05, P < 0.05), and the number of spermatogenic cells and sperms in seminiferous tubules reduced on the 105th day in the experimental group, the counts of sperms (46.08 +/- 6.56 vs 32.53 +/- 3.41) and the percentage of swelled sperm (60.60 +/- 5.86 vs 48.60 +/- 3.85) significantly lower (P < 0.05), while the level of serum T significantly higher than that in the control group (P < 0.05).</p><p><b>CONCLUSION</b>FSH autoantibody might cause testis dyszoospermia.</p>

Safety and Efficacy of Intravesical Keyhole-Limpet Hemocyanin Therapy for Superficial Transitional Cell Carcinoma: A Prospective, Multicenter Study / 대한비뇨기과학회지

PURPOSE: We wanted to determine the safety and efficacy 1 year after intravesical keyhole-limpet hemocyanin (KLH) therapy for the patients suffering with superficial transitional cell carcinoma. MATERIALS AND METHODS: A total of 60 patients with bladder cancer who had undergone transurethral resection were treated with a one-year protocol (once a week for 6 weeks and 12 further treatments every 4 weeks) of KLH 20mg. The local and systemic safety and efficacy were evaluated. RESULTS: In terms of the safety of KLH, 4 patients (6.7%) reported dizziness and slight fever, and 2 (3.3%) experienced urgency. The remaining 54 patients (90.0%) did not report any local side effects during the treatment period. In terms of efficacy, recurrence developed in 32 patients (53.3%) at a mean of 4.5 months after transurethral resection; 28 patients (46.7%) were disease free after 14 months. The univariate analysis showed a greater level of efficacy in the patients suffering with bacille Calmette-Gu rin (BCG)-refractory disease (p=0.0150). No significant differences were found between the low, the intermediate-risk and the high-risk patients in terms of efficacy. CONCLUSIONS: The results of this study have confirmed the good tolerability of intravesical KLH and better efficacy in the BCG-refractory patients. However, in terms of overall efficacy, intravesical KLH therapy had no superiority over that of other intravesical treatments in terms of the overall efficacy.

Antitumor Activity of Keyhole Limpet Hemocyanin on N-butyl-N-(4-hydroxybutyl) Nitrosamine Induced Bladder Carcinoma in Mice / 대한비뇨기과학회지

Purpose: The antitumor activity and toxicity of keyhole limpet hemocyanin (KLH) on bladder carcinomas induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) were investigated. Materials and Methods: Sixty 6 to 8-week-old female C3H2 mice were divided into three groups. Bladder carcinomas were induced by the addition of 0.05% BBN to the drinking water for 8 weeks. Group A received an intravesical instillation of 0.25ml KLH-free buffer, Group B received 0.5mg KLH in 0.1ml buffer subcutaneously and Group C received an intravesical instillation of 2mug KLH in 0.25ml buffer twice weekly for 8 weeks (day 15 to day 70). On day 91, all the animals were sacrificed. The tumor incidence, bladder weight and toxicity were evaluated. Results: The incidences of cancer in groups A, B and C were 80, 40 and 60%, respectively. The incidence of cancer was significantly reduced in group B compared to group A (p<0.05). The average bladder weights were 93.63+/-17.746, 71.5+/-7.540 and 77.5+/-9.530mg in groups A, B and C, respectively. The bladder weights in groups B and C were significantly reduced compared to group A (p<0.05). There was no liver, kidney or bone marrow toxicities in groups B and C. Conclusions: These results suggest that KLH act as an effective and safe immunotherapeutic agent for bladder cancer. Prospective randomized clinical trials should be used to evaluate the role of KLH as an immunotherapeutic agent in the prophylaxis of recurrent bladder cancer.

Effect of Inhibitor of TNF-alpha and Oxatomide on Experimental Immune Mediated Otitis Media with Effusion / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Local immune response, one of the pathogenesis of middle ear effusion (MEE) is associated with the development and persistence of effusion in the middle ear cavity and inflammatory mediators play a major role in the production of MEE. The purpose of this study was to determine the effects of TNF-alpha antagonist and oxatomide on the outcome of immune mediated otitis media with effusion (OME) in rats. MATERIALS AND METHOD: Otitis media was induced by injecting KLH (Keyhole lympet hemocyanin) transtympanically 7 days after systemic immunization. Phosphate-buffered saline solution was used as control. Other groups were pretreated with TNF-alpha antagonist (soluble TNF receptor type I, sTNF RI) or oxatomide respectively before transtympanic injection of KLH. Seventy-two hours after the transtympanic injection, temporal bones in each group were examined histopathologically and vascular permeability of the middle ear mucosa was measured by the Evans blue vital dye technique. RESULTS: In the KLH, sTNF RI and oxatomide groups, MEE was developed in 83%, 0%, 66% of the ears, respectively. The oxatomide group and sTNF RI group showed significant decrease in inflammation, mucosal thickening and vascular permeability as compared with KLH group and those parameters of sTNF RI group showed lower values than those of oxatomide group. CONCLUSION: Transtympanic administration of sTNF RI and oxatomide appears to suppress the development of immune mediated OME. In terms of inhibiting MEE, sTNF RI was more effective than oxatomide. This study suggests that TNF-alpha antagonist and oxatomide may have a adjunctive role in the treatment and prevention of otitis media with effusion.

Influence of immunization dose schemes on the immune response to anti-tetrodotoxin vaccine / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the relationship between the immune response of anti-tetrodotoxin vaccine, including its dose-response, and to select optimal immunization dose so as to enhance antitoxic effect of the anti-tetrodotoxin vaccine.</p><p><b>METHODS</b>Tetrodotoxin (TTX) was coupled to Tachypleus tridentatus hemocyanin (TTH) chemically to form artificial antigen (TTX-TTH), and with which Balb/c mice were immunized. Influence of different immunization doses [100 microg as the higher (H) and 25 microg as the lower (L) dose group] on the protective effects of TTX vaccine was compared. The quality of antisera and effects of vaccine in anti-TTX poisoning were observed.</p><p><b>RESULTS</b>The sera antibody quality increased more quickly in group L than that in group H after immunization. The dose at which the half of immunized mice survived when challenged once with TTX were 16 x LD (1 LD = 13.5 microg/kg, i.p.) in group L and 11 x LD in group H. When TTX was used time and again, the half of immunized mice could tolerate as high as 40 x LD and 22 x LD of accumulated dose, and the maximum tolerable cumulated dose was 104 x LD and 90 x LD for group L and H respectively. The scheme L was better both in antibody quality and effect of protecting against TTX toxicity than that in scheme H.</p><p><b>CONCLUSIONS</b>The experimental vaccine of TTX could effectively protect animal from TTX intoxication. The lower immunization dose in this study is selected as the optimal immunization scheme.</p>

Influence of the pituitary gland on the immune response in young rats

The response of hypophysectomized (HYPOX) and sham-operated (S-HYPOX) female and male Wistar young rats (8 weeks old) to antigenic stimulation was compared. Humoral antigenic responses against hemocyanin were measured by ELISA. [3H]thyimidine incorporation into cultured splen cells was used to determine proliferative response to concanavalin A (ConA) or antigenic stimulation. Anti-hemocyanin serum titers in the HYPOX animals was about half of that observed in control S-HYPOX rats. Similarly, the cellular proliferative response was significantly decreased in HYPOX animals when compared to S-HYPOX rats; the blastogenic response to hemocyanin in UC rats (which did not receive the antigen injection) was close to zero. S-HYPOX control rats responded to direct ConA stimulation as UC controls. Body weight and the weight of pituitary target organs (adrenal, thyroid, ovary and testes) was about 1/4 of that of controls. Hypophysectomy also resulted in a striking reduction in spleen weight. These results indicate that the pituitary gland is involved in cellular and humoral immune regulation in young rats.

Acute schistosomiasis: clinical, diagnostic and therapeutic features

Three distinct syndromes caused by schistosomiasis have been described: cercarial dermatitis or swimmer's itch, acute schistosomiasis or Katayama fever, and chronic schistosomiasis. Complications of acute schistosomiasis have also been reported. The absence of a serological marker for the acute stage has hindered early diagnosis and treatment. Recently, an ELISA test using KLH (keyhole limpet haemocyanin) as antigen, has proved useful in differentiating acute from chronic schistosomiasis mansoni. Clinical and experimental evidence indicate that steroids act synergistically with schistosomicides in the treatment of Katayama syndrome. In this paper, clinical, diagnostic and therapeutic features of acute schistosomiasis are updated.

Permeabilidade vascular nas reacoes de hipersensibilidade cutanea basofilica. / Vascular permeability in cutaneous basophillic hypersensitivity

Reacoes de hipersensibilidade cutanea basofilica (HCB) provocadas em cobaias com ovoalbumina e hemocianina apresentaram aumento de permeabilidade vascular quando estes antigenos foram injetados localmente nas lesoes. Animais imunizados concomitantemente com os dois antigenos (ovoalbumina e hemocianina) apresentaram desgranulacao dos bosafilos apos injecao de hemocianina nas reacoes provocadas pela ovoalbumina.Quando estes antigenos alteraram a permeabilidade vascular nas reacoes de HCB, houve paralelamente extravasamento de proteinas plasmaticas na pele normal, embora em menor grau. Os resultados deste trabalho sugerem que as reacoes de HCD causadas pela ovoalbumina e apresentam caracteristicas semelhantes e que extrusao dos granulos dos basofilos nestas reacoes do tempo de desenvolvimento da lesao e da presenca de anticorpos especificos