Team-Based Learning in a Pipeline Course in Medical Microbiology for Under-Represented Student Populations in Medicine Improves Learning of Microbiology Concepts.

As part of an undergraduate pipeline program at our institution for students from underrepresented minorities in medicine backgrounds, we created an intensive four-week medical microbiology course. Team-based learning (TBL) was implemented in this course to enhance student learning of course content. Three different student cohorts participated in the study, and there were no significant differences in their prior academic achievement based on their undergraduate grade point average (GPA) and pre-course examination scores. Teaching techniques included engaged lectures using an audience response system, TBL, and guided self-directed learning. We hypothesized that more active learning exercises, irrespective of the amount of lecture time, would help students master course content. In year 2 as compared with year 1, TBL exercises were decreased from six to three with a concomitant increase in lecture time, while in year 3, TBL exercises were increased from three to six while maintaining the same amount of lecture time as in year 2. As we hypothesized, there was significant (p < 0.01) improvement in performance on the post-course examination in years 1 and 3 compared with year 2, when only three TBL exercises were used. In contrast to the students' perceptions that more lecture time enhances learning of course content, our findings suggest that active learning strategies, such as TBL, are more effective than engaged lectures in improving student understanding of course content, as measured by post-course examination performance. Introduction of TBL in pipeline program courses may help achieve better student learning outcomes.

Protection against porcine reproductive and respiratory syndrome virus (PRRSV) infection through passive transfer of PRRSV-neutralizing antibodies is dose dependent.

Previous work in our laboratory demonstrated that passive transfer of porcine reproductive and respiratory syndrome virus (PRRSV)-neutralizing antibodies (NA) protected pregnant sows against reproductive failure and conferred sterilizing immunity in sows and offspring. We report here on the dose requirement for protection by passive transfer with NA in young weaned pigs. The presence of a 1:8 titer of PRRSV-NA in serum consistently protected pigs against viremia. Nevertheless, their lungs, tonsils, buffy coat cells, and peripheral lymph nodes contained replicating PRRSV similar to the infected control group. Likewise, these animals excreted infectious virus to sentinels similar to the infectivity control animals. In an attempt to reach complete protective immunity equivalent to that previously observed in sows, the pigs were transferred with a higher titer of PRRSV-NA (1:32), and even then apparent sterilizing immunity was attained in only 50% of the animals. In conclusion, the presence of anti-PRRSV-NA in serum with a titer of 1:8 is enough to block viremia but not peripheral tissue seeding and transmission to contact animals. While a relatively low level of NA in blood is capable of conferring sterilizing immunity against PRRSV in sows, the amount of NA necessary to obtain full protection of a young weaned pig would be significantly higher, suggesting that differences exist in the PRRSV pathogenesis between both age groups. In addition, the titer of NA could be a helpful parameter of protection in the assessment of PRRSV vaccines.

Role of neutralizing antibodies in PRRSV protective immunity.

Little has been known about the components of the immune system that are effective in the protection of a pig against PRRSV infection. Although antibodies were initially perceived as a deleterious, ineffective component of the PRRSV-specific immune response, neutralizing antibodies (NA) are now considered to be an important correlate of protective immunity against PRRSV. This paper reviews the current knowledge on arterivirus-specific NA, the role that NA have in protection against infection with PRRSV, as well as the viral molecular structures that are responsible for the production of this type of antibodies by the pig. This information should prove central to the design of new generation vaccines against PRRSV.

B-cell epitopes in the immunodominant p34 antigen of mycobacterium avium ssp. paratuberculosis recognized by antibodies from infected cattle.

Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) causes Johne's disease, a chronic and fatal enteritis in ruminants. In the last stage of the disease, antibody titres rise and levels of interferon-gamma decrease, suggesting that the host-immune response is switching from a T helper 1 (Th1) to a Th2 profile. In infected cattle, the membrane protein p34 elicits the predominant humoral response against M. paratuberculosis. To map the B-cell epitopes of this antigen, affinity-purified bovine antibodies against the carboxy-terminal region of p34 were used to screen a 12-mer phage display library. Several phage clones carrying peptides resembling fragments of p34 were affinity selected. Based on the predicted amino acid sequence, peptides were chemically synthesized, which demonstrated reactivity with serum from naturally infected and p34-vaccinated cattle. Immunization of mice with these peptides elicited an anti-p34 antibody response. Two B-cell epitopes were identified and characterized. Based on the reactivity and the type of immune response elicited, epitope A was determined to be conformational, whereas epitope B was demonstrated to be sequential. Both epitopes were shown to be present in p34 proteins from M. avium ssp. avium or M. paratuberculosis but absent from M. intracellulare, the other member of the M. avium complex. Furthermore, both epitopes were mapped to regions of p34 that display high variability when compared to homologous proteins from other mycobacterial species of public and animal health importance. We hypothesize that these variable regions of p34 may play a role in the immunobiology of M. paratuberculosis infections.

Genotypic variability at the major histocompatibility complex (B and Rfp-Y) in Camperos broiler chickens.

Evidence for the importance of major histocompatibility complex (MHC) genotype in immunological fitness of chickens continues to accumulate. The MHC B haplotypes contribute resistance to Marek's and other diseases of economic importance. The Rfp-Y, a second cluster of MHC genes in the chicken, may also contribute to disease resistance. Nevertheless, the MHC B and Rfp-Y haplotypes segregating in broiler chickens are poorly documented. The Camperos, free-range broiler chickens developed in Argentina, provide an opportunity to evaluate MHC diversity in a genetically diverse broiler stock. Camperos are derived by cross-breeding parental stocks maintained essentially without selection since their founding. We analysed 51 DNA samples from the Camperos and their parental lines for MHC B and Rfp-Y variability by restriction fragment pattern (rfp) and SSCP typing methods for B-G, B-F (class Ia), B-Lbeta (class II) and Y-F (class Ib) diversity. We found evidence for 38 B-G genotypes. The Camperos B-G patterns were not shared with White Leghorn controls, nor were any of a limited number of Camperos B-G gene sequences identical to published B-G sequences. The SSCP assays provided evidence for the presence of at least 28 B-F and 29 B-Lbeta genotypes. When considered together B-F, B-L, and B-G patterns provide evidence for 40 Camperos B genotypes. We found even greater Rfp-Y diversity. The Rfp-Y class I-specific probe, 163/164f, revealed 44 different rfps among the 51 samples. We conclude that substantial MHC B and Rfp-Y diversity exists within broiler chickens that might be drawn upon in selecting for desirable immunological traits.

Identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain.

After infection of swine with porcine reproductive and respiratory syndrome virus (PRRSV), there is a rapid rise of PRRSV-specific nonneutralizing antibodies (NNA), while neutralizing antibodies (NA) are detectable not sooner than 3 weeks later. To characterize neutralizing epitopes, we selected phages from a 12-mer phage display library using anti-PRRSV neutralizing monoclonal antibody (MAb) ISU25-C1. In addition, phages carrying peptides recognized by swine antibodies with high seroneutralizing titer were isolated after subtracting from the library those clones binding to swine anti-PRRSV serum with no neutralizing activity. Two epitopes located in the ectodomain of PRRSV GP5 were identified. One of these epitopes, which we named epitope B, was recognized both by neutralizing MAb ISU25-C1 and swine neutralizing serum (NS) but not by swine nonneutralizing serum (NNS), indicating that it is a neutralizing epitope. Epitope B is sequential, conserved among isolates, and not immunodominant. Antibodies directed against it are detected in serum late after infection. In contrast, the other epitope, which we named epitope A, is hypervariable and immunodominant. Antibodies against it appear early after infection with PRRSV. This epitope is recognized by swine NNA but is not recognized by either neutralizing MAb ISU25-C1 or swine NA, indicating that it is not involved in PRRSV neutralization. During infection with PRRSV, epitope A may act as a decoy, eliciting most of the antibodies directed to GP5 and delaying the induction of NA against epitope B for at least 3 weeks. These results are relevant to the design of vaccines against PRRSV.

Use of a single VH family and long CDR3s in the variable region of cattle Ig heavy chains.

We have analyzed transcripts encoding the variable regions of Ig heavy chains from adult and fetal bovine splenocytes and bovine x mouse heterohybridomas. The 13 adult, seven fetal and two heterohybridomas transcripts as well as the six genes that were sequenced had > 83% identity to each other in the VH-encoded regions (FRs 1-3 and CDRs 1 and 2). By this criterion, all the bovine sequences were assigned to one family, which corresponds to the bovine homolog of the murine Q52 family. Southern blot analysis of genomic DNA demonstrated that homologs of other murine VH families such as 7183, S107 and 36-60 were present in the genome, but transcripts from these families were not detected in rapid amplification of cDNA ends (RACE)-PCR amplified products or in individual clones. The sequences of the adult transcripts using the mu isotype showed extensive somatic mutation indicating that the process of somatic hypermutation begins earlier in development of the bovine B cell. The length of CDR3 from V(D)J rearrangements averaged 21 amino acids, which is larger than other mammalian CDR3s. Analysis of CDR3s from 23 fetal transcripts revealed a preference for a reading frame in the putative D genes which is rich in glycine and tyrosine, and is also extensively mutated in adults. The bovine immune system appears to utilize Ig VH genes of a single family, but generates antibody diversity by extensive somatic mutation and long CDR3s which are subsequently hypermutated.

Bovine herpesvirus type-4 (BHV-4) persistently infects cells of the marginal zone of spleen in cattle.

Bovine Herpes virus type 4 (BHV-4) has the ability to persist during long post-infection periods in spleen and other lymphoreticular tissues of cattle and laboratory rabbits. Our previous studies indicated that splenic macrophages are the main reservoir of this persistent herpesvirus infection in rabbits. Now we report the use of in situ hybridization (ISH) and cell separation methods to characterize the cellular localization of persistent BHV-4 in cattle. Using cloned sub-genomic probes of BHV-4 DNA labelled with 35S, we detected BHV-4 nucleic acids in cells of the marginal zone of spleen from persistently infected cattle and rabbits. In addition, cell separation studies indicated that a non-T, non-B cell population of the bovine spleen harbours BHV-4. This association requires cell integrity and in vitro co-cultivation for re-expression of the persistent virus. We were also able to detect BHV-4 by explantation/co-cultivation from several other tissues of cattle including trigeminal ganglia, urinary bladder, kidney, lung and several lymphoid tissues including lymph nodes and thymus.

Epilepsy with continuous spike-waves during slow wave sleep: a clinical and electroencephalographic study.

We report four children with epilepsy with "continuous spike-waves during slow wave sleep" (CSWSS). The main clinical features were partial motor seizures, mental retardation and motor deficit. The EEG findings were characterized by nearly continuous (> 85%) diffuse slow spike and wave activity in two patients, and localized to one hemisphere in two other cases during non-REM sleep. The treatment was effective in improving the clinical seizures, but not the EEG pattern. We believe that this epileptic syndrome has been overlooked and routine sleep EEG studies on epileptic children may disclose more cases of CSWSS.

Evaluation of PCR for diagnosis of bovine viral diarrhea virus in tissue homogenates.

Tissue homogenates from 60 specimens submitted to the Veterinary Diagnostic Center were evaluated by polymerase chain reaction (PCR) for detection of bovine viral diarrhea virus (BVDV). Conventional virus isolation procedures showed the specimens contained BVDV. The BVDV RNA was extracted from the homogenates and subjected to a reverse transcription reaction followed by PCR amplification. The PCR product was blotted onto a nylon membrane and hybridized with a 30-base pair oligonucleotide probe labeled with 32P. One set of PCR primers detected BVDV in 46/60 (77%) of the tissue homogenates. An additional set of primers was used to detect 10/11 samples that had escaped detection with the first set of primers. The results indicate that BVDV can be detected by PCR directly out of tissue homogenates generated in a diagnostic setting.

Presence of bovine viral diarrhoea virus in lymphoid cell populations of persistently infected cattle.

Cattle infected in utero with bovine viral diarrhoea virus (BVDV) often develop a lifelong persistent infection (PI). During this PI, BVDV infects many cell types including peripheral blood mononuclear cells (PBMNC). To define the lymphoid cell populations in which BVDV persists PBMNC subpopulations were separated using monoclonal antibodies to cell surface markers. Separated cells were analysed by a sensitive PCR assay for BVDV, in conjunction with flow cytometry to identify antigen-containing cells and with viral infectivity assays. The results indicate that BVDV establishes a productive PI in monocytes and T cells bearing the marker BoCD4, BoCD8 or gamma-delta T cell receptor. BVDV was not detected in B cells as a productive nor a latent infection.

Comparison of the immune response elicited by infectious and inactivated foot-and-mouth disease virus in mice.

The immune response to foot-and-mouth disease virus (FMDV) elicited by infection or immunization with inactivated virus in adult mice was examined. A model of adoptive transfer of immunocompetent cells was used for this purpose. The results presented here indicate that both short- and long-term secondary immune responses elicited by high doses of inactivated virus are indistinguishable, at the humoral or cellular level, from that observed after infection. The responses to inactivated or infectious virus were both efficiently mediated by B cells. However, immunization with low doses of inactivated virus induced a response which, although effective in aborting infection, was fully dependent on FMDV-specific T cell cooperation. These findings suggest that the different immune responses observed after infection and immunization are mainly the result of the different viral mass presented to the immune system in each case.

Rapid detection of bovine viral diarrhea virus by polymerase chain reaction.

The polymerase chain reaction was used to detect genomic sequences of the positive-stranded RNA of bovine viral diarrhea virus (BVDV), a member of the family Togaviridae. Using a set of 20-bp primers located within the conserved 3' region of the BVDV genome, we were able to consistently amplify a 205-bp target sequence from BVDV cDNA. BVDV RNAs from cell culture-propagated BVDV reference strains, diverse unrelated cytopathic and noncytopathic field isolates, and clinical serum samples were transcribed to cDNA by using avian myeloblastosis virus reverse transcriptase and further specifically amplified by using the polymerase chain reaction assay. The amplification assay was sensitive enough to detect one molecule of cloned BVDV cDNA. Reconstitution experiments conducted by adding decreasing amounts of BVDV (NADL strain) to BVDV-free serum indicated that the threshold of sensitivity of the assay was less than or equal to 1 50% tissue culture infective dose. These results show that the polymerase chain reaction may be used for the rapid detection of diverse strains of BVDV in cell cultures, biological products, and clinical specimens from cattle.

Immune response to foot-and-mouth disease virus in an experimental murine model. II. Basis of persistent antibody reaction.

A murine model was used to study the mechanisms involved in the prolonged immune response to live and inactivated foot-and-mouth disease virus (FMDV). The antibody response elicited by the infection persisted throughout the entire life of the animal, while immunization with inactivated virus induced a transient response. The administration of inactivated virus in a water-in-oil emulsion increased antibody titres to values as high as those obtained by infection. There was a high correlation between neutralizing antibody titre and transfer of immunity with primed cells, and the protection afforded against challenge with infectious virus. It appears that the mechanism involved in the induction of prolonged immune memory in infected animals is not due to viral persistence. Nude mice infected with FMDV also evidenced a prolonged immune response, showing marked differences in antibody levels but equal effectiveness against challenge when nu/nu and nu/+ animals were compared. Furthermore, athymic and euthymic littermates were efficient in conferring protection when cells were transferred to irradiated animals. It is concluded that there is an effective, T-cell-independent, prolonged immune memory against FMDV in this murine model, and that the difference in the immune responses to live and inactivated virus is due mainly to differential antigenic processing rather than to a difference in the degree of sensitization of effector cells.

[Gastric adenoacanthoma. Report of a case and review of the literature]. / Adenoacantoma gástrico. Presentación de un caso y revisión de la literatura.

Squamous and adenosquamous carcinomas of the stomach are extremely rare, having a reported incidence of between 0.04 and 0.7%. Up to date, only two cases have been published in argentine literature by W. G. Lange, 1955. This is the reason of presenting this 68 year old woman, whose disease begins as an inflammatory process of the gallbladder and pancreas, later studies showed that it was an adenosquamous carcinoma of the stomach. Its pathogenesis is most probably from an area of preexisting squamous metaplasia, but the origin from a totipotencial cell (stem cell), from an area of ectopic stratified squamous epithelium or by squamous metaplasis developing within a preexisting adenocarcinoma has not as yet been established.

Adenoacantoma gastrico. Presentacion de un caso y revision de la literatura. / Gastric adenocarcinoma.Report of a case and review of the literature

Los carcinomas pavimentosos y adenoacantomas gastricos son tumores extremadamente raros, habiendose reportado una incidencia del 0.04% al 0.7%. Hasta el presente solo se han publicado 2 casos en la literatura argentina por W.G. Lange, 1955. Ello motiva la presentacion de esta mujer de 68 anos de edad que debuta clinicamente como un proceso inflamatorio colecisto-pancreatico y que los estudios posteriores evidenciaron tratarse de un adenoacantoma gastrico. Existe una tendencia del carcinoma pavimentoso y del adenoacantoma del estomago a presentarse en edades mas tempranas que las demostradas para los adenocarcinomas en general. 60% ocurren antes de los 60 anos de edad, mientras que en numerosas series de adenocarcinomas gastricos el 65% ocurren despues de los 60 anos. Su patogenia muy probablemente seria a partir de un area de metaplasia pavimentosa preexistente; pero el origen a partir de una celula totipotencial, de un area de epitelio pavimentoso estratificado o de una adenocarcinoma preexistente, todavia no ha sido establecido aun

Adenoacantoma gástrico. Presentación de un caso y revisión de la literatura. / [Gastric adenoacanthoma. Report of a case and review of the literature]

Squamous and adenosquamous carcinomas of the stomach are extremely rare, having a reported incidence of between 0.04 and 0.7

. Up to date, only two cases have been published in argentine literature by W. G. Lange, 1955. This is the reason of presenting this 68 year old woman, whose disease begins as an inflammatory process of the gallbladder and pancreas, later studies showed that it was an adenosquamous carcinoma of the stomach. Its pathogenesis is most probably from an area of preexisting squamous metaplasia, but the origin from a totipotencial cell (stem cell), from an area of ectopic stratified squamous epithelium or by squamous metaplasis developing within a preexisting adenocarcinoma has not as yet been established.

Adenoacantoma gastrico. Presentacion de un caso y revision de la literatura. / Gastric adenocarcinoma.Report of a case and review of the literature

Los carcinomas pavimentosos y adenoacantomas gastricos son tumores extremadamente raros, habiendose reportado una incidencia del 0.04% al 0.7%. Hasta el presente solo se han publicado 2 casos en la literatura argentina por W.G. Lange, 1955. Ello motiva la presentacion de esta mujer de 68 anos de edad que debuta clinicamente como un proceso inflamatorio colecisto-pancreatico y que los estudios posteriores evidenciaron tratarse de un adenoacantoma gastrico. Existe una tendencia del carcinoma pavimentoso y del adenoacantoma del estomago a presentarse en edades mas tempranas que las demostradas para los adenocarcinomas en general. 60% ocurren antes de los 60 anos de edad, mientras que en numerosas series de adenocarcinomas gastricos el 65% ocurren despues de los 60 anos. Su patogenia muy probablemente seria a partir de un area de metaplasia pavimentosa preexistente; pero el origen a partir de una celula totipotencial, de un area de epitelio pavimentoso estratificado o de una adenocarcinoma preexistente, todavia no ha sido establecido aun