Isolation and characterization of high affinity and highly stable anti-Chikungunya virus antibodies using ALTHEA Gold Libraries™.

BACKGROUND: More than 3 million infections were attributed to Chikungunya virus (CHIKV) in the 2014-2016 outbreak in Mexico, Central and South America, with over 500 deaths directly or indirectly related to this viral disease. CHIKV outbreaks are recurrent and no vaccine nor approved therapeutics exist to prevent or treat CHIKV infection. Reliable and robust diagnostic methods are thus critical to control future CHIKV outbreaks. Direct CHIKV detection in serum samples via highly specific and high affinity anti-CHIKV antibodies has shown to be an early and effective clinical diagnosis. METHODS: To isolate highly specific and high affinity anti-CHIKV, Chikungunya virions were isolated from serum of a patient in Veracruz, México. After purification and characterization via electron microscopy, SDS-PAGE and binding to well-characterized anti-CHIKV antibodies, UV-inactivated particles were utilized as selector in a solid-phase panning in combination with ALTHEA Gold Libraries™, as source of antibodies. The screening was based on ELISA and Next-Generation Sequencing. RESULTS: The CHIKV isolate showed the typical morphology of the virus. Protein bands in the SDS-PAGE were consistent with the size of CHIKV capsid proteins. UV-inactivated CHIKV particles bound tightly the control antibodies. The lead antibodies here obtained, on the other hand, showed high expression yield, > 95% monomeric content after a single-step Protein A purification, and importantly, had a thermal stability above 75 °C. Most of the antibodies recognized linear epitopes on E2, including the highest affinity antibody called C7. A sandwich ELISA implemented with C7 and a potent neutralizing antibody isolated elsewhere, also specific for E2 but recognizing a discontinuous epitope, showed a dynamic range of 0.2-40.0 mg/mL of UV-inactivated CHIKV purified preparation. The number of CHIKV particles estimated based on the concentration of E2 in the extract suggested that the assay could detect clinically meaningful amounts of CHIKV in serum. CONCLUSIONS: The newly discovered antibodies offer valuable tools for characterization of CHIKV isolates. Therefore, the strategy here followed using whole viral particles and ALTHEA Gold Libraries™ could expedite the discovery and development of antibodies for detection and control of emergent and quickly spreading viral outbreaks.

Nanocochleates containing N-Octylglicoside extracted Vibrio cholerae antigens elicited high vibriocidal antibodies titers after intragastric immunization in a mice model.

Biological detergents are used in research laboratories, to extract or solubilize proteins from cell membranes. In order to evaluate the ability to extract antigens from the bacterial cell surface of the wild Vibrio cholerae strain C7258 and study their immunogenic potential by forming proteoliposomes and cochleate and preserving their immunogenicity, the non-ionic detergent, n-Octylglucoside (n-OG), and the Zwitterionic detergent (3-cholamidopropyl dimethylammonio 1-propanesulfonate; CHAPS) were tested in concentrations between 5 and 15%. The anionic detergent sodium deoxycholate (DOC) was used as a reference. Electrophoretic, immunochemical and electron microscopy techniques have characterized the extracts and their chromatographic fractions. With CHAPS and n-OG detergents in concentrations between 5 and 15%, a higher yield was obtained in the extraction of proteins and lipopolysaccharides (LPS) and other components from the bacterial surface compared to 10% DOC. When using 10% DOC, 15% CHAPS and n-OG between 5 and 15%, stable proteoliposomes were formed, of average size between 82 and 93 nm in diameter, with known proportions of proteins, LPS and other components. In some of the concentrations, liposomes were formed with almost pure proteins. Some cholera outer membrane proteins like the 17 kDa protein, which corresponds to the mannose-sensitive hemagglutinin (MSHA), which mediates the adhesion to the brush border of the small intestine and the outer membrane protein U (OMPU) were identified with monoclonal antibodies (mAbs) and purified. The fundamental components of liposomes, proteins and LPS, retained their molecular weights, when compared with known standards and by processing programs of electrophoretic profiles and their antigenicity, without alterations due to the extraction procedure, as could be verified by immune identification techniques with monoclonal antibodies in the case of LPS, significant antigens in this pathogen. The main purpose of the present work was to show that a new anticholera vaccine formulation based on cochleates, containing selected protein and LPS fraction extracted by detergents, is able to elicit protective high titers of bactericidal antibodies after intragastric immunization in the mice model. The objective was achieved.

Linfoma no Hodgkin primario de mama: reporte de un caso / Primary non-Hodgkin lymphoma of the breast: a case report

El linfoma no Hodgkin primario de la mama es una patología poco frecuente, constituye menos del 0,5 por ciento de todos los tumores mamarios malignos, debido a que carecen de características propias, tanto clínicas, mamográficas como ultrasonográficas. Es difícil el diagnóstico preoperatorio, la citología mediante BAAF tiene mejor rendimiento que el estudio con material congelado ya que este último tiende a confundirse con el carcinoma. En la actualidad se prefiere el tratamiento con quimioterapia tanto para el tratamiento local como para el regional. Presentamos el caso de un linfoma no Hodgkin primario de la mama en una paciente de 72 años. (AU)

Primary non-Hodgkin lymphoma of the breast is a rare entity. It represents less than 0.5 percent of all breast cancer malignancies. No features at clinical presentation distinguish patients with lymphoma from those with carcinoma of the breast. There are both mammographic and sonographic difficulties to establish the preoperative diagnosis. Contemporary frozen sections can be mistaken with breast carcinoma. In this article we present a case of a primary non-Hodgkin lymphoma of the breast in 72 years old women.(AU)

Resorbable synthetic mesh supported with omentum flap in the treatment of giant hiatal hernia.

Covering a large hiatal hernia with a mesh has become a basic procedure in the last few years. However, mesh implants are associated with high complication rates (esophageal erosion, perforation, fistula, etc.). We propose using a synthetic resorbable mesh supported with an omental flap as a possible solution to this problem. A 54-year-old female patient with a large hiatal defect (9 cm) was laparoscopically implanted with a synthetic resorbable mesh supported with an omental flap. The surgical procedure was successful and the patient was discharged on postoperative day 2. On a follow-up examination 6 months after surgery, she remained free of relapse or complication signs. Supporting an implanted resorbable mesh with an omental flap may be a solution to the problems posed by large esophageal hiatus defects. However, more studies based on larger patient samples and longer follow-up periods are necessary.

Psychological modulation in patients surgically intervened for gastroesophageal reflux disease.

Gastroesophageal reflux disease (GERD) has been related with certain psychological dimensions. The influence of mood, emotional intelligence, and perceived quality of life on clinical symptoms and outcome of antireflux surgery was evaluated in GERD patients with and without hiatal hernia. The study included 61 patients who were diagnosed with GERD between 2003 and 2008: 16 of them without hiatal hernia (group A) and 45 of them with hiatal hernia (group B). All of these patients had undergone laparoscopic antireflux surgery. Patients were clinically examined and evaluated with the following instruments: Short Form (SF)-36 Health Survey, Gastrointestinal Quality of Life Index, Hospital Anxiety and Depression (HAD) Scale, and Trait Meta-Mood Scale (TMMS)-24. Proportions were compared by using the chi-squared test; averages were compared by using the Student's t-test (with Bonferroni's correction). In general, our patients intervened for GERD showed results lower than normal or close to the lower limit of normal in the administered tests. Patients in the group without hernia were younger (P < 0.001) and with lower American Society of Anaesthesiologists risk. They showed higher scores in the SF-36 dimensions: Physical Functioning, Physical Role and Emotional Role, and lower scores in the Social Role (P < 0.001). They showed lower scores in the Emotional dimension of Gastrointestinal Quality of Life Index (P = 0.0068) and worse results in the Hospital Anxiety and Depression subscales of Anxiety (P < 0.001) and Depression (not significant). Men in the group without hernia showed higher scores than men in the group with hernia in the TMMS subscales corresponding to Emotional Clarity and Emotional Repair (P < 0.001). Women in the group with hernia showed higher scores than women in the group without hernia regarding Emotional Clarity (P = 0.0012). GERD patients showed poor results in all the tests, and patients without hiatal hernia compared with patients with hernia showed higher levels of anxiety, which interfered with their social life. Moreover, they showed lower tolerance to stress and higher frustration, fear, and worry. On the basis of such unfavorable phychoemotional results observed with GERD patients (especially those without hernia) in the different tests, we propose that improving our knowledge of the psychological profile of GERD patients - particularly those without hiatal hernia - could help in designing individualized medical and psychological therapies and increase success rates.

Metástasis esplénica metacrónica de cáncer de colon. Presentación de un caso infrecuente / Metachronous splenic metastasis from colon cancer. An infrecuent case

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CD26, adenosine deaminase, and adenosine receptors mediate costimulatory signals in the immunological synapse.

Adenosine deaminase (ADA), a protein whose deficit leads to severe combined immunodeficiency, binds to the cell surface by means of either CD26, A(1) adenosine receptors, or A(2B) adenosine receptors. The physiological role of these interactions is not well understood. Our results show that by a 3-fold reduction in the EC(50) for the antigen, ADA potentiated T cell proliferation in autologous cocultures with antigen-pulsed immature or mature dendritic cells. Costimulation was not due to the enzymatic activity but to the interaction of ADA-CD26 complexes in T cells with an ADA-anchoring protein in dendritic cells. From colocalization studies, it is deduced that ADA colocalizing with adenosine receptors on dendritic cells interact with CD26 expressed on lymphocytes. This costimulatory signal in the immunological synapse leads to a marked increase (3- to 34-fold) in the production of the T helper 1 and proimmflamatory cytokines IFN-gamma, TNF-alpha, and IL-6.

[Expert opinion on the basic surgical technique for laparoscopic ventral hernia repair]. / Técnica quirúrgica básica consensuada para el tratamiento por vía laparoscópica de las hernias ventrales.

Laparoscopic ventral hernia repair is currently the subject of intense debate, even though it provides a series of advantages over open surgery and is feasible and safe. Various studies have shown this technique to be as effective as open repair with a lower recurrence rate. Despite the excellent results of laparoscopic repair of ventral hernias, there are numerous controversies associated with this procedure. These controversies concern the indications and contraindications of the procedure, and technical aspects such as how to create the pneumoperitoneum, perform adhesiolysis, manage the hernia sac, and insert and fix the mesh to the anterior abdominal wall. Also controversial are outcome, complications related to postoperative seroma, and which type and size of mesh should be used. The present article aims primarily to address many of these issues, based on the experience of distinct surgeons with expertise in this approach, in order to provide data to establish a consensus on how laparoscopic ventral hernia repair should be performed.

Generation of Human Monoclonal Antibodies from HIV-Infected Individuals / Generación de anticuerpos monoclonales humanos a partir de individuos infectados por VIH

Recently, the protective role of anti-HIV-1 neutralising antibodies has been «re-discovered». Few broadly protective epitopes on the HIV envelope proteins are known, which were defined by few human monoclonal antibodies (mAbs) derived many years ago from non-progressor HIV-infected individuals. It is of interest to try to generate new neutralising human mAbs both to identify new protective epitopes for vaccine design and as potential passive immunotherapy agents. In addition, it is now known that rather than recognising HIV envelope proteins, some neutralising antibodies might be autoantibodies directed to the receptor (CD4) or co-receptors (CCR5 or CXCR4) on the HIV target cells. In that context, we attempted to generate neutralising human mAbs from few HIV-1-infected individuals who, after structured antiretroviral therapy interruptions, showed good virologic and immunologic responses, including serum HIV neutralising activity. We employed the classical heterohybridoma technology and found that the best screening strategy is a primary selection of IgG-producing hybridomas, followed by secondary screenings with different antigens and assays such as HIV neutralisation activity, direct binding to HIV components, binding to HIV target cells, and to cell lines transfected with human HIV receptors and co-receptors. From a single subject, 61 IgG-producing hybridomas out of 5,760 primary wells were obtained, and preliminary data indicate the presence of six (out of 23 tested) neutralising antibodies of the X4 strain, eight anti-p24 antibodies and two antibodies that bind to the MT-2 cell line, the target of X4 strains. None of the 61 mAbs obtained reacted with cells expressing CD4, CXCR4 or CCR5. Other screening tests such as neutralisation assay with the R5 strain, binding to HIV gp120-CD4 covalent complex, and to whole chemically (aldrithiol-2)-inactivated HIV are in progress. The screening strategy is also a means to “open” the repertoire of IgG antibodies of circulating B cells in HIV-infected individuals permitting to assess the frequency of HIV-related IgG antibodies and the IgV genes encoding them, an issue largely unknown (AU)

Recientemente se ha «re-descubierto» el carácter protectivo de los anticuerpos anti-VIH neutralizantes. Se conocen sólo unos pocos epitopos protectivos en las proteínas de la envoltura del VIH definidos gracias a unos pocos anticuerpos monoclonales humanos (mAbs) generados hace ya tiempo en pacientes infectados asintomáticos. Tiene interés intentar generar otros mAbs humanos neutralizantes que puedan definir nuevos epitopos protectivos para el diseño de vacunas, y ser útiles en inmunoterapia pasiva. Además, actualmente está claro que podrían existir anticuerpos neutralizantes que en vez de ir dirigidos contra la envoltura del VIH, reconocieran al receptor (CD4) o coreceptores (CCR5 o CXCR4) en la membrana de las células diana del VIH. En este contexto, hemos intentado obtener mAbs neutralizantes a partir de aquellos pacientes VIH+ que, tras un protocolo de interrupciones estructuradas de la terapia antiretroviral, mostraron buena respuesta virológica e inmunológica, con aumento de actividad sérica neutralizante. Utilizamos la metodología de heterohybridomas convencional y hallamos que la mejor estrategia de escrutinio es la selección primaria de hibridomas secretores de IgG, y luego el escrutinio con distintos ensayos para actividad neutralizante, unión a proteínas víricas, unión a la membrana de las células diana y a células transfectadas con receptores y co-receptores del VIH. A partir de un paciente, de 5.760 microcultivos primarios, se obtuvieron 61 hibridomas productores de IgG entre los que, según datos preliminares, hay seis mAbs (de 23 probados) neutralizantes contra cepas X4, ocho anti-p24 del VIH, y dos que se unen a la componentes de la membrana de la línea MT-2, diana de las cepas X4. Ningún mAb de los 61 obtenidos se unía a CCR5, CD4 o CXCR4. Hay otros escrutinios en curso como neutralización frente a cepa R5, la unión a complejo covalente gp120-CD4 y a VIH total inactivado químicamente con aldritiol-2. El escrutinio utilizado es también un modo de abrir el repertorio de anticuerpos IgG de los linfocitos B circulantes en individuos VIH+ y averiguar la frecuencia de los anticuerpos de especificidad relacionada con el VIH y los genes IgV que los codifican, un aspecto apenas estudiado (AU)

Expression of factor VII in the liver of patients with liver disease: correlations with the disease severity and impairment in the hemostasis.

Factor VII (FVII) plasma levels in patients with liver disease may be below the normal range. However, no data are available on FVII expression in liver biopsies from patients with liver diseases other than cirrhosis. We have analyzed the expression of FVII by in situ hybridization in liver biopsies from 50 patients in comparison with the procoagulant activity of FVII, and with the plasma levels as activated FVII (FVIIa) and FVII antigen. The level of FVIIa was significantly lower in stage 4 liver fibrosis patients than in the remaining ones (P < 0.05). The percentage of hepatocytes expressing FVII was significantly lower in stage 4 liver fibrosis patients (4.1+/-1.3%) than in stage 3 (22.7+/-6.1%), stage 2 (31.5+/-6.1%), stage 1 (43.7+/-8.2%) and stage 0 patients (63.8+/-4.4%) (P < 0.001). These percentages correlated inversely in a statistically significant way with the histological activity index and the liver function tests. We have demonstrated that the FVIIa plasma levels in patients with chronic liver disease other than cirrhosis may be below the normal range in the absence of blood coagulation impairment. The percentage of hepatocytes expressing FVII decreases as the severity of liver damage increases.

Sarcoma of the thyroid region mimicking Riedel's thyroiditis.

Because sarcomas of the anterior lower neck region occur so infrequently, they are not usually considered in the differential diagnosis of Riedel's thyroiditis. Riedel's thyroiditis itself may be confused on clinical grounds alone with malignant neoplasms because of its invasive features. Sarcomatoid carcinoma is the main entity to be discarded in this regard. This is accomplished through histological examination by the finding of carcinomatous areas and/or reactivity with epithelial markers. These features also set apart sarcomatoid carcinoma from true sarcomas. This report concerns a patient with a sarcoma of the anterior lower neck region which was initially suspected to be Riedel's thyroiditis or sarcomatoid carcinoma on clinical and radiological grounds. A peroperative biopsy was interpreted by two independent pathologists as consistent with Riedel's thyroiditis. The subsequent clinical course and postmortem examination demonstrated a high grade sarcoma with metastasis to both lungs and the pleura, and invasion of adjacent neck structures. Nevertheless, some areas of the postmortem material showed a microscopic pattern similar to mediastinal fibrosis, raising the possibility of the malignant transformation of a fibrosclerotic lesion.

[Acute kidney failure induced by rifampicin]. / Fracaso renal agudo inducido por rifampicina.

A case of acute renal failure requiring dialysis and associated with a characteristic, fulminant clinical course following the intermittent administration of rifampicin is presented. Renal biopsy showed severe tubular injury and a mild interstitial mononuclear cell infiltrate. Withdrawal of rifampicin led to a compete resolution of renal injury. We review the literature on the pathogenesis and treatment of this syndrome and we discuss the different substrates for acute renal failure induced by rifampicin.

Distribution of hepatitis C virus infection in liver biopsies from children and adults with chronic hepatitis C.

Chronic hepatitis C in children is characterized by milder forms of liver damage than those found in adults. Such a difference has been attributed to a low viral load in children that may lead to poor recognition of infected cells by the immune system. One approach that could be used to confirm this hypothesis may be to examine the number of infected hepatocytes in liver biopsies. Paraffin embedded liver biopsies from 21 children and 15 adults with chronic hepatitis C virus (HCV) infection (with a similar duration of the infection) were hybridized in situ and the percentage of infected hepatocytes was correlated with the histological activity index, alanine aminotransferase levels and HCV viraemia levels. Histological activity index and HCV viraemia levels were statistically higher (P < 0.05 and P < 0.01 respectively) in adults than in children, and the percentage of infected hepatocytes was higher in adults (11.0 +/- 19.7%) than in children (4.6 +/- 3.6%), although it did not reach statistical significance. Also, the percentage of infected hepatocytes correlated with HCV-RNA concentration in serum in both children (r = 0.683, P = 0.001) and adults (r = 0.768, P = 0.001). The results show that liver damage in children with chronic hepatitis C is not related to the extent of infection in the liver. This findings support the hypothesis of that liver injury in chronic HCV infection is mediated by the host immune response.

Primary cutaneous large B-cell lymphoma: the relation between morphology, clinical presentation, immunohistochemical markers, and survival.

The histogenesis, morphology, immunophenotype, and clinical behavior of cutaneous large B-cell lymphomas (CLBCL) are largely a matter of controversy. We performed an investigation to determine whether CLBCL have features that differentiate them from other large B-cell lymphomas and whether CLBCL is itself a heterogeneous group. To this end, we reviewed the main characteristics of a series of 32 cases of LBCL found in the skin. We reviewed the clinical findings and paraffin sections of the tumors from these 32 patients. The immunohistochemical study performed included p53, MIB1, Bcl2, Bcl6, and CD10 markers. We carried out statistical analysis of these data (univariate and multivariate), seeking an association between the features of the tumors and clinical outcome, as defined by failure-free survival time. Only one patient died as a consequence of the lymphoma. Nevertheless, the accumulated probability of survival without failure at 48 months was 0.46. The number, type, and localization of the lesions were not associated with variations in either survival or failure-free survival. The expression of p53 was negative in this group of CLBCL, whereas Bcl-2 expression or localization in the lower leg did not relate to any other significant feature. Histologic examination of the cases disclosed three different groups: Grade III follicular lymphomas (FLs), monomorphous large B-cell lymphomas (LBCL type I), and LBCL with an admixed component of small B-lymphocytes (LBCL type II). Grade III FL (11 cases) tended to be found in the head and neck and showed CD10 expression in a majority of cases. A higher probability of lymph node relapses was associated with cases located in the head and neck and with CD10+ tumors. Cutaneous large B-cell lymphomas are indolent tumors, but follow an insidious course. Our data support the interpretation that CLBCL is a heterogeneous condition; comprises some LBCL derived from CD10+ germinal center cells which manifests more frequently as tumors in the head and neck region, with an increased probability of relapse in lymph nodes [1] and has some distinctive morphologic features. The existence of a component of small B-cells within the other CLBCL could lend support to the theory that some of these tumors, more than arise de novo, may have originated in preexistent small B-cell lymphomas, but no firm evidence of this is provided in this study.

In situ detection of hepatitis C virus RNA in salivary glands.

Chronic hepatitis C virus (HCV) infection has been associated with several extrahepatic manifestations, among these, to diseases with oral manifestations such as Sjögren's syndrome or sialadenitis. HCV-RNA has been detected in saliva and in salivary glands from patients with sialadenitis by polymerase chain reaction. However, morphological evidence of HCV replication in salivary gland cells is needed to support a role for HCV in causing sialadenitis or Sjögren's syndrome. We have used in situ hybridization and immunohistochemistry to analyze the presence of HCV-RNA of sense and antisense polarity and HCV core antigen, respectively, in salivary gland biopsies from 19 patients with chronic sialadenitis or Sjögren's syndrome (eight anti-HCV-positive; 11 anti-HCV-negative). HCV-RNA of both positive and negative polarity as well as HCV core antigen were detected in the epithelial cells of the salivary gland biopsies from all of the anti-HCV-positive patients but in none of the anti-HCV-negative cases. The percentage of HCV-infected cells ranged from 25 to 48.8% in the patients studied. In conclusion, we have shown that HCV infects and replicates in the epithelial cells from salivary glands of patients with Sjögren's syndrome or chronic sialadenitis. However, its implication in the pathogenesis of these diseases deserves future research.

Fracaso renal agudo inducido por rifampicina / Rifampicin induced acute renal failure

Presentamos un caso de fracaso renal agudo que precisó diálisis, asociado a un característico cuadro clínico fulminante directamente relacionado con el uso intermitente de rifampicina. La biopsia renal mostró una lesión fundamentalmente tubular, con un discreto infiltrado inflamatorio intersticial. La suspensión de la rifampicina condujo a una resolución completa del cuadro. Revisamos la literatura sobre la patogenia y tratamiento de este síndrome y discutimos las distintas formas de fracaso renal agudo asociado a rifampicina (AU)

Detection of TT virus DNA in liver biopsies by in situ hybridization.

A novel hepatitis-associated virus named TT virus (TTV) has been isolated. However, its hepatotropism has not been proven. We have retrospectively analyzed the presence of TTV-DNA by polymerase chain reaction (PCR) and in situ hybridization in liver biopsies from 30 patients with liver disease (15 TTV-DNA-positive and 15 TTV-DNA-negative in serum), and prospectively in serum and liver from eight patients with normal liver histology. TTV-DNA was detected by PCR in the liver from the 15 patients with serum TTV-DNA and in serum and liver of two of the eight patients without liver disease. TTV-DNA titers in liver were 10 times higher than in serum, although no correlation between TTV-DNA titers in serum and liver were observed. In situ hybridization shows positive signals in the hepatocytes of the 17 patients infected by TTV but in none of the TTV-DNA-negative patients by PCR. No morphological changes were observed in the hepatocytes showing hybridization signals. The percentage of positive hepatocytes ranged from 2.1% to 30% and correlated with the TTV-DNA titers in liver (r = 0.54; P = 0.037). In conclusion, our results show that TTV is able to infect liver cells although they do not support a role for TTV in causing liver disease.

Hepatitis C virus RNA in kidney biopsies from infected patients with renal diseases.

Hepatitis C virus (HCV) infection has been associated with several renal pathologies, including membranoproliferative and membranous glomerulonephritis. Although the presence of HCV proteins has been reported, there are no data concerning detection of the viral RNA in renal cells from HCV-infected patients with kidney disease. In this report we analysed, by in situ hybridization, the presence of HCV RNA in renal biopsies from 10 patients who were positive for antibodies to HCV (anti-HCV) and serum HCV RNA positive, and from four patients without HCV infection, with different renal disease. HCV RNA was detected in the renal biopsies from all of the 10 HCV-infected patients. Hybridization signals were detected in the tubular and capillary endothelial cells. No hybridization signals were found in the renal biopsies of the four anti-HCV-negative patients. In conclusion, our results demonstrate that HCV RNA is common in kidney cells of patients with renal diseases who are infected with HCV. The presence of HCV RNA is not necessarily associated with a pathogenetic consequence.