Technical approaches to evaluate the surfactant-enhanced biodegradation of biodiesel and vegetable oils.

This research compared the effects of biosurfactant on the biodegradation of biodiesel and vegetable oils while validating two conceptually diverging methodologies. The two experimental setups were successfully modeled towards the effects of biosurfactants during biodegradation. We established the equivalence of both methodologies from the data output. As expected, the biosurfactants caused an increased oil uptake, thus increasing biodegradation performance. Cooking oils were favored by the microbial consortium as a carbon source when compared with biodiesel fuel, especially after use in food preparation. However, we found that biodiesel substrate standout with the highest biodegradation rates. Our results might indicate that a rapid metabolic change from the original compound initially favored biodiesels during the assimilation of organic carbon for a set specialized microbial inoculum. The data output was successfully combined with mathematical models and statistical tools to describe and predict the actual environmental behavior of biodiesel and vegetable oils. The models confirmed and predicted the biodegradation effectiveness with biosurfactants and estimated the required timeframe to achieve satisfactory contaminant removal.

Muscular dystrophy in dogs: does the crossing of breeds influence disease phenotype?

Golden Retriever (GR) muscular dystrophy is an inherited degenerative muscle disease that provides an excellent model for Duchenne muscular dystrophy in humans. This study defined the histopathologic lesions, including the distribution of type I and II muscle fibers (FTI and FTII), in 12 dystrophic and 3 nondystrophic dogs between 7 and 15 months of age. The authors were interested in studying the influence on disease phenotype from crossing the base GR breed with Yellow Labrador Retrievers. The dystrophic dogs were divided according to breed: GRs and Golden Labrador Retrievers (GLRs). On hematoxylin and eosin staining, histopathologic lesions were more severe in GRs than GLRs. Six of eight GR muscles (75%) had a severe lesion grade (grade 3). In contrast, seven GLR muscles (87.5%) had mild lesions (grade 2), and only one had severe lesions (grade 3). Changes in fiber-type distribution were more pronounced in GRs versus GLRs. FTI:FTII ratio inversion was observed in three dystrophic GRs but only one GLR. The mean diameter of FTI and FTII was smaller in GRs and GLRs than in nondystrophic dogs (P < .01). The FTI of five GR muscles (62.5%) were larger than those of GLRs, whereas only one GLR muscle was larger (P < .05). The differential was less pronounced for FTII, with four GR muscles being larger and three GLR being larger. Observations indicate that crossing the base GR breed with Labrador Retrievers lessened the severity of the GR muscular dystrophy phenotype.

Effects of glycosaminoglycan polysulphate on the organisation of collagen fibres in experimentally induced tendonitis in horses.

An inflammatory process was induced by intratendinous injection of bacterial collagenase into the superficial digital flexor tendon (SDFT) of the left thoracic limb of 10 horses. One week later, the tendons in five of the horses (group 1) were treated with glycosaminoglycan polysulphate (GAGPS), and the tendons of the other five (group 2) were treated with saline solution. The horses were euthanased 150 days after the collagenase injections, and samples of the SDFTs were frozen at -14 degrees C, sectioned at 5 to 7 mum longitudinally and transversely, and stained by the picrosirius red method. Morphometric analysis was used to quantify the organised and disorganised bundles of collagen in the samples from groups 1 and 2. Significantly more organised bundles of collagen were observed in the tendons treated with GAGPS.

A new antigen system expressed in human endothelial cells.

Kidney transplant recipients were previously found to have antibodies that reacted with cells isolated from the endothelium of umbilical cord veins and which were not cytotoxic for lymphocytes from the same donors. Results of the present experiments indicate that endothelial (E) antigens are different from previously known HLA antigens and also from Ia-like antigens of bone marrow-derived (B) lymphocytes. Attempts to absorb E antibodies with lymphocytes from E-positive donors failed in most cases. Antigen redistribution experiments showed that E antigens were located in separate molecules from the products of HLA-A, B, and C. Thus, E cells treated with E antibody became resistant to lysis by the antibody used, but remained susceptible to the effects of typing sera for alleles of HLA-A, B, and C. Antibodies to E cells were also cytotoxic for blood monocytes. Moreover, monocytes were able to absorb E-antibody reactions, indicating that similar antigens were expressed in both cells. E antibodies did not react with B lymphocytes isolated from peripheral blood. In that regard E antibodies were different from antibodies to human Ia-like antigens which reacted with E cells, monocytes, and isolated B lymphocytes. Thus it appears that E antigens constitute a system of human alloantigens which has not been previously identified. The possibility that these antigens play a role in kidney allograft rejection should now be investigated since matching can be performed using monocytes isolated from the blood of recipients and donors.

Desmoglein-1-specific T lymphocytes from patients with endemic pemphigus foliaceus (fogo selvagem).

Fogo selvagem (FS), the endemic form of pemphigus foliaceus, is a cutaneous autoimmune disease characterized by subcorneal blistering of the epidermis and the production of autoantibodies against the desmosomal antigen desmoglein-1 (Dsg1). Previously, we showed that mice injected with autoantibodies from FS patients develop a skin disease that reproduces the clinical, histological, and immunological features of FS, indicating that autoantibodies play an essential role in the development of this disease. The purpose of this study was to characterize the autoimmune T-cell response associated with FS. We provide here the first evidence, to our knowledge, that the great majority of FS patients have circulating T lymphocytes that specifically proliferate in response to the extracellular domain of Dsg1. Long-term T cells developed from these patients also responded to Dsg1, and this antigen-specific response was shown to be restricted to HLA-DR molecules. These Dsg1-reactive FS T cells exhibited a CD4-positive memory T-cell phenotype and produced a T helper 2-like cytokine profile. These findings represent the initial steps in defining the role of T cells in FS autoimmunity.

An active focus of high prevalence of fogo selvagem on an Amerindian reservation in Brazil. Cooperative Group on Fogo Selvagem Research.

Fogo Selvagem (FS) is an autoimmune disease characterized by subcorneal vesicles and antidesmoglein-1 autoantibodies. Previous epidemiologic data have linked the onset of FS to exposure to an environmental antigen(s). This investigation describes a unique human settlement with an extraordinarily high prevalence of FS. This community is made up of Amerindians belonging to the Terena tribe, which has settled on the Limao Verde reservation in the state of Mato Grosso do Sul in Brazil. Twenty-six well-characterized FS cases have been identified within a total population of 998, yielding a prevalence of 2.6%. Seventeen of the patients (65 %) were males, and over 50% were older than 30 y of age. The incidence of the disease shows temporal periodicity, i.e., years with several cases of FS alternating with years with no cases. Over one-half of the cases occurred in genetically related family members. Another Terena reservation, the Ipegue/Taunay, located 90 km west of the Limao Verde reservation, was also evaluated as a control group. This reservation, with a population of 2203, had no recorded cases of FS. Thus, the Limao Verde reservation represents a new focus of FS in which the disease exhibits temporal, geographic, and familial clustering. These results suggest that the environmental antigen or antigens precipitating FS are endemic to the Limao Verde reservation. This reservation appears to be an ideal population for carrying out sero-epidemiologic, genetic, and environmental studies aimed at disclosing the etiology of FS.

Prediction of early kidney transplant rejection by a crossmatch with donor skin.

Immunofluorescence staining of donor skin with recipient serum was performed in 25 patients undergoing kidney transplantation. Transplants were performed when cytotoxicity T cell crossmatches with long incubation and with antiglobulin enhancement were negative. In 20 patients the skin crossmatch was negative and all had an uneventful course. In 5 other recipients, the crossmatch with skin was positive and all developed severe rejection 1-8 days after transplantation. The correlation between skin crossmatch and early rejection was statistically highly significant (P less than 0.00001). Rejection in the skin-crossmatch-positive group led to graft loss in 3, and chronic rejection with poor residual function in the other 2 patients. Serum creatinine at one month was in the 2.6-5.0 mg/dl range in these two patients, while in the group with negative skin crossmatches 15 had serum creatinine levels of less than 1.6, and 5 were in the 1.6-2.5 mg/dl range. Although the nature of the skin antigen is unknown, it appears that this crossmatch procedure was highly accurate in predicting early kidney transplant rejection.

Alloantibodies against donor epidermis and early kidney transplant rejection.

We have previously observed that transplant recipient sera with endothelial antibodies are bound to epidermal cells, as shown by immunofluorescence on sections of skin. It was also reported that early kidney failures that occurred despite negative T cell crossmatches were associated with, and could have been predicted by, a crossmatch with donor skin. Ninety patients undergoing kidney transplantation have now been evaluated using this technique. A few other patients were excluded from the analysis because of the presence of autoantibodies staining autologous skin. In 12 the crossmatch with donor skin was positive, and 9 of them had severe rejection within the first 10 days after transplantation. The three patients with a positive skin crossmatch and a benign course had not been tested with autologous skin and therefore autoantibodies could not be excluded. Only 7 of the 78 patients with negative skin crossmatches had early rejection. The correlation between skin crossmatch and early rejection was statistically highly significant (P less than 0.0001). Studies by flow cytometry have shown that these antigens are found on the surface of epidermal and endothelial cells, and are modulated by gamma interferon. When tested against a panel of skin donors, skin alloantibodies gave different patterns of positive and negative reactions, suggesting polymorphism.

DNA typing for class II HLA antigens with allele-specific or group-specific amplification. V. Typing for subsets of HLA-DR1 and DR'Br'.

Three DRB1 alleles of the DR1 group including DRB1*0101, DRB1*0102, and DRB1*0103 are currently recognized. The first two of these are defined as HLA-DR1 by serologic typing and as either Dw1 or Dw20 by typing with T cells. DRB1*0103, previously called DR'Br' or DR'BON', is not detectable by serology. Little information exists about the population frequencies of DRB1*0102 and DRB1*0103. In the present study we have used the polymerase chain reaction (PCR) and allele-specific oligonucleotide probes to determine these alleles. To avoid cross-hybridization with other DRB genes having the same DNA sequences as those of the region to be analyzed, we performed group-specific PCR to amplify only DR1 DRB1 genes. This was accomplished using a 21-nucleotide-long primer, homologous to the first hypervariable region common to the DR1 DRB1 genes, and which under appropriate conditions amplified only DRB1 genes of the DR1 group. Five oligonucleotide probes, one matching the second hypervariable region, two spanning the third hypervariable region, and two covering codons 82 through 89, were used to determine the three alleles. DRB1*0101 (Dw1) was found to be the major type of DR1 in North American Caucasians. In North American black and Brazilian mestizo populations DRB1*0102 (Dw20) was more prevalent. DRB1*0103 (DR'Br') was detected in only six individuals in the present study.

Evolution of HLA-class I compared to HLA-class II polymorphism in Terena, a South-American Indian tribe.

We have studied the HLA alleles of 60 unrelated healthy Terena and 10 Terena families. They are members of an isolated Brazilian tribe located in Mato Grosso do Sul (South Central Brazil). Six novel alleles were found in this population: HLA-A*0219 (gf = 0.02), A*0222 (gf = 0.15), HLA-B* 3520 (gf = 0.01), B*3521 (gf = 0.03), B*3912 (gf = 0.03) and B*4803 (gf = 0.16). Five of the six novel alleles differ from their putative progenitors by amino acid replacements in residues that contribute to the pockets of the peptide-binding site. Many of the variants defined by molecular methods were not identified correctly by serological typing. We calculated heterozygosity values (H) for HLA-A, -B, -C, DRB1, DQB1 and DPB . The highest values were observed at the HLA-B locus, followed by HLA-A, -DRB1 and DQB1. Residue positions 9, 24, 45, 62, 67, 95, 114, 116, 156, and 163 of HLA class I showed heterozygosity values greater than 0.50. Nine of them contribute to the peptide-binding specificity pockets and one to the T cell receptor binding site. If HLA antigens are useful for defense against pathogenic agents, heterozygosity would offer an advantage by allowing binding of a larger repertoire of peptides to the class I molecules. Individuals that are heterozygous at these positions would probably have a wider repertoire of peptide presentation to T cells. The observed results including the presence of novel alleles in the class I HLA loci suggest a functionally significant, more rapid evolution of class I compared to class II loci in this South American isolated population.

Unrelated peripheral blood stem cell transplantation for Fanconi anemia.

The Brazilian unrelated bone marrow donor program began in 1993 and an unrelated matched donor was found for a Fanconi anemia patient without a sibling match. An 11-year-old female recipient received FTBI (6.0 Gy) and cyclophosphamide (40 mg/kg) as conditioning. The 41-year-old female unrelated donor received G-CSF at 5 micrograms/kg x 5 days, and on day 6 and 7 postmobilization, peripheral blood stem cells were harvested. Engraftment was seen on day 19 post-BMT and she remains alive and well on day 191+. This case supports the potential role of harvesting G-CSF-stimulated PBSC for unrelated bone marrow transplantation.

Clinical relevance of antibodies to non-HLA antigens in organ transplantation.

This article summarizes work that focuses on alloantibodies that bind to endothelial cells and attempts to provide an evaluation of the apparent role that antigens distinct from classic HLA alleles, and not expressed in lymphocytes, may play in kidney transplant rejection. The most recent experience with the donor-skin crossmatch in prediction of early kidney allograft rejection also is described.

Effect of a persistent inflammatory process on experimental heterotopic ossification. The influence of macrophages.

1. We investigated the effect of a persistent carrageenin- or nystatin-induced inflammatory reaction on heterotopic ossification produced by the subcutaneous implant of a demineralized bone matrix in female Swiss mice (25 to 35 g). 2. Subcutaneous carrageenin injection (0.3 ml of a 2% solution in saline) into mice induced an inflammatory reaction characterized by a mature granuloma predominantly of macrophages containing particles of the irritant in their cytoplasm and which remained unchanged until the end of the experiment (40th day). 3. Subcutaneous nystatin inoculation (30,000 IU in 0.3 ml saline) induced an inflammatory reaction consisting initially of macrophages (4th day) but later turning into an epithelioid granuloma (7th day) consisting predominantly of epithelioid cells and which was present up to the 21st day when it was gradually replaced by adipocytes up to the 30th day. 4. An intramuscular implant of demineralized bone matrix (DBM, approximately 10 mg) induced the formation of cartilage and bone tissue and of hemopoietic bone marrow (heterotopic ossification) in 100% of the control animals (N = 5). An intramuscular DBM implant in animals that received carrageenin (N = 19) or nystatin (N = 21) induced heterotopic ossification in 100 and 57% (P < 0.01) of the animals, respectively. 5. The response to a dorsal subcutaneous DBM implant was essentially negative in control animals (N = 5), whereas implants performed near the site injected with carrageenin (N = 28) or nystatin (N = 31) produced a response in 71 (P < 0.01) and 36% (P < 0.01) of the animals, respectively. A DBM implant into the contralateral (control) dorsal subcutaneous tissue of the same animals that received carrageenin (N = 25) or nystatin (N = 29) resulted in heterotopic ossification in 64 (P < 0.01) and 7% of the animals, respectively. 6. The results suggest that the macrophages present in the mature granuloma induced by carrageenin somehow favored the development of metaplastic plates after subcutaneous DBM implant and that this effect may be systemic since the same response was observed in contralateral subcutaneous tissue.

Participation of macrophages in chloramphenicol-potentiated carrageenin-induced peritonitis in rats.

1. We investigated the possible potentiating effect of chloramphenicol succinate (30 mg/kg, every 12 h for 4 days, ip) on the response of polymorphonuclear neutrophils to carrageenin (150 micrograms, ip) or dextran (100 micrograms, ip) in the peritoneal cavity of male Wistar rats (180-230 g; N = 12 in each group). 2. Chloramphenicol potentiated the cell migration induced by carrageenin (35%) but not that induced by dextran. Previous macrophage depletion in the peritoneal cavity by washing with sterile saline abolished the cell response, whereas a previous thioglycollate-induced increase in macrophage numbers enhanced the potentiating effect (60%). 3. These results suggest that the potentiating effect on polymorphonuclear neutrophil migration induced by chloramphenicol may be related to chemotactic factors released by macrophages.

Effect of alloxan diabetes and adrenalectomy on carrageenin-induced pleurisy in the rat.

1. The effect of alloxan diabetes and adrenalectomy on the acute inflammatory response was investigated in rats using the carrageenin-induced pleurisy model. 2. Diabetic rats had reduced exudate volume (-85%) and inflammatory cell accumulation (-50%), especially polymorphonuclear leucocytes (-60%) in the pleural cavity 24 h after stimulation in relation to control values. Subcutaneous insulin administration 2 h after the inflammatory stimulus restored these parameters to normal levels. In contrast, all three measures of the response were potentiated 1.5 to 4-fold in adrenalectomized animals. 3. Results obtained by other investigators using different experimental models point to a possible pro-inflammatory function of insulin for the vascular component of inflammation. The present data show that insulin also has a significant effect on the cellular component of inflammation, especially the polymorphonuclear one, by increasing its accumulation at the affected site.

Do lymphocytes play a pro-inflammatory role in any pharmacological type of rat paw edema?

1. The hypothesis of a pro-inflammatory activity in lymphocytes related to the development of nonimmune acute inflammation was tested in leucopenic rats injected subcutaneously with various stimuli representing different pharmacological classes of paw edema-inducing agents into the subplantar area of one of the hind paws. 2. Amethopterin-induced leucopenia rendered animals hyporeactive to carrageenin and trypsin, but not to kaolin or dextran. 3. Administration of splenic lymphocytes to leucopenic rats to correct lymphopenia partially restored the inhibited responses to carrageenin and the early phase of responses to trypsin. 4. These results suggest that lymphocytes may play a pro-inflammatory role in nonimmune acute inflammation, which is more demonstrable in some pharmacological classes of paw edema than in others.

Human endothelial cell antigens: molecular independence from HLA and expression in blood monocytes.

Human alloantisera identified antigens in endothelial cells from umbilical cord veins that were not detectable in peripheral blood lymphocytes. Results of antigen redistribution experiments by the resistance to lysis induction method suggested that E-antigens were present in separate molecules of the endothelial cell surface. Monocytes from peripheral blood reacted like endothelial cells in cytotoxicity tests and were able to absorb the endothelial cytotoxic antibodies. Comparison of concordant and discordant monocyte reactions in first-degree relatives indicated that the antigens involved were inherited in linkage with HLA.

Separate Ia-like determinants in human lymphocytes and macrophages.

Some human alloantisera cytotoxic for B-lymphocyte preparations lost their reactivity when the number of contaminating monocytes was decreased by treatment with carbonyl iron. Such sera were found to react with the corresponding monocyte preparations isolated by adherence to Petri dishes. Macrophage antibodies were present in 38% of random sera from multiparous women and in 70% of sera from kidney transplant recipients. They could not be absorbed with B-lymphocyte preparations, thus suggesting that determinants expressed in macrophages were not present in B lymphocytes. Macrophages stimulate in mixed lymphocyte culture and participate in immune reactions. It is possible that alloantigens of macrophages, as observed in the present experiments, are products of genes associated with these histocompatibility-controlled functions.

Almost 50,000 volunteers participate at Redome, the Brazilian Bone Marrow Donor Registry.

Striking progress has been observed in the number of volunteer donors for hematopoietic stem cell transplantation in the last years in Brazil. By the end of 1998, the number of donors barely reached 4200 but it has grown progressively. It was close to 48,000 by the end of May 1993. It is possible to notice a steady increase from the first (1993 to 2000) to the last years (2001 to 2003). The regulation of each procedure by the Brazilian Health System, with the collaboration of the Hematology Societies, was essential for the success of Redome and for the stem cell transplantation program in Brazil. However, when analyzing these results some problems were detected: 95% of Redome donors come from the south and southeastern regions of the country, while few donors are from the north, northeast, and central parts of Brazil. The different miscegenation of races in different regions and states of Brazil makes this an important issue: to represent the whole Brazilian population, Redome must improve the donor search in such places. It also became clear that several other centers involved in unrelated hematopoietic transplantation must be accredited to avoid a long line of patients with compatible donors a waiting transplantation.