Extracellular matrix modulates depot-specific adipogenic capacity in adipose tissue of dairy cattle.

Adipose tissue (AT) expands through both hyperplasia and hypertrophy. During adipogenesis, adipose stromal and progenitor cells (ASPCs) proliferate and then accumulate lipids, influenced by the local AT microenvironment. Increased adipogenic capacity is desirable as it relates to metabolic health, especially in transition dairy cows where excess free fatty acids in circulation can compromise metabolic and immune health. Our aim was to elucidate the depot-specific adipogenic capacity and ECM properties of subcutaneous (SAT) and visceral (VAT) AT of dairy cows and define how the ECM affects adipogenesis. Flank SAT and omental VAT samples were collected from dairy cows in a local abattoir. Tissue samples were utilized for transcriptome analysis, targeted RT-qPCR for adipogenic markers, adipocyte sizing, assessment of viscoelastic properties and collagen accumulation, and then decellularized for native ECM isolation. For in vitro analyses, SAT and VAT samples were digested via collagenase, and ASPCs cultured for metabolic analysis. Adipogenic capacity was assessed by adipocyte size, quantification of ASPCs in stromal vascular fraction (SVF) via flow cytometry, and gene expression of adipogenic markers. In addition, functional assays including lipolysis and glucose uptake were performed to further characterize SAT and VAT adipocyte metabolic function. Data were analyzed using SAS (version 9.4; SAS institute Inc., Cary, NC) and GraphPad Prism 9. Subcutaneous AT adipogenic capacity was greater than VAT's, as indicated by increased ASPCs abundance, increased magnitude of adipocyte ADIPOQ and FASN expression during differentiation, and higher adipocyte lipid accumulation as shown by an increased proportion of larger adipocytes and abundance of lipid droplets. Rheologic analysis revealed that VAT is stiffer than SAT, which led us to hypothesize that differences between SAT and VAT adipogenic capacity were partly mediated by depot-specific ECM microenvironment. Thus, we studied depot-specific ECM-adipocyte crosstalk using a 3D model with native ECM (decellularized AT). Subcutaneous AT and VAT ASPCs were cultured and differentiated into adipocytes within depot-matched and mis-matched ECM for 14d, followed by ADIPOQ expression analysis. Visceral AT ECM impaired ADIPOQ expression in SAT cells. Our results demonstrate that SAT is more adipogenic than VAT and suggest that divergences between SAT and VAT adipogenesis are partially mediated by the depot-specific ECM microenvironment.

Controlling bovine leukemia virus in dairy herds by identifying and removing cows with the highest proviral load and lymphocyte counts.

The objective of this field trial was to reduce bovine leukemia virus (BLV) transmission and prevalence in commercial dairy herds using proviral load (PVL) and lymphocyte count (LC) measurements as indicators of the most infectious animals for culling or segregation. Bovine leukemia virus causes lymphoma in <5% of infected cattle, and increased lymphocyte counts (lymphocytosis) in about one-third. Recent research has shown that dairy cows infected with BLV have altered immune function associated with decreases in milk production and lifespan. Recent findings show that a minority of infected cattle have PVL concentrations in blood and other body fluids of over 1,000 times that of other infected cattle. In combination with a high LC, these animals are thought to be responsible for most transmission of BLV in a herd. Milk or blood samples from adult cows in our 3 Midwestern dairy farm field trials were tested semiannually with ELISA for BLV antibodies, and ELISA-positive cattle were then retested using a blood LC and a quantitative PCR test for PVL to identify the animals presumed to be most infectious. Herd managers were encouraged to consider PVL and LC status when making cull decisions, and to segregate cows with the highest PVL and LC from their BLV ELISA-negative herd mates where possible. After 2 to 2.5 yr of this intervention, the incidence risk of new infections decreased in all 3 herds combined, from 13.8 to 2.2, and the overall herd prevalence decreased in all 3 herds combined from 62.0 to 20.7%, suggesting that this approach can efficiently reduce BLV transmission as well as prevalence. This is encouraging, because a very low prevalence of BLV infection would make it economically feasible to cull the remaining ELISA-positive cattle, as was achieved in national eradication programs in other countries decades ago.

HLA polymorphism in a Guarani-Indian population from Paraguay and its usefulness for the Hispano-Indian admixture study in Paraguay.

In this study we investigated the human leucocyte antigen-A (HLA-A), -B and DRB1 polymorphism of Native American population of Paraguay, the Guarani Indians. We found that the HLA variability consisted of 5 HLA-A, 7 HLA-B and 6 HLA-DRB1 groups of alleles and of several specific alleles (B*1504, B*3505, B*3912, B*4004, B*5104, DRB1*0411, DRB1*1413) common in other Native American populations. The comparison of the HLA polymorphism of the Guaranis from Paraguay with the «Mestizos¼ of Paraguay and the Spaniards showed that the «Mestizos¼ of Paraguay are genetically very distant from the Guarani Indians of Paraguay but much more close to the Spaniards. This can be explained, at least in part, by the history of the country. Our results are of importance in transplantation, in particular in the search for an unrelated donor for a Paraguayan patient requiring hematopoietic stem cell transplantation.

Analysis of the HLA population data (AHPD) submitted to the 15th International Histocompatibility/Immunogenetics Workshop by using the Gene[rate] computer tools accommodating ambiguous data (AHPD project report).

During the 15th International Histocompatibility and Immunogenetics Workshop (IHIWS), 14 human leukocyte antigen (HLA) laboratories participated in the Analysis of HLA Population Data (AHPD) project where 18 new population samples were analyzed statistically and compared with data available from previous workshops. To that aim, an original methodology was developed and used (i) to estimate frequencies by taking into account ambiguous genotypic data, (ii) to test for Hardy-Weinberg equilibrium (HWE) by using a nested likelihood ratio test involving a parameter accounting for HWE deviations, (iii) to test for selective neutrality by using a resampling algorithm, and (iv) to provide explicit graphical representations including allele frequencies and basic statistics for each series of data. A total of 66 data series (1-7 loci per population) were analyzed with this standard approach. Frequency estimates were compliant with HWE in all but one population of mixed stem cell donors. Neutrality testing confirmed the observation of heterozygote excess at all HLA loci, although a significant deviation was established in only a few cases. Population comparisons showed that HLA genetic patterns were mostly shaped by geographic and/or linguistic differentiations in Africa and Europe, but not in America where both genetic drift in isolated populations and gene flow in admixed populations led to a more complex genetic structure. Overall, a fruitful collaboration between HLA typing laboratories and population geneticists allowed finding useful solutions to the problem of estimating gene frequencies and testing basic population diversity statistics on highly complex HLA data (high numbers of alleles and ambiguities), with promising applications in either anthropological, epidemiological, or transplantation studies.

[Multiple sclerosis and nephrotic syndrome in minimal change disease]. / Esclerosis múltiple y síndrome nefrótico por enfermedad glomerular de cambios mínimos: coincidencia de dos enfermedades autoinmunes.

The coinciding suffering of two or more autoimmune diseases has been occasionally reported during the last years. We report a multiple sclerosis case in a young male patient who presented minimal-change nephrotic syndrome with frequent relapses seven months later. New relapses have been prevented with cyclosporine therapy in a dose of 3,8 mg/kg bw/day associated to prednisone 10 mg/day during eighteen months. Documentation and investigation of such cases will help to the best understanding of the immunopathogenesis of those diseases.

Difference in kinetics of hematopoietic reconstitution between ALL and ANLL after autologous bone marrow transplantation with marrow treated in vitro with mafosfamide (ASTA Z 7557).

The kinetics of hematopoietic recovery after autologous bone marrow transplantation (ABMT) reflect the hematopoietic capacity of the infused marrow. In vitro treatment of marrow with high doses of mafosfamide (ASTA Z 7557) alters the hematopoietic regenerative capacity of the graft. Thirty-two patients with acute leukemia (12 acute lymphoblastic leukemia (ALL) and 20 acute non-lymphoblastic leukemia (ANLL] with 27 in complete remission and five in partial remission were consolidated with cyclophosphamide (60 mg/kg x 2) and total body irradiation (10 Gy), followed by reinfusion of autologous marrow treated in vitro with mafosfamide. The marrow of each patient had been incubated with the highest tolerable dose of mafosfamide, individually predetermined from a preincubation test. We report here that the kinetics of engraftment are strikingly different in ANLL and ALL patients. In the ANLL group recovery to 0.1% reticulocytes took a median of 20.5 days (range 14-32) versus 15 (11-28) in the ALL group; 33.5 days (18-45) versus 19 (15-30) for leukocytes to reach 1.0 x 10(9)/l; 35 (19-60) versus 20.5 (15-30) for neutrophils to reach 0.5 x 10(9)/l; 110+ (45-480+) versus 50 (23-90) for platelets to reach 50 x 10(9)/l (p less than 0.01 and p less than 0.05). Detection of granulocyte-macrophage progenitors (CFU-GM) regeneration in marrow aspirates post-ABMT was delayed in ANLL (p less than 0.05). Neither the nature of the previous induction therapy, nor the status of the blood or bone marrow at the time of collection (CFU-GM and erythroid burst-forming units/ml) nor the stem cell sensitivity to mafosfamide, nor the doses of progenitor cells infused could explain these differences. We interpreted these observations as suggesting that the engraftment potential has been more severely altered in ANLL than in ALL, which may reflect both the intensity of the in vitro treatment and the intrinsic fragility of the stem cell pool in ANLL.

[Oxidative stress in neurodegeneration]. / Estrés oxidativo en las neurodegeneraciones.

INTRODUCTION: The aetiology of the neuronal death which occurs in neurodegenerative diseases is still unknown. These disorders are of insidious onset and follow an inexorable, gradually progressive course. The best known and most frequent are Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). DEVELOPMENT: Advances in molecular genetics and neurobiochemistry towards the understanding of processes involved in cell death, suggest the association of phenomena of excito-toxicity and oxidation damage in the selective degeneration of neuronal populations, characteristic of these disorders. CONCLUSION: The evidence presented here suggests that the species reactive to oxygen (SRO) play a direct part in the aetiology and/or pathogenesis of neurodegenerative disorders, although it is still very difficult to establish whether these reactive species represent the primary etiological factor, or are toxic products secondary to tissue damage.

[The action of epidermal growth factor on the development of cultured striatum cells]. / Acción del factor de crecimiento epidérmico sobre el desarrollo de las células estriatales en cultivo.

INTRODUCTION: Epidermic growth factor (EGF) has a neurotrophic mitogenic effect on different cell populations in the nervous system. This is modulated by the stage of development and microenvironment of the cells. OBJECTIVE AND METHODS: In this paper we describe the action of EGF on embryonic striatum cells of a culture system dissociated from neurons and glias. The cell culture is prepared from 16-17 day rat embryos. In the system used, the cell population was cultured for 20-24 hours in a medium containing serum. This medium was later replaced by a mixture of specific nutrients and treated for 6 days with 20 mg/ml of EGF. RESULTS AND CONCLUSIONS: The substitution of serum during the initial period of development led to an appreciable reduction in the living cells in the treated cultures and in the controls. The surviving cells were mainly cellular precursors, taking into account their morphological characteristics and capacity for proliferation. The effect of EGF was seen in an increase in the number of cells and was shown to be a stimulus to the proliferation of neuronal and astrocyte precursors. The specific activity of choline acetyl-transferases determined in the cultures at 16 days showed differentiation of a cholinergic neurons subpopulation, which responded to treatment with nerve growth factor with an increase in the activity of this enzyme.

[Classification of acute leukemias according to the first latin-american consensus conference for the immunophenotyping of leukemias]. / Clasificación de las leucemias agudas de acuerdo con el consenso de la primera conferencia latinoamericana en la tipificación inmunológica de las leucemias.

OBJECTIVE: To evaluate the recommendations of the First Latinamerican Consensus Conference for the Immunophenotyping of Acute Leukemia in untreated patients with de novo disease immunologically classified employing flow cytometry and an extended panel of monoclonal antibodies. MATERIAL AND METHODS: In that conference it was recommended the use of the following antibodies: cytoplasmic CD79a (cCD79a) and CD19 to define B-progenitor acute lymphoblastic leukemia (B-ALL); cCD3 and CD7 for T-cell ALL (T-ALL), and CD13, CD33 and myeloperoxidase (cMPO) for acute myeloblastic leukemia (AML). We analyzed the expression of these cellular antigens in 91 non-consecutive patients classified with the extended panel as: B-ALL 28 cases; T-ALL 7; B-T-ALL 2; AML 47; and mixed-lineage acute leukemia 7 cases. RESULTS: All 28 B-ALL cases were positive with each of the two recommended antibodies cCD79a and CD19, whereas in 24 AML cases (the expression of cCD79a was not assayed in 23 cases) and in 7 T-ALL patients both antigens were absent. cCD3 and CD7 antigens were identified in 71% and 100% of T-ALL, respectively. CD7 antigen was not detected in any of the 28 patients with B-ALL but it was expressed in 6 of 47 AML cases, while none of 75 B-ALL and AML cases were positive to cCD3. Forty nine percent of AML were positive for the three recommended markers: cMPO, CD13 and CD33, and 51% of AML cases reacted with one or two of these three monoclonal antibodies. Six out of 28 cases of B-ALL had aberrant expression of myeloid antigen (CD33 in 3 cases and CD13 in 3 cases). CONCLUSIONS: There was no difference in the definition of AL lineage between employing the extended antibody panel and that recommended by the Latinamerican consensus.

[A loud second heart sound]. / Een luide tweede harttoon.

When a loud second heart sound is heard in a child, the possibility of pulmonary hypertension must be borne in mind. The second heart sound is also loud in patients with congenitally corrected transposition (CGT) because of the anterior position of the aortic valve. Therefore, it is difficult to distinguish the relatively rare isolated CGT from cardiac disease with high pressure in the pulmonary artery. We discuss this diagnostic problem on the basis of a two year old boy with a loud second heart sound.

Response of the exocrine pancreas to the CCK on offspring rats of ethanol dams. Effects of folic acid.

AIMS: The aim of this study was to study the reverse effect of folic acid administered during gestation and lactation to ethanol-treated dams, on cholecystokinin Cholecystokinin (CCK) stimulus-secretion coupling in pancreatic exocrine secretion in offspring rats. METHODS: Animals were randomized into three groups: Control group (C) received water and basic diet during pregnancy and lactation period; ethanol-treated rats (E) received ethanol and basic diet; the ethanol+folic acid group (EF) received folic acid supplement concomitantly with ethanol administration. RESULTS: Body and pancreatic weight was lower in offsprings after ethanol treatment. Folic acid supplementation increased these parameters with respect to ethanol rats. After CCK stimulation, a significant decrease in amylase, lipase and chymotrypsin activities in the duodenal juice were detected in ethanol, this trend was partially corrected with folate supplementation. CONCLUSION: Ethanol exerts its action on exocrine pancreatic secretion by two pathways: 'per se' and diminishing the folic acid content, because a folic acid supplement in rats during pregnancy and lactation periods produces an advantageous effect on amylase, lipase and chymotrypsin secretion in their offspring. Although extrapolation from animal studies may be tenuous, the present findings may explain the use of folic acid in the prevention of ethanol-induced damage by increasing the enzyme levels to adequate physiological concentrations.

[Patterns in the household acceptance of oral rehydration therapy in a cohort of rural mothers]. / Patrones de aceptación de terapia de hidratación oral a nivel domiciliario en una cohorte de madres rurales.

The household acceptance of oral rehydration solution salts was studied in a cohort of 75 rural mothers over a two year period. The study was carried out in a rural Mexican village between August 1985 and February 1988. There were 636 episodes of diarrhea, with an average of four episodes per child per year. There were no deaths due to diarrhea or its complications and only 2% of these episodes required the use of parenteral solutions. Acceptance of oral rehydration remained around 50% during subsequent bouts of diarrhea. Variables such as a medium high socioeconomic level of the family, and the reading of journals and newspapers by the mother and the presence of water supply in the home were significantly associated with the acceptance of oral rehydration treatment. On the other hand, families classified as traditional rejected the procedure with significantly higher frequency. The data presented indicates the need for redesigning oral rehydration, treatment programs to increase their impact at the community level.

[A comparative study between the use of the Spicer-Edwards method and the coagglutination reaction for identifying Salmonella antigens]. / Estudio comparativo entre el uso del método de Spicer-Edwards y la reacción de coaglutinación para identificación de antígenos de Salmonella.

A comparative study between tube agglutination using Spicer-Edwards antisera and a coagglutination method using Staphylococcus aureus Cowan 1 (NCTC 8530) protein A to detect Salmonella flagellar antigens was carried out. A total of 39 Salmonella strains belonging to eight serogroups of the Kauffmann-White scheme were typed by both methods. Each serogroup include monophasic and biphasic strains. Statistical analysis of the results showed that the coagglutination method was more sensitive and specific that the classic tube agglutination method normally used in different laboratories. The use of less antiserum in the coagglutination method to detect Salmonella antigens without loss of specificity or sensitivity represents an important saving for clinical laboratories and for epidemiologic research that require the identification of these antigens.

[Etiology of bloody diarrhea in children from a rural community]. / Etiología de diarrea con sangre en niños de una comunidad rural.

The etiology of bloody diarrhea was investigated in a cohort of 75 children followed longitudinally from birth during the first two years of life in a rural Village of Central Mexico. Of a total of 636 episodes of diarrhea, 71 (11%) showed presence of blood. A single associated pathogen was isolated in 59 (83%) of 71 children; 35% showed the presence of enteroaggregative E. coli (EAEC), 11% of E. coli producing Shiga-like toxins (SLT) I and/or II; 13% of Shigella, 7% of Campylobacterjejuni or enterotoxigenic E. coli (ETEC) and 4% enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), Salmonella, Giardia lamblia or Hymenolepis nana. Mixed cultures were found in 16% of cases during the first year and in 18% during the second. These were mainly combinations of C. jejuni, with ETEC or EAEC. In 78% of children with bloody diarrhea the episode was single event during the first two years of life, lasting on average seven days. Epidemiologically, the incidence of bloody diarrhea rose steadily during the first two years of life, with a significant decrease after the tenth month. Prevalence was affected by seasonality in all pathogens, except for EAEC that had an endemic frequency. In the case of Shigella and SLT-producing E. coli clustering of cases and isolation of serologically identical strains indicated that infections were the result of common source outbreaks.

Rotavirus-inhibitory activity in serial milk samples from Mexican women and rotavirus infections in their children during their first year of life.

A total of 75 children born in rural Mexico were followed for diarrheal diseases and rotavirus (RV) excretion during the first year of life. For 18 children, an average of 14 serial breast milk samples were obtained between days 2 and 360 after delivery and were tested for RV-inhibitory activity. Of these samples, 70, 62, and 85% showed inhibitory activity against serotype (ST) 1 human RV, ST4 human RV, and ST3 simian RV, respectively; the median titers were 10, 10, and 20, respectively. Some 89% of the milk samples showed RV-specific antibodies in an enzyme-linked immunosorbent assay (median titer, 20). Surprisingly, 98% of the milk samples inhibited ST6 bovine RV. ST6, but not ST1, RV-inhibitory activity survived heat treatment (10 min at 80 degrees C). Of the 18 children tested, 13 children experienced 23 episodes of diarrhea (enterotoxigenic [n = 8] and enteropathogenic [n = 3] Escherichia coli, Campylobacter jejuni [n = 4], Shigella flexneri [n = 2], RV [n = 1]) and 5 children experienced 6 RV infections. Only one RV infection was associated with diarrhea. The five RV excretors did not differ from the nonexcretors with respect to the RV-inhibitory activity in the breast milk fed to them. The RV-inhibitory titers were too low in the majority of the studied Mexican milk samples to indicate an important effect of breast-feeding on the take rate of oral human, simian, or reassortant RV vaccines. Breast-feeding might, however, inhibit the take rate of a bovine RV vaccine.

Short electropherotype rotaviruses isolated longitudinally from a cohort of Mexican infants.

Stool specimens obtained longitudinally during the first year of life of a cohort of 75 rural infants were tested for the presence of rotavirus with an enzyme-linked immunosorbent assay. Thirty four infants showed the presence of rotaviruses during the first year of life. Only 41% of these episodes were associated with diarrhea. Extraction of rotavirus RNA was still possible in 12 of 34 stool samples, ten of them obtained during episodes of associated-diarrhea. Electropherotyping of these rotavirus showed that nine had a short electropherotype. One diarrheal and two asymptomatic samples had a long electropherotype. Subgrouping the viruses with monoclonal antibodies showed that five of the nine rotaviruses with short electropherotype belonged to subgroup I, and two of the three with long electropherotype belonged to subgroup II. The rest of rotaviruses could not be subgrouped. This study confirms the prevalence of only few rotavirus strains in any defined geographic region over a specific period of time, in this case, a rotavirus with a short electropherotype.

Immunoglobulin A antibodies directed against Campylobacter jejuni flagellin present in breast-milk.

We studied the relationship between IgA anti-campylobacter flagellin antibodies in breast milk samples and protection of breastfed infants living in a rural Mexican village from campylobacter infection. There were fewer episodes of campylobacter infection (symptomatic and asymptomatic combined) in infants breastfed with milk containing specific anti-flagellin antibodies (1.2/child/year, 95% CI 0.6-1.8) versus non-breastfed children (3.3/child/year, 95% CI 1.8-4.8; P < 0.01). Infants breastfed with milk that was anti-flagellin antibody negative by ELISA also had fewer episodes of infection compared with non-breastfed children, but the difference did not reach statistical significance (1.8/child/year, 95% CI 0.7-3.0 versus 3.3/child/year, 95% CI 1.8-4.8, P > 0.05). Breastfeeding has a protective effect against campylobacter infection and is associated with the presence of specific antibodies directed against campylobacter flagellin.

Hispano-Indian admixture in Paraguay studied by analysis of HLA-DRB1 polymorphism.

The genetic polymorphism of the Paraguayan population results from the admixture between South American Indians named Guaranis and Spaniards. In order to evaluate the genetic predominance in the Paraguayan population, we typed 50 healthy Paraguayans for HLA-DRB1 by molecular biology and compared their HLA-DRB1 polymorphism to that of the Guaranis and of two Spanish populations. Six significant differences of alleles frequencies were observed between Paraguayans and Guaranis--DRB1*01, 06 (13, 14), 15, 16, 07--whereas only one difference was observed with the Spaniards (DRB1*14). The DRB1*14 frequency was higher in Paraguayan than in the Spanish populations essentially due to the presence of DRB1*1402 related alleles (1402,06,13). These alleles are extremely rare in the Spanish populations whereas frequent in the Guaranis from Brazil and in South American Indian tribes living in the lymitrophe regions of Paraguay (Toba, Wichi and Terena). Thus, the presence of the DRB1*1402 related alleles (6%) in the Paraguayan population constitutes the major Indian contribution to the HLA-DR polymorphism of the Paraguayan population. The genetic distances between Paraguayans and the two Spanish populations were closer (.494 and .415) than that between Paraguayans and Guaranis (.958). Altogether these results suggest the predominance of the Spanish genetic in the Paraguayan population. Historical events are discussed to explain this predominance.