Papers by marcela salazar
Developmental and Comparative Immunology, 2007
Subtractive suppressive hybridization was used to identify differentially expressed genes in subc... more Subtractive suppressive hybridization was used to identify differentially expressed genes in subcuticular tissues from white spot syndrome virus(WSSV)-infected shrimp kept at different temperatures. Subtractive libraries I and II contained genes expressed at 26 and 33 degrees C, respectively. Three hundred and seventy-nine insert positive clones were selected to confirm differential expression by dot-blot hybridization. Twenty-two clones from library I and eight from library II were sequenced. All sequences from Library I corresponded to white spot syndrome virus genes. From library II, five clones were homologous with previously reported expressed sequence tags of Litopenaeus vannamei, two had similarity with beta-actin and one transcript represented an unknown gene. Over-expression of VP15 in shrimp at 26 degrees C was further confirmed by real-time polymerase chain reaction (PCR), whereas beta-actin expression was similar in animals kept at both temperatures. Together, our results show that hyperthermia reduces the expression of WSSV genes on shrimp subcuticular epithelial cells.
Cryobiology, 2004
Although the cryopreservation of penaeid prawn sperm or embryos has definite applications in the ... more Although the cryopreservation of penaeid prawn sperm or embryos has definite applications in the aquaculture industry, there is no protocol routinely used for this procedure. One of the main problems relies on the limitations for the determination of sperm cell viability. In this study, we evaluated the toxicity and cryoprotectant effect of four agents, at three different concentrations, in sperm suspension, spermatic mass, and complete spermatophore of the marine shrimp Litopenaeus vannamei. Cells were frozen by fast and slow cooling rates. After thawing, they were analyzed by optical microscopy and flow cytometry, which was also utilized to determine spermatic viability by DNA staining with propidium iodine. Considering viability by morphotype analysis, the best result was obtained when the spermatic mass was frozen by slow cooling rate in the presence of methanol (61.6%). There was a positive correlation between morphotype analysis and flow cytometry, although the percentage of viable cells was always lower when determined by the later. These results show that flow cytometry is a valuable tool to evaluate sperm cell viability in decapod species and it is more sensitive technique than optical microscopy.
Tissue Antigens, 1996
Pemphigus vulgaris (PV) is a blistering disease of the skin and mucous membranes characterized by... more Pemphigus vulgaris (PV) is a blistering disease of the skin and mucous membranes characterized by an autoantibody response against a keratinocyte adhesion molecule, desmoglein 3, causing acantholysis and blister formation. We compared high resolution MHC class II alleles and haplotype frequencies (HLA-DRB, DQA1 and DQB1) in 37 patients with PV to 89 haplotypes of normal relatives from New Delhi and Ahmedabad. We found that PV patients had significantly increased frequencies of DRB1*1404 (P<0.0001), DQA1*0101 (P=0.001), and DQB1*0503 (P<0.0001). These associations were due to the increased frequencies of the haplotype HLA-DRB 1 * 1404, DRB3*0202, DQA1*0101, DQB1*0503 in patients compared to control haplotypes (p<0.0001). Also, patients from Ahmedabad had a significant increase in HLA-DQB 1*0302 (p=0.03). An identical amino acid sequence (Leu-Leu-Glu-Arg-Arg-Arg-Ala-Glu), in positions 67–74 of the P domain of DRB alleles is restricted to some DR14 alleles. Therefore, there are three possible explanations for class II allele involvement in autoantibody in PV patients with class II haplotypes marked by HLA-DR14. First, the class II alleles could be markers for an unidentified susceptibility gene in linkage disequilibrium with them. Second, the primary association could be with DQB 1*0503 and the association with HLA-DR14 alleles would be the result of linkage disequilibrium. Third, the HLA-DRB 1 locus susceptibility could involve a specific amino acid sequence in the third hypervariable region shared by several HLA-DR14 alleles.
Tissue Antigens, 1992
Abstract: We have used a PCR-RFLP method with one generic amplification of HLA-DPB1 second exon a... more Abstract: We have used a PCR-RFLP method with one generic amplification of HLA-DPB1 second exon and 6 endonucleases to differentiate the 19 HLA-DPB1 alleles and 171 heterozygous combinations. The set of primers used in our studies produced fragment sizes different from those published before (1). The HLA-DPB1 alleles in Caucasians showed a higher frequency of DPB 1*0401 and DPB 1*0402, when compared to a small group of Colombians who showed a higher frequency of DPB 1*0402 and DPB 1*0201. We found three HLA-DPB1 alleles associated with two HLA haplotypes that result from non-random association of alleles: DPB1*0401 with HLA-A26, B38, DR4, DQA1*0301 and DPB1*0101 and DPB1*0401 with HLA-A1, B8, DR3, DQA1*0501. We also report that 70% of combinations between HLA (generic A,B,C,DR) and DQA1-identical MLC-unreactive cell mixtures showed HLA-DPB1 mismatches, suggesting that HLA-DPB1 differences are not important in MLC reactivity.
Tissue Antigens, 1997
Until recently, the majority of HLA class I typing has been performed by serology. Expensive comm... more Until recently, the majority of HLA class I typing has been performed by serology. Expensive commercial typing trays are frequently used for testing non-Caucasian subjects and new strategies using DNA-based methods have been adopted for improving clinical histocompatibility testing results and adapted as supplements in proficiency testing. A double-blind comparison of the typing of HLA-B specificities in 40 samples was carried out between serology and two polymerase chain reaction (PCR) methods, PCR amplification with sequence-specific primers (PCR-SSP) and PCR amplification and subsequent hybridization with sequence-specific oligonucleotide probes (PCR-SSOP). The results demonstrated 22.5% misassignments of HLA-B antigens by serology. There was complete concordance between the results obtained with the two PCR based typing methods. A second panel of 20 donor samples with incomplete or ambiguous serologic results was analyzed by PCR-SSP and SSOP. Both PCR methods identified correctly the HLA-B antigens. Our results suggest that more accurate typing results can be achieved by complementing serologic testing with DNA-based typing techniques. The level of resolution for HLA-B antigen assignment can be obtained by this combination of serology and limited DNA-based typing is equivalent to the HLA-B specificities defined by the WHO-HLA Committee. This level of resolution cannot routinely be achieved in clinical histocompatibility testing or in proficiency testing using serologic reagents only.
Immunogenetics, 1995
Lymphocytes from nonresponders to HBsAg fail to proliferate in vitro in the presence of HBsAg-pul... more Lymphocytes from nonresponders to HBsAg fail to proliferate in vitro in the presence of HBsAg-pulsed antigen presenting cells. We studied four pairs of major histocompatibility complex (MHC)-matched, mixed lymphocyte reaction-negative individuals discordant for HBsAg response. For each pair, responder lymphocytes proliferated in the presence of nonresponder antigen-pulsed antigen presenting cells. Respondera nd nonresponder antigen presenting cells were equally effective. There was no evidence for inhibition of responder T-cell proliferation by nonresponder lymphocytes or antigen presenting cells. The defect is thus in the helper T cells of nonresponders and not in the antigen processing or binding of processed peptides to MHC molecules on antigen presenting cells.
Tissue Antigens, 1992
Abstract: We have developed a new PCR-RFLP method for HLA-DQB1 typing. This method was easy to fo... more Abstract: We have developed a new PCR-RFLP method for HLA-DQB1 typing. This method was easy to follow, requiring only one DQB1 generic amplification and 5 endonucleases to assign 14 out of 15 HLA-DQB1 alleles. In addition, we determined that by using one generic amplification and two enzymes (Sau96 I and Hae III) it was possible to type the generic specificities: DQw2, DQw4, DQw5, DQw6, and DQw7, DQw8-9, providing a practical alternative for serological HLA-DQ generic typing. We also performed a side-by-side correlation with a PCR-SSO typing method and found an almost 100% concordance between the methods. The limitations of these methods were: 1) the PCR-RFLP method did not allow the differentiation between the HLA-DQB1 *0602 and *0603 alleles; 2) the PCR-SSO method gave crosshybridization signals in the detection of *0302 or *0303 alleles. Our results suggested that both methods, PCR-RFLP and PCR-SSO, are useful alternatives for HLA-DQB1 typing.
Tissue Antigens, 1996
Serology has been routinely used for class I HLA typing for the selection of donors for allotrans... more Serology has been routinely used for class I HLA typing for the selection of donors for allotransplantation. However, serology is not adequate for the assignment of all class I specificities especially when testing non-Caucasians subjects and it is necessary to adopt new strategies for routine testing. At the present time the extent of incorrect serologic HLA-A assignments in clinical testing is not known. The polymerase chain reaction (PCR) based techniques have become useful standard clinical typing methods of HLA class II alleles but most laboratories still use serology for class I typing. In this report we have compared two PCR based techniques, PCR amplification with sequencespecific primers (PCR-SSP) and PCR amplification and subsequent hybridization with sequence-specific oligonucleotide probes (PCR-SSOP), for the assignment of HLA-A specificities in 56 blood samples from patients and families serologically typed for HLA-A. This side-by-side comparison of PCR methods showed 100% correlation between them. However, serology showed 7.1% misassignments and, in an additional panel of 19 cells where serology produced equivocal results, the PCR-SSP and SSOP methods identified the correct HLA-A specificity. Our results emphasize the need to complement routine serologic testing of HLA specificities with a small number of primers designed to test HLA-A34, A36, A43, A66, A74 and A80, that are not detected with high precision by serology. We concluded that the PCR-SSP and -SSOP methods can be used in routine HLA-A typing of patients and donors for transplantation with a greater precision than serology.
Human Immunology, 1997
Peripheral blood lymphocytes from nonresponders to hepatitis B vaccine (HBsAg) failed to undergo ... more Peripheral blood lymphocytes from nonresponders to hepatitis B vaccine (HBsAg) failed to undergo a proliferative response to recombinant HBsAg in vitro, whereas cells from responders proliferated vigorously. The lack of proliferative response was not due to defective antigen presentation in that MHC-identical responder and nonresponder antigen presenting cells were equally effective in stimulating responder T cells. Nonresponder T cells did not proliferate in response to antigen-pulsed MHC identical responder antigen presenting cells. The present study demonstrated that: 1) there were no detectable (1 in Ͻ20 ϫ 10 4 ) HBsAg-precursor T cells in any of the nonresponders, while in responders the frequency of HBsAg-precursor T cells ranged from 1 in 3.2 ϫ 10 3 to 1 in 40 ϫ 10 3 ; 2) nonresponder cell cultures did not secrete IL-2 in response to HBsAg stimulation; 3) exogenous recombinant IL-2 did not restore the proliferative response of the T cells in HBsAg-pulsed cultures of nonresponders.
Human Immunology, 1994
HLA class Il (DRB1, DQA1, DQBl and DPB1) genotyping was performed in 57 unrelated Uygur indwidual... more HLA class Il (DRB1, DQA1, DQBl and DPB1) genotyping was performed in 57 unrelated Uygur indwiduals inhabiting the northwestern China area by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Among 98 DRBl alleles tested, 23 alleles were detected, and DRB1*0701 (16.7%) and DRB1*0301 (14.0%) were the most and the second most common alleles, respectively. In 8 DQAl alleles detected, DQA1*05 (26.3%), DQAl*03 (21.9%) and DQAl*0201 (21.1%) were very frequent. Of 21 DQBl alleles tested, 13 were observed. Among them, DQBl*O2 was highly predominant with the gene kequency of 32.5%. Of 46 DPBl alleles tested, 15 were detected, among which DPB1*0401 (31.6%) was the most frequent. Two haplotypes predominate clearly; DRB1*0701-DQA1*0201-DQBl*O2 (15.5%) and DRB1*0301-DQA1*05-DQB1*02 (12.6%). The dendrogram constructed by the neighbour-joining (NJ) method based on the allele frequencies of the DRB1, DQA1, DQBl and DPBl genes of 13 representative populations all over the world suggested that Uygur belonged to the Asian group and lay at the closest genetic distance to a Kazak population inhabiting the same area.
Tissue Antigens, 1997
We are reporting the results of HLA-A typing by PCR-SSOP complemented by PCR-SSP of samples obtai... more We are reporting the results of HLA-A typing by PCR-SSOP complemented by PCR-SSP of samples obtained from the National Marrow Donor Program (NMDP). These samples were a representative group from 2486 tested in duplicate by serology. A total of 390 samples gave HLA-A discrepant results. Comparing the molecular typing results of 238 samples (samples with available DNA) with the serological typing results, 54 homozygotes and 184 heterozygotes produced a total of 422 assignments by molecular methods. We found assignment discrepancies in 147/422 (35%) in laboratory 1 and 144/422 (34%) in laboratory 2 (a combined group of 4 NMDP laboratories; laboratory 1 is not included). The serological discrepancies found were of 3 categories: a) false negatives, b) incomplete typing (discrepancies due to the level of resolution within a cross-reactive or CREG group) and c) false positives. Major problems were identified using serology for typing HLA-A antigens: a) inability to identify all WHO-recognized specificities, more frequently in non-Caucasians or in HLA-A specificities known to be found more frequently in non-Caucasians for laboratory 1 and incorrect assignments of A19 specificities in laboratory 2, b) incorrect assignments in cells with poor viability and c) false-positive assignments in homozygotes. We propose a possible strategy to type HLA-A specificities with two steps: a) a minimum of serology for typing specificities for common CREG groups: A1, A2, A3, A11, A9, A10, A28, A19. However, a given laboratory can determine the level of serological assignments needed as a first step. And b) molecular methods to identify splits: A23, A24, A29, A30, A31, A32, A33, A34, A36, A66, A74 and A80. The technique described is useful for large-scale bone marrow donor typings for cells with poor viability, and for resolving ambiguous results including false-positive assignments of homozygous cells.
Human Immunology, 1992
In our study of rheumatoid arthritis (RA) patients, we observed a decrease of tetanus toxoid anti... more In our study of rheumatoid arthritis (RA) patients, we observed a decrease of tetanus toxoid antigen-presenting capacity of synovial fluid (SF) adherent cells to autologous T cells of either SF or peripheral blood. Additionally, we found a higher capacity of adherent synovial cells to stimulate autologous T-lymphocytes. Our results suggest that antigen-presenting cells of the SF of RA patients have defects that may play a role in defective presentation of antigens in joints and may account for other abnormal functions important in the pathogenesis of RA.
Tissue Antigens, 1998
Abstract: We have developed a DNA based typing method to detect 38 known B*15 alleles using seque... more Abstract: We have developed a DNA based typing method to detect 38 known B*15 alleles using sequence-specific primers (PCR-SSP). This method involves 38 primers and 39 PCR-SSP reactions with results that can be obtained in 3 hours. The method is easy, fast and suitable for clinical typing for bone marrow and organ transplantation. We have typed 106 HLA-B15 samples using this method. For homozygous HLA-B15 samples, some B*15 allele combinations need to be resolved by additional PCR reactions not included in this article. The method allows the detection of potential new alleles requiring sequencing for confirmation, and it is useful to resolve unusual serological reaction patterns for different HLA-B15 serological specificities. In addition, it could be used to resolve ambiguous PCR-SSOP typing results and for recognition of mismatches in serologically matched unrelated individuals.
Diseases of Aquatic Organisms, 2006
We have previously reported that white spot syndrome virus-infected Penaeus vannamei (also called... more We have previously reported that white spot syndrome virus-infected Penaeus vannamei (also called Litopenaeus vannamei ) maintained at 32°C show higher survival rates and a significant increase in number of apoptotic cells when compared to infected shrimp kept at 26°C. As apoptosis plays an important part in the antiviral response of invertebrates, we hypothesized that this process would reduce WSSV replication, allowing the shrimp to control the disease and survive. To test this hypothesis, shrimp were orally infected and maintained at either 26°C (Group 1) or 32°C (Group 2), DNA was extracted from haemolymph collected at various times from 6 to 216 h postinfection, and the number of viral units was quantified by real time PCR using SYBR Green. In parallel, histological examination was carried out to confirm the WSSV infection and to rule out concomitant diseases. Linear regression of real time PCR units (rtPCRU) of WSSV from Group 1 showed a significant increase with time post-infection (r 2 = 0.7383; p < 0.001). Conversely, there was no increase in rtPCRU with time post-infection in Group 2 (r 2 = 0.142), indicating that hyperthermia inhibited, either directly or indirectly, viral replication. In addition, comparison between the groups showed no difference in WSSV rtPCRU up to 48 h post-infection. After 72 h, shrimp from Group 1 had a significantly higher viral rtPCRU (ANOVA, p < 0.001). We conclude that hyperthermia-associated WSSV rtPCRU reduction could reflect either an increase in the shrimp antiviral response, or a direct negative effect on viral replication, or both.
Journal of The World Aquaculture Society, 2001
This study was conducted to examine the effect of increasing seawater temperature on White Spot S... more This study was conducted to examine the effect of increasing seawater temperature on White Spot Syndrome Virus (WSSV) infection in juvenile Pacific White shrimp (Litopenaeus vannamei). Infection by WSSV was achieved using two methods, intramuscular injection and per os (oral) administration. Forty injected and 20 per os infected animals were kept in heated tanks at 32.3 ± 0.8 C, and the same number of WSSV infected animals were maintained in tanks at ambient temperature (25.8 ± 0.7 C). Despite the route of exposure, there were no survivors among the animals kept at ambient temperature; whereas, in heated tanks the survival of the WSSV infected juvenile shrimp was always above 80%, suggesting the existence of a beneficial effect from hyperthermia that mitigated the progression of WSSV disease. Moreover, this beneficial effect was not attributable to viral inactivation. Infected animals kept at 32 C had histologically detectable lymphoid organ spheroids suggestive of a chronic viral infection but were PCR negative (hemolymph) for WSSV. These findings might be related to low viral replication in WSSV-infected shrimp held at the higher environmental temperature. When the WSSV-infected shrimp were transferred from 32 C to ambient temperature, the mortality from WSSV ensued and was always 100%. Although the mechanism related to the beneficial effect of heating was not determined, our results indicate that increasing the water temperature modifies dramatically the natural history of the WSSV disease and the survival curves of WSSV-infected juvenile Pacific White shrimp.
Tissue Antigens, 1991
Abstract: We have described a practical and inexpensive method whereby any individual can be type... more Abstract: We have described a practical and inexpensive method whereby any individual can be typed and assigned to any of the 14 generic HLA-DR types: DR1, DRwl5, DRwl6, DRwl7, DRwl8, DR4, DRwll, DRwl2, DRwl3, DRwl4, DR7, DRw8, DR9 and DRw10. Previous methods to type these specificities include - among others - serology, conventional RFLP, PCR-oligonucleotide typing, and a PCR-RFLP method useful for typing homozygous individuals. In the method reported here, DNA is amplified with a set of group-specific primers and then restricted with a number of endonucleases. Six group-specific pairs of primers have been chosen to avoid cross-amplification with other DRB alleles, including DRB3, DRB4 and DRB5 alleles, and to anneal at uniform temperature: 61deg;C. Endonucleases were chosen to generate unique patterns of easily recognizable bands that led to unequivocal assignments of HLA-DR generic types in heterozygous as well as homozygous individuals. This technique involves two steps: 1) Amplification of DNA with six different pairs of primers where DR1, DR4 and DRw10 types can be assigned at once, and 2) Endonuclease digests of amplified DNA to assign DR7, DRw8, DR9, DRw11, DRw12, DRw13, DRw14, DRw15, DRw16, DRw17 and DRw18 types. Individuals carrying any combination of all alleles published so far can be typed by this method. The ease, low cost and reliability of this method are discussed.
, Venezuela, by using different second-and third-generation enzyme immunoassays (EIAs) and immuno... more , Venezuela, by using different second-and third-generation enzyme immunoassays (EIAs) and immunoblot assays. HCV antibodies were detected in 162 patients (71%) by the recombinant-based secondgeneration assays (Abbott and Ortho) and in 161 patients by the synthetic peptide-based EIA (UBI). Of the 162 HCV antibody-positive serum samples, 161 were confirmed to be positive by RIBA 3. HCV RNA was detected in 49 of 68 (72%) of the seropositive patients and in 5 of 21 (24%) of the seronegative ones. HCV RNA was not always correlated with an increase in alanine aminotransferase (ALT) levels. Among 20 patients positive for HCV RNA and for HCV antibodies (without any hepatitis B virus [HBV] marker), only 10 had elevated ALT levels. The possible interference of HBV for HCV replication was evaluated. No significant difference was found between the presence of HCV RNA and the presence of any HBV serological markers. The possible routes of transmission of HCV in hemodialysis patients are multiple, and some of them are still controversial.
Proceedings of The National Academy of Sciences, 1995
Survival, T-cell functions, and postmortem histopathology were studied in H-2 congenic strains of... more Survival, T-cell functions, and postmortem histopathology were studied in H-2 congenic strains of mice bearing H-2b, H-2k, and H-2d haplotypes. Males lived longer than females in all homozygous and heterozygous combinations except for H-2d homozygotes, which showed no differences between males and females. Association of heterozygosity with longer survival was observed only with H-2b/lH-2 and
International Journal of Cardiology, 2000
We performed HLA Class I and Class II typing in 16 patients (15 women, one man) with a confirmed ... more We performed HLA Class I and Class II typing in 16 patients (15 women, one man) with a confirmed diagnosis of Takayasu arteritis. We did not find any of the previously described associations with HLA-B52, and/or HLA-DRB1*1301 alleles. However, in our patients, HLA-DRB1*1602 and HLA-DRB1*1001 were significantly increased. The association of Takayasu arteritis with Amerindian and Asian HLA-DRB1 alleles (DRB1*1602 and DRB1*1001) in the Colombian mestizo patients reported here, and with HLA-B*3906 previously reported in Mexicans, suggest the possibility that some HLA and disease associations are markers for ethnicity of a population carrying a disease gene which is present in an admixed population with the disease.
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Papers by marcela salazar