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Supplementary Figures 1-6 from Antibody-Dependent Cell Cytotoxicity Synapses Form in Mice during Tumor-Specific Antibody Immunotherapy

Supplementary Table 1 from Antibody-Dependent Cell Cytotoxicity Synapses Form in Mice during Tumor-Specific Antibody Immunotherapy

Supplementary Methods, Figure Legends 1-6 from Antibody-Dependent Cell Cytotoxicity Synapses Form in Mice during Tumor-Specific Antibody Immunotherapy

Background: COVID-19, an infectious disease caused by SARS-CoV-2 virus, can provoke a vast range of clinical manifestations, ranging from asymptomatic to potentially life-threatening complications. At the beginning, convalescent plasma therapy has been proposed as an effective alternative to treat severe cases. The aim of this study was to follow over time a two-time renal transplanted COVID-19 severe patient treated with convalescent plasma from an immunological and virologic perspective.Case presentation: A 42-year-old female patient, two-time kidney transplanted was hospitalized with COVID-19. Due to worsening of respiratory symptoms, she was admitted to the intensive care unit where she received two doses of convalescent plasma. Conclusion: We analyzed the dynamics of viral load in nasopharyngeal swab, saliva and tracheal aspirate samples, before and after convalescent plasma transfusion. Pro-inflammatory cytokines and antibody titers were also measured in serum samples. A post-treatment decrease in viral load was observed to be sharp in saliva and nasopharyngeal swab samples, and slight in tracheal aspirate samples. Furthermore, we evidenced an increase of antibody titers post transfusion, accompanied with a decrease of several cytokines responsible of the cytokine storm.

The transition from pregnant non lactating condition to non pregnant lactating status is a period of dramatic changes for the cow, which has to adapt its metabolism to the strong requirements for milk production. From the equilibrium that the cow resolves this period will depend the capacity to maximize milk production and quality, to evade metabolic diseases and ensure the following pregnancy. The nutritional improvement, genetic selection and animal management have increase milk production in the last decades and this is associated with a decrease in the reproductive performance and in the increase of health diseases. This review summarizes the studies performed in the last years in Uruguay, with emphasis in nutritional management, ingestive behaviour, endocrine and molecular mechanisms of nutrient partitioning and its relation with fertility in dairy cows. Studies that investigate the productive efficiency of different dairy biotypes and breeds are reported. Studies in the cow´s health that identify the transition period as a risk factor for metabolic, infections and traumatic diseases are included. We conclude that studies that integrate problems of national relevance are the appropriate methodology to investigate complex biological systems as is the dairy cow during the transition period under grazing conditions.

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has proven to be a successful strategy for prevent severe infections. CoronaVac and BNT162b2 are the most used vaccines worldwide, but their use in heterologous vaccination schedules is still subjected to evaluation.Fifty healthy individuals who received heterologous prime-boost vaccination with CoronaVac and BNT162b2 were enrolled in a post-vaccination serological follow-up longitudinal prospective study. We evaluated specific serum anti-receptor binding domain (RBD) IgG antibody levels, and their capacity to block RBD-ACE2 interaction with a surrogate neutralization assay. In 20 participants, we assessed antibody binding kinetics by surface plasmon resonance, and Fc-mediated functions by ADCC and ADCP reporter assays.Our baseline seronegative cohort, displayed seroconversion after two doses of CoronaVac and an important decrease in serum anti-RBD IgG antibodies levels 80 days post-second dose. These levels increased significantly early after the third dose with BNT162b2, but 73 days after the booster we found a new fall. Immunoglobulin functionalities showed a similar behavior.The heterologous prime-boost vaccination with CoronaVac and BNT162b2 generated an impressive increase in serum anti-RBD specific antibody levels followed by a drop. Nevertheless, these titers remained well above those found in individuals only vaccinated with CoronaVac in the same elapsed time. Serum IgG levels showed high correlation with antibody binding analysis, their capacity to block RBD-ACE2 interaction, and Fc-effectors mechanisms. Our work sheds light on the humoral immune response to heterologous vaccination with CoronaVac and BNT162b2, to define a post-vaccination correlate of protection against SARS-CoV-2 infection and to discuss the scheduling of future vaccine boosters in general population.

Severe COVID-19 is associated with hyperinflammation and weak T cell responses against SARS-CoV-2. However, the links between those processes remain partially characterized. Moreover, whether and how therapeutically manipulating T cells may benefit patients are unknown. Our genetic and pharmacological evidence demonstrates that the ion channel TMEM176B inhibited inflammasome activation triggered by SARS-CoV-2 and SARS-CoV-2–related murine β-coronavirus. Tmem176b −/− mice infected with murine β-coronavirus developed inflammasome-dependent T cell dysfunction and critical disease, which was controlled by modulating dysfunctional T cells with PD-1 blockers. In critical COVID-19, inflammasome activation correlated with dysfunctional T cells and low monocytic TMEM176B expression, whereas PD-L1 blockade rescued T cell functionality. Here, we mechanistically link T cell dysfunction and inflammation, supporting a cancer immunotherapy to reinforce T cell immunity in critical β-coronavirus disease.

Breast cancer is a public health concern and is currently the fifth cause of mortality worldwide. Identification of different biological subtypes is essential for clinical management, therefore, the role of pathologists is essential and useful tools for immunohistochemistry diagnosis are needed. Polypeptide-GalNAc-transferases are emerging novel biomarkers related to cancer behavior and GalNAc-T13, correlated with aggressiveness in some tumors, is an interesting candidate. Few monoclonal antibodies reacting with native proteins, and not affected by fixation and paraffin embedding, have been reported. The aim of this work was to develop a useful monoclonal antibody anti-GalNAc-T13 and to assess its potential significance in breast cancer diagnosis. We evaluated 6 human breast cancer cell lines, 338 primary breast tumors and 48 metastatic lymph nodes and looked for clinical significance correlating GalNAc-T13 expression with patients’ clinical features and survival. We found high GalNAc-T13 expression in 43.8% of the cases and observed a significant higher expression in metastatic lymph nodes, correlating with worse overall survival. We hypothesized several possible molecular mechanisms and their implications. We conclude that GalNAc-T13 may be a novel biomarker in breast cancer, useful for routine pathological diagnosis. Elucidation of molecular mechanisms related to aggressiveness should contribute to understand the role of GalNAc-T13 in breast cancer biology.

Material complementario: https://www.frontiersin.org/articles/10.3389/fviro.2021.756559/full#supplementary-material Bovine leukemia virus (BLV) is an oncogenic deltaretrovirus that infects cattle worldwide. In Uruguay, it is estimated that more than 70% of dairy cattle are infected, causing serious economic losses due to decreased milk production, increased calving interval, and livestock losses due to lymphosarcoma. Several attempts to develop vaccine candidates that activate protective immune responses against BLV were performed, but up to date, there is no vaccine that ensures efficient protection and/or decreased viral transmission. The development and application of new vaccines that effectively control BLV infection represent amajor challenge for countries with a high prevalence of infection. In this study, we generated two Drosophila melanogaster S2 stable cell lines capable of producing BLV virus-like particles (BLV-VLPs). One of them, BLV-VLP1, expressed both Gag and Env wild-type (Envwt) full-length proteins, whereas BLV-VLP2 contain Gag together with a mutant form of Env non-susceptible to proteolytic maturation by cellular furin type enzymes (EnvFm).We showed that Envwt is properly cleaved by cellular furin, whereas EnvFm is produced as a full-length gp72 precursor, which undergoes some partial cleavage. We observed that said mutation does not drastically affect its expression or its entry into the secretory pathway of S2 insect cells. In addition, it is expressed on the membrane and retains significant structural motifs when expressed in S2 insect cells. Morphology and size of purified BLV-VLPs were analyzed by transmission electron microscopy and dynamic light scattering, showing numerous non-aggregated and approximately spherical particles of variable diameter (70–200 nm) as previously reported for retroviral VLPs produced using different expression systems. Furthermore, we identified two N-glycosylation patterns rich in mannose in EnvFm protein displayed on VLP2. Our results suggest that the VLPs produced in Drosophila S2 cells could be a potential immunogen to be used in the development of BLV vaccines that might contribute, in conjunction with other control strategies, to reduce the transmission of the virus. CSIC I+D 2014 ANII: ALI_1_2016_2_129851; POS_NAC_2015_1_109471 PEDECIBA-FOCEM: COF 03/11 CAP: BFPD_2020_1#28143834

Neospora caninum causes neosporosis, a leading cause of bovine abortion worldwide. Uruguay is a developing economy in South America that produces milk to feed seven times its population annually. Naturally, dairy production is paramount to the country's economy, and bovine reproductive failure impacts it profoundly. Recent studies demonstrated that the vast majority of infectious abortions in dairy cows are caused by N. caninum. To delve into the local situation and contextualize it within the international standing, we set out to characterize the Uruguayan N. caninum strains. For this, we isolated four distinct strains and determined by microsatellite typing that these represent three unique genetic lineages, distinct from those reported previously in the region or elsewhere. An unbiased analysis of the current worldwide genetic diversity of N. caninum strains known, whereby six typing clusters can be resolved, revealed that three of the four Uruguayan strains group closely with regional strains from Argentina and Brazil. The remaining strain groups in an unrelated genetic cluster, suggesting multiple origins of the local strains. Microsatellite typing of N. caninum DNA from fetuses opportunistically collected from local dairy farms correlated more often with one of the isolates. Overall, our results contribute to further understanding of genetic diversity among strains of N. caninum both regionally and worldwide.

BackgroundSouth America has become the new epicenter of the COVID-19 pandemic with more than 1.1M reported cases and >50,000 deaths (June 2020). Conversely, Uruguay stands out as an outlier managing this health crisis with remarkable success.MethodsWe developed a molecular diagnostic test to detect SARS-CoV-2. This methodology was transferred to research institutes, public hospitals and academic laboratories all around the country, creating a “COVID-19 diagnostic lab network”. Uruguay also implemented active epidemiological surveillance following the “Test, Trace and Isolate” (TETRIS) strategy coupled to real-time genomic epidemiology.ResultsThree months after the first cases were detected, the number of positive individuals reached 826 (23 deaths, 112 active cases and 691 recovered). The Uruguayan strategy was based in a close synergy established between the national health authorities and the scientific community. In turn, academia rapidly responded to develop national RT-qPCR tests. Consequently, Uruguay was able to perform ∼1,000 molecular tests per day in a matter of weeks. The “COVID-19 diagnostic lab network” performed more than 54% of the molecular tests in the country. This, together with real- time genomics, were instrumental to implement the TETRIS strategy, helping to contain domestic transmission of the main outbreaks registered so far.ConclusionsUruguay has successfully navigated the first trimester of the COVID-19 health crisis in South America. A rapid response by the scientific community to increase testing capacity, together with national health authorities seeking out the support from the academia were fundamental to successfully contain, until now, the COVID-19 outbreak in the country.

International audience; PknG from Mycobacterium tuberculosis is a multidomain Serine/Threonine protein kinase that regulates bacterial metabolism as well as the pathogen's ability to survive inside the host by still uncertain mechanisms. To uncover PknG interactome we developed an affinity purification-mass spectrometry strategy to stepwise recover PknG substrates and interactors; and to identify those involving PknG autophosphorylated docking sites. We report a confident list of 7 new putative substrates and 66 direct or indirect partners indicating that PknG regulates many physiological processes, such as nitrogen and energy metabolism, cell wall synthesis and protein translation. GarA and the 50S ribosomal protein L13, two previously reported substrates of PknG, were recovered in our interactome. Comparative proteome analyses of wild type and pknG null mutant M. tuberculosis strains provided evidence that two kinase interactors, the FHA-domain containing protein GarA and the enzyme glutamine synthetase, are indeed endogenous substrates of PknG, stressing the role of this kinase in the regulation of nitrogen metabolism. Interestingly, a second FHA protein was identified as a PknG substrate. Our results show that PknG phosphorylates specific residues in both glutamine synthetase and FhaA in vitro, and suggest that these proteins are phosphorylated by PknG in living mycobacteria.

Because of resistance development by cancer cells against current anticancer drugs, there is a considerable interest in developing novel antitumor agents. We have previously demonstrated that CIGB-552, a novel cell-penetrating synthetic peptide, was effective in reducing tumor size and increasing lifespan in tumor-bearing mice. Studies of protein-peptide interactions have shown that COMMD1 protein is a major mediator of CIGB-552 antitumor activity. Furthermore, a typical serine-protease degradation pattern for CIGB-552 in BALB/c mice serum was identified, yielding peptides which differ from CIGB-552 in size and physical properties. In the present study, we show the results obtained from a comparative analysis between CIGB-552 and its main metabolites regarding physicochemical properties, cellular internalization, and their capability to elicit apoptosis in MCF-7 cells. None of the analyzed metabolites proved to be as effective as CIGB-552 in promoting apoptosis in MCF-7. Taking into account these results, it seemed important to examine their cell-penetrating capacity and interaction with COMMD1. We show that internalization, a lipid binding-dependent process, is impaired as well as metabolite-COMMD1 interaction, key component of the apoptotic mechanism. Altogether, our results suggest that features conferred by the amino acid sequence are decisive for CIGB-552 biological activity, turning it into the minimal functional unit. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Pretargeted imaging, based on the highly reactive process between [1,2,4,5]tetrazines with trans-cyclooctene (TCO), appears as an attractive strategy to overcome disadvantages associated with traditional radioimmunoconjugates. To be successful, the radiolabeled component should react in vivo with the conjugated antibody and the non reactive excess clear fast from the organism. Herein, we explore the in vivo effects of hydrophilic linker incorporation into [1,2,4,5]tetrazine systems bearing a 6-hydrazinonicotinyl (HYNIC) moiety for technetium-99m coordination. Incorporation of a polypeptide chain containing hydrophilic aminoacids, resulted in a derivative with renal clearance. Pretargeted bevacizumab imaging was used as proof of concept.

International audience; Antibody-drug conjugates (ADC), combining the specificity of tumor recognition by monoclonal antibodies (mAb) and the powerful cytotoxicity of anticancer drugs, are currently under growing interest and development. Here, we studied the potential of Chi-Tn, a mAb directed to a glyco-peptidic tumor-associated antigen, to be used as an ADC for cancer treatment. First, we demonstrated that Chi-Tn specifically targeted tumor cells in vivo. Also, using flow cytometry and deconvolution microscopy, we showed that the Chi-Tn mAb is rapidly internalized - condition necessary to ensure the delivery of conjugated cytotoxic drugs in an active form, and targeted to early and recycling endosomes. When conjugated to saporin (SAP) or to auristatin F, the Chi-Tn ADC exhibited effective cytotoxicity to Tn-positive tumor cells in vitro, which correlated with the level of tumoral Tn expression. Furthermore, the Chi-Tn mAb conjugated to auristatin F also exhibited efficient antitumor activity in vivo, validating for the first time the use of an anti-Tn antibody as an effective ADC.

Because of resistance development by cancer cells against current anticancer drugs, there is a considerable interest in developing novel antitumor agents. We have previously demonstrated that CIGB-552, a novel cell-penetrating synthetic peptide, was effective in reducing tumor size and increasing lifespan in tumor-bearing mice. Studies of protein-peptide interactions have shown that COMMD1 protein is a major mediator of CIGB-552 antitumor activity. Furthermore, a typical serine-protease degradation pattern for CIGB-552 in BALB/c mice serum was identified, yielding peptides which differ from CIGB-552 in size and physical properties. In the present study, we show the results obtained from a comparative analysis between CIGB-552 and its main metabolites regarding physicochemical properties, cellular internalization, and their capability to elicit apoptosis in MCF-7 cells. None of the analyzed metabolites proved to be as effective as CIGB-552 in promoting apoptosis in MCF-7. Taking into account these results, it seemed important to examine their cell-penetrating capacity and interaction with COMMD1. We show that internalization, a lipid binding-dependent process, is impaired as well as metabolite-COMMD1 interaction, key component of the apoptotic mechanism. Altogether, our results suggest that features conferred by the amino acid sequence are decisive for CIGB-552 biological activity, turning it into the minimal functional unit. Copyright © 2016 European Peptide Society and John WileySons, Ltd.

Nitric oxide ((*)NO) has been implicated in multiple physiological and pathological immune processes. Different methods have been developed to detect and quantify (*)NO, where one of the principal difficulties are the accurately detection in cellular system with low levels of (*)NO production. The choice of the (*)NO detection method to be used depends on the characteristics of the experimental system and the levels of (*)NO production which depend on either the organism source of samples or the experimental conditions. Recently, high sensitive methods to detect and image (*)NO have been reported using 4,5-diaminofluorescein-based fluorescent probes (DAF) and its derivate 4,5-diaminofluorescein diacetate (DAF-2 DA). This work was aimed to adapt and optimize the use of DAF probes to detect and quantify the (*)NO production in systems of high, moderate and low out-put production, especially in human PBMC and their subpopulations. Here, we report an original experimental design which is useful to detect and estimate (*)NO fluxes in human PBMC and their subpopulations with high specificity and sensitivity.

International audience; Protein kinase G of Mycobacterium tuberculosis has been implicated in virulence and in regulation of glutamate metabolism. Here we show that this kinase undergoes a pattern of autophosphorylation that is distinct from that of other M. tuberculosis protein kinases characterized to date and we identify GarA as a substrate for phosphorylation by PknG. Autophosphorylation of PknG has little effect on kinase activity but promotes binding to GarA, an interaction that is also detected in living mycobacteria. PknG phosphorylates GarA at threonine 21, adjacent to the residue phosphorylated by PknB (T22), and these two phosphorylation events are mutually exclusive. Like the homologue OdhI from Corynebacterium glutamicum, the unphosphorylated form of GarA is shown to inhibit alpha-ketoglutarate decarboxylase in the TCA cycle. Additionally GarA is found to bind and modulate the activity of a large NAD(+)-specific glutamate dehydrogenase with an unusually low affinity for glutamate. Previous reports of a defect in glutamate metabolism caused by pknG deletion may thus be explained by the effect of unphosphorylated GarA on these two enzyme activities, which may also contribute to the attenuation of virulence.

International audience; The Tn antigen (GalNAcα-O-Ser/Thr) is a well-established tumor-associated marker which represents a good target for the design of anti-tumor vaccines. Several studies have established that the binding of some anti-Tn antibodies could be affected by the density of Tn determinant or/and by the amino acid residues neighboring O-glycosylation sites. In the present study, using synthetic Tn-based vaccines, we have generated a panel of anti-Tn monoclonal antibodies. Analysis of their binding to various synthetic glycopeptides, modifying the amino acid carrier of the GalNAc(*) (Ser* vs Thr*), showed subtle differences in their fine specificities. We found that the recognition of these glycopeptides by some of these MAbs was strongly affected by the Tn backbone, such as a S*S*S* specific MAb (15G9) which failed to recognize a S*T*T* or a T*T*T* structure. Different binding patterns of these antibodies were also observed in FACS and Western blot analysis using three human cancer cell lines (MCF-7, LS174T and Jurkat). Importantly, an immunohistochemical analysis of human tumors (72 breast cancer and 44 colon cancer) showed the existence of different recognition profiles among the five antibodies evaluated, demonstrating that the aglyconic part of the Tn structure (Ser vs Thr) plays a key role in the anti-Tn specificity for breast and colon cancer detection. This new structural feature of the Tn antigen could be of important clinical value, notably due to the increasing interest of this antigen in anticancer vaccine design as well as for the development of anti-Tn antibodies for in vivo diagnostic and therapeutic strategies.

The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3α) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins - the trifunctional enzyme (TFP), the ATP synthase and the sulfide quinone oxidoreductase - and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhibited by a specific phosphatase inhibitor. Moreover, PtpA wt was capable of dephosphorylating immunopurified human TFP in vitro supporting that TFP may be a bona fide PtpA susbtrate. Overall, these results suggest a novel scenario where PtpA-mediated dephosphorylation may affect pathways involved in cell energy metabolism, particularly the beta oxidation of fatty acids through modulation of TFP activity and/or cell distribution.

Retroviral capsids in their native form Capsid proteins of retroviruses form protective lattices around viral RNA molecules. The precise molecular details of how individual, full-length capsid proteins assemble to shield the viral genome; however, are not well understood. Obal et al. and Gres et al. now report high resolution crystal structures of the full length capsid proteins from Bovine Leukemia Virus and HIV-1, respectively. The two studies complement each other to reveal the dynamic nature of capsid protein assembly and of how individual capsid proteins interact in the lattice. The findings may have relevance for drug design. Science , this issue p. 95 ; see also p. 99

Tryparedoxin peroxidase from Trypanosoma cruzi (TcTXNPx) belongs to the family of typical 2-Cys peroxiredoxins. These enzymes function as antioxidants through their peroxidase and peroxynitrite reductase activities. In T. cruzi, as in all trypanosomatids, this enzyme is the final electron acceptor of a unique system for detoxifying hydroperoxides, constituting a relevant target for drug design. We have determined the crystal structure of TcTXPNx in the reduced active state. The structure comprises 10 subunits in the asymmetric unit, associated to form a decamer of toroidal shape obeying 52 (D5) point group symmetry. We have analyzed the structure of TcTXNPx by comparing it with other structures of typical 2-Cys peroxiredoxins in both redox states, and have identified key residues in the structural rearrangement taking place in the enzymatic cycle. This is the first report of the structure of an active peroxiredoxin that has peroxidase and peroxynitrite reductase activity, and it is noteworthy that it is from a human parasite. This knowledge is of interest for further understanding peroxide metabolism in these parasites, and in the design of new trypanosomatidal drugs against Chagas disease.

Trypanosoma cruzi, the causative agent of Chagas disease, has evolved particular mechanisms of gene regulation. Gene expression is regulated firstly at post-transcriptional level. This feature makes proteomic methods a promising tool for studying adaptative changes in these parasites. In this work we generated a reproducible method for protein analysis by two-dimensional electrophoresis coupled to mass spectrometry, and a protein map for T. cruzi. Western-blot analysis supported the identity of some of the proteins. This work points to proteomic approach as a powerful tool to study differential expression, stress response or drug resistance in T. cruzi.

Although antibodies have been assumed to bind a specific antigen, evidence exists showing that a single antibody can bind to multiple unrelated antigens. We previously studied a human monoclonal antibody expressing a mutated form of the V(H)3-73 gene and displaying anti-tubulin activity in a patient suffering from an immunocytic lymphoma. Despite its expression of a V(H)3 family member, this immunoglobulin failed to react with protein A (SpA), suggesting that somatic mutations could account for its change in specificity. To examine this possibility, we produced recombinant Ig expressing germ-line (IgM kappa-Germ) or the mutated form (IgM kappa-PER) of the V(H)3-73 fragment. Comparison of the respective affinities of the two Ig demonstrated that IgM kappa-Germ restores its SpA-binding capacity, and shows a moderate decrease in its affinity for tubulin. Interestingly, IgM kappa-Germ displayed polyreactive specificity for different autoantigens, which contrasted to the monospecific binding of IgM kappa-PER to tubulin. These results suggest that the monoreactive IgM kappa-PER antibody may be derived from a natural polyreactive antibody through somatic mutation. In addition, both temperature modification and mild denaturation succeeded in recovering the polyreactivity of IgM kappa-PER, which favors the view that conformational modifications of the tertiary structure of antibodies may play a key role in the genesis of polyreactivity.

Peroxynitrite-dependent tyrosine nitration has been postulated to be involved in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Evidence supporting this supposition includes the appearance of both free and protein-linked 3-nitro-l-tyrosine (nitrotyrosine) in both sporadic and familial ALS, as well as of increased free nitrotyrosine levels in the spinal cord of transgenic mice expressing ALS-linked superoxide dismutase mutants at symptom onset. Here we demonstrate that incubation with clinically relevant concentrations of nitrotyrosine induced apoptosis in motor neurons cultured with trophic factors. Nitrotyrosine was bound to proteins, but it was not incorporated into alpha-tubulin, as previously demonstrated for other cell types. Neither inhibition of nitric oxide production nor scavenging of superoxide and peroxynitrite prevented increases in cell nitrotyrosine immunoreactivity or motor neuron death, suggesting that these effects are not due to the endogenous formation of reactive nitrogen species. In contrast, some populations of astrocytes incorporated nitrotyrosine into alpha-tubulin, but free nitrotyrosine had no effect on the viability and phenotype of astrocytes in culture, as evaluated by glial fibrillary acidic protein immunoreactivity, cell growth and morphology. Co-culture of motor neurons on astrocyte monolayers delayed, but did not prevent, nitrotyrosine-induced motor neuron death. These results suggest that free nitrotyrosine may play a role in the induction of motor neuron apoptosis in ALS.

Functional inducible NOS (iNOS) may be involved in the prolonged lifespan of chronic lymphocytic leukemia cells (B-CLL), although the exact mechanisms implicated remain elusive as yet. In this work, we have examined iNOS expression in normal B lymphocytes and B-CLL cells in pro- and antiapoptotic conditions. Our results demonstrate: (1) The existence of a new splice variant characterized by a complete deletion of exon 14 (iNOS 13-16(14del)), which was preferentially detected in normal B lymphocytes and may represent an isoform that could play a role in the regulation of enzyme activity. (2) The existence of another alternatively spliced iNOS mRNA transcript involving a partial deletion of the flavodoxin region (iNOS 13-16(neg)) was correlated to a decreased B-CLL cell viability. The 9-beta-D-arabinofuranosyl-2-fluoradenine or fludarabine (F-ara) treatment induced iNOS 13-16(neg) transcript variants, whereas IL-4 enhanced both the transcription of variants, including these exons (iNOS 13-16(pos)), and the expression of a 122 kDa iNOS protein. These results suggest that in B-CLL, a regulation process involving nitric oxide (.- NO) levels could occur by a post-transcriptional mechanism mediated by soluble factors. Our results also provide an insight into a new complementary proapoptotic action of F-ara in B-CLL by the induction of particular iNOS splice variants, leading to the activation of a caspase-3-dependent apoptotic pathway.

Reverse genetics can be used in the bovine leukemia virus (BLV) system to characterize mechanisms of viral persistence and pathogenesis. The question addressed here pertains to the role of glycans bound to the BLV envelope glycoprotein (SU). A commonly accepted hypothesis is that addition of carbohydrates to the SU protein potentially creates a structure called « glycan shield » that confers resistance to the virus against the host immune response. On the other hand, glycosylation can also modulate attachment of the virus to the cell membrane. To unravel the role of SU glycosylation, three complementary strategies were developed: pharmacological inhibition of different glycosylation pathways, interference with glycan attachment and site-directed mutagenesis of N-glycosylation sites in an infectious BLV provirus. The different approaches show that glycosylation is required for cell fusion, as expected. Simultaneous mutation of all 8 potential N-glycosylation sites destroys infectivity. Surprisingly, mutation of the asparagine residue at position 230 creates a virus having an increased capacity to form syncytia in vitro. Compared to wild-type BLV, mutant N230 also replicates at accelerated rates in vivo. Collectively, this data thus illustrates an example of a N-glycosylation site that restricts viral replication, contrasting with the hypothesis supported by glycan shield model.

International audience; Using synthetic Tn (GalNAc-O-Ser/Thr) glycopeptide models and a biosensor based on surface plasmon resonance spectroscopy we have determined that isolectin B4 from Vicia villosa (VVLB4) binds to one Tn determinant whereas the anti-Tn monoclonal antibodies 83D4 and MLS128 require at least two Tn residues for recognition. When an unglycosylated amino acid is introduced between the Tn residues, both antibodies do not bind. MLS128 affinity was higher on a glycopeptide with three consecutive Tn residues. These results indicate that Tn residues organized in clusters are essential for the binding of these antibodies and indicate a different Tn recognition pattern for VVLB4.

Summary. The excess risk of chronic lymphocytic leukaemia (CLL) in the first-degree relatives of affected patients suggests that familial CLL might constitute a useful model to study the pathogenesis of this disease, as has been demonstrated in numerous other neoplastic disorders. Previous studies have shown non-random utilization of immunoglobulin genes in CLL, some germline in sequence and others containing numerous somatic mutations. To investigate whether familial cases of CLL exhibit similarities in the composition of the B-cell receptor repertoire to the pattern expressed by CLL patients as a whole, we have studied 25 CLL patients belonging to 12 different families (four French and eight Italian), each of which contained at least two affected members. Among familial cases, VH gene segment utilization proved non-random and diverged from the frequencies previously reported among unrelated patients with CLL. Specifically, although the 4-34 and 5-51 gene segments were found repeatedly, the 1-69 and 4-39 gene segments were used sparingly and the 3-23 gene segment presented with increased frequency. Following the pattern detected in studies of unrelated patients, the single 1-69 expressing CLL contained an unmutated H chain sequence and included a long HCDR3 interval. In contrast, 3-23 containing H chains all used JH4, retained at most 93% homology with germline sequence, and included only short HCDR3 intervals. The vast majority of the CLL variable domains contained a high degree of somatic mutation and exhibited an excess of replacement mutations in the CDR intervals. These findings suggest that familial CLL cases may preferentially derive from B-cell progenitors that have responded to antigen.

International audience; We report here the first amino acid sequence of an anti-Tn monoclonal antibody raised against human breast cancer cells and show that a single chain Fv fragment of this IgM retains the Tn-binding specificity as defined by functional assays with asialo-OSM and membrane extracts from MCF-7 cells. Sequence comparisons and molecular modeling of 83D4 indicate that the antibody combining site displays a cavity-like feature primarily defined by the CDR H1 and H2 loops. This pocket could accommodate a single Tn molecule, thus, suggesting a structural explanation for the predominant expression of a particular VH gene segment in a group of antibodies that recognize tumor-associated antigens arising from an aberrant O-glycosylation.

MARCKS (Myristoylated Alanine-Rich C Kinase substrate) is a natively unfolded protein that interacts with actin, Ca(2+)-Calmodulin, and some plasma membrane lipids. Such interactions occur at a highly conserved region that is specifically phosphorylated by PKC: the Effector Domain. There are two other conserved domains, MH1 (including a myristoylation site) and MH2, also located in the amino terminal region and whose structure and putative protein binding capabilities are currently unknown. MH2 sequence contains a serine that we described as being phosphorylated only in differentiating neurons (S25 in chick). Here, Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy were used to characterize the phosphorylated and unphosphorylated forms of a peptide with the MARCKS sequence surrounding S25. The peptide phosphorylated at this residue is recognized by monoclonal antibody 3C3 (mAb 3C3). CD and NMR data indicated that S25 phosphorylation does not cause extensive modifications in the peptide structure. However, the sharper lines, the absence of multiple spin systems and relaxation dispersion data observed for the phosphorylated peptide suggested a more ordered structure. Surface Plasmon Resonance was employed to compare the binding properties of mAb 3C3 to MARCKS protein and peptide. SPR showed that mAb 3C3 binds to the whole protein and the peptide with a similar affinity, albeit different kinetics. The slightly ordered structure of the phosphorylated peptide might be at the origin of its ability to interact with mAb 3C3 antibody, but this binding did not noticeably modify the peptide structure.

La transición del estado preñada no lactante al no preñado lactante es un período de cambios drásticos para la vaca, la cual debe adaptar su metabolismo a las fuertes exigencias que le demanda la producción. Del equilibrio con que la vaca resuelva este proceso dependerá la capacidad de maximizar la producción y la calidad de la leche, de evitar enfermedades metabólicas y asegurar la siguiente preñez. La mejora nutricional, la selección genética y el manejo animal han aumentado la producción de leche en las últimas décadas, y esto se asocia a una disminución del desempeño reproductivo y al aumento de problemas sanitarios. Esta revisión resume trabajos realizados en los últimos años en Uruguay, enfatizando en el manejo diferencial de nutrientes, la respuesta en comportamiento ingestivo, los mecanismos endocrino-moleculares de la partición de nutrientes y su relación con la fertilidad en vacas lecheras. Se reportan experimentos que estudian la eficiencia productiva de diferentes biotipos lecheros y razas. Se describen trabajos en salud que identifican el período de transición como período de riesgo a enfermedades metabólicas, infecto-contagiosas y traumáticas. Se concluye que los estudios integrados en problemas de relevancia nacional es la respuesta necesaria a sistemas biológicos complejos como lo es la vaca lechera durante el período de transición en pastoreo The transition from pregnant non lactating condition to non pregnant lactating status is a period of dramatic changes for the cow, which has to adapt its metabolism to the strong requirements for milk production. From the equilibrium that the cow resolves this period will depend the capacity to maximize milk production and quality, to evade metabolic diseases and ensure the following pregnancy. The nutritional improvement, genetic selection and animal management have increase milk production in the last decades and this is associated with a decrease in the reproductive performance and in the increase of health diseases. This review summarizes the studies performed in the last years in Uruguay, with emphasis in nutritional management, ingestive behaviour, endocrine and molecular mechanisms of nutrient partitioning and its relation with fertility in dairy cows. Studies that investigate the productive efficiency of different dairy biotypes and breeds are reported. Studies in the cow´s health that identify the transition period as a risk factor for metabolic, infections and traumatic diseases are included. We conclude that studies that integrate problems of national relevance are the appropriate methodology to investigate complex biological systems as is the dairy cow during the transition period under grazing conditions

It is widely accepted that the majority of cancers result from multiple cellular events leading to malignancy after a prolonged period of clinical latency, and that the immune system plays a critical role in the control of cancer progression. Bovine leukemia virus (BLV) is an oncogenic member of the Retroviridae family. Complete genomic sequences of BLV strains isolated from peripheral blood mononuclear cells (PBMC) from cattle have been previously reported. However, a detailed characterization of the complete genome of BLV strains directly isolated from bovine tumors is much needed in order to contribute to the understanding of the mechanisms of leukemogenesis induced by BLV in cattle. In this study, we performed a molecular characterization of BLV complete genomes from bovine B-cell lymphosarcoma isolates. A nucleotide substitution was found in the glucocorticoid response element (GRE) site of the 5' long terminal repeat (5'LTR) of the BLV isolates. All amino acid substitutions in Tax previously found to be related to stimulate high transcriptional activity of 5'LTR were not found in these studies. Amino acid substitutions were found in the nucleocapsid, gp51 and G4 proteins. Premature stop-codons in R3 were observed. Few mutations or amino acid substitutions may be needed to allow BLV provirus to achieve silencing. Substitutions that favor suppression of viral expression in malignant B cells might be a strategy to circumvent effective immune attack.

Prolymphocytic teukemia (PLL] is a chronic lymphoproliferative disorder, characterized by prominent splenomegaly, prolymphocytes accounting for more than 55% of circulating lymphocytes, and short-term survival. To better characterize the nature of the cellular origin in this disease, we analyzed Ig heavy chain variable region (V,) genes in eleven cases of de novo PLL. Leukemic cells expressed a skewed repertoire characterized by predominant use of the V3 family members (73%). with preferential use of the V3-23 gene (50% of the VH3 genes). All sequences from expressed Vn genes diverged from their putative germline counterpart, and in eight cases ROLYMPHOCYTIC leukemia (PLL) is a relatively rare P form of chronic lymphoproliferative disease, first described by Galton in 1974.' According to French-AmericanBritish (FAB) diagnostic criteria, the hallmark of the disease is the presence of more than 55% circulating morphologically identified prolymphocytes.' It is a distinct clinicopathological entity characterized by massive splenomegaly without lymph node enlargement, frequent marked hyperleukocytosis (often >lo0 x 109/L; the majority of which are prolymphocytes), and associated anemia and thrombopenia. Morphologically, prolymphocytes are large cells with condensed nuclear chromatin and a prominent central nucleolus. The vast majority of PLL are from the B-lineage origin and express high density monotypic surface immunoglobulin (sIg), usually IgM with or without IgD, and the CD19, CD20, and CD22 B cell marker^.^ In contrast to chronic lymphocytic leukemia (CLL), they often express FMC7, but rarely CD23. About half of the cases express the CD5 antigen. Although PLL is considered to be distinct from CLL by many criteria, the delineation between the two is not clearcut because some cases of CLL can transform into PLL: Furthermore, mixed forms of CLLPL have been identified in which the number of prolymphocytes is between 10% and 55%.5 Analysis of Ig genes has provided important information in understanding the biology of B-cell tumors6 After completing a series of complex DNA rearrangements of the Ig genes in the bone marrow, virgin B cells emigrate to the lymphoid organs. Further maturation of B lymphocytes occurs within the germinal centers of these organs, including the introduction of point mutations in the Ig variable (V) regions by the poorly understood mechanism of somatic hypermutation.' On stimulation by local antigens, those cells expressing V genes with mutations leading to higher affinity are selected and expand preferentially. This process results in the generation of memory B cells and high-affinity antibody producing cells. Consequently point mutations are found preferentially in nucleotides coding for amino acid residues critical for antigen-antibody interactions, the complementarity determining regions (CDR). In contrast, polyclonal stimulation not mediated by antigen results in mutations that are randomly distributed throughout the V regions.8 Analysis of V region nucleotide sequences can therefore the divergence was greater than 5%. In seven cases, which expressed the V3-23 gene and V,,4 family members, nucleotide substitutions could be confidently attributed to somatic mutations. The type and distribution of these mutations clearly indicated that in three cases the cells had been subjected to an antigen selection process. Taken together, these results suggest that B-PLL cells display a skewed repertoire of Ig VH regions and probably represent, at least in some instances, expansion of postgerminal center cells that have undergone antigen driven selection.

Although the switch process is frequently associated with affin- ity maturation, the constant region is not assumed to play a role in Ag-Ab binding. In the present work, we demonstrate that two clonally related human monoclonal Igs sharing iden- tical V H and V L sequences, but expressing different isotypes (IgA1 k PER and IgG1 k PER ), bind tubulin with significantly dif- ferent affinities. This difference was mainly accounted for by a disparity in the association rate constants. These results sug- gest that affinity maturation of this clone could be achieved through class switching in the absence of further somatic mu- tations. Since the differences observed were found at the Fab level, they also suggest a role for the C H 1 domain in struc- turing the Ag-binding site into a more kinetically competent form. ( J. Clin. Invest. 1996. 98:2235-2243.) Key words: im- munoglobulinsisotypeswitchingC H 1 • affinity

International audience; Despite being the most abundant class of immunoglobulins in humans and playing central roles in the adaptive immune response, high-resolution structural data are still lacking for the antigen-binding region of human isotype A antibodies (IgAs). The crystal structures of a human Fab fragment of IgA1 in three different crystal forms are now reported. The three-dimensional organization is similar to those of other Fab classes, but FabA1 seems to be more rigid, being constrained by a hydrophobic core in the interface between the variable and constant domains of the heavy chain (VH-CH1) as well as by a disulfide bridge that connects the light and heavy chains, influencing the relative heavy/light-chain orientation. The crystal structure of the same antibody but with a G-isotype CH1 which is reported to display different antigen affinity has also been solved. The differential structural features reveal plausible mechanisms for constant/variable-domain long-distance effects whereby antibody class switching could alter antigen affinity.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in Western countries mainly affecting individuals older than 50 years. It follows an extremely variable course, with survival ranging from months to decades. Available treatments often induce remissions, though almost all patients relapse and CLL remains an incurable disease [1]. However, recent advances in molecular biology have enabled us to better understand the disease physiopathology and together with the development of new therapeutic agents have made the management of the disease more rational and more effective.

As an approach to determining the aetiology of chronic lymphocytic leukaemia (CLL), we searched for a virus expressed in human CLL B-cells by combining high-throughput sequencing and digital subtraction. Pooled B-cell mRNA transcriptomes from five CLL patients and five healthy donors were sequenced with 454 Life Sciences technology. Human reads were excluded by BLAST (Basic Local Alignment Search Tool) and BLAT (BLAST-like alignment tool) searches. Remaining reads were screened with BLAST against viral databases. Purified B-cells from two CLL patients, with and without stimulation by phorbol-esters, were sequenced using Illumina technology to achieve depth of sequencing. Burrows-Wheeler Aligner mapping and BLAST searches were used for the Illumina data. Pyrosequencing resulted in about 400 similar to 000 reads per sample. No viral candidate could be found. Illumina single-end sequencing for 115 cycles yielded an average of 26 +/- 2.5million filtered reads per sample, of which 2.2 +/- 0.6 similar to million remained unmapped to human references. BLAST searches of these reads against viral and human databases assigned nine reads to an Epstein-Barr virus origin, in one sample following phorbol-ester stimulation. Other reads showing a putative viral origin were dismissed after further analysis. Despite an in-depth analysis of the CLL transcriptome reaching more than 100 similar to million sequences, we have not found evidence for a putative viral candidate in CLL.