Chronic mastitis is associated with altered ovarian follicle development in dairy cattle.

Connection between mastitis and fertility is multifaceted; therefore, several aspects need more elucidation. In particular, the aim was to investigate if naturally occurring chronic mastitis has an effect on ovarian function. At the time of slaughter, a milk sample and both ovaries were collected from 68 cows. The presence and intensity of chronic mastitis was diagnosed by the combined evaluation of bacteriological examination and somatic cell count of the milk of each individual quarter according to the measures of the National Mastitis Council. Animals were divided into 4 groups characterized by a low (n=15), mild (n=14), intense (n=19), or severe (n=16) degree of infection. A count of visible follicles on each ovary was followed by a quantitative analysis of microscopic traits on a selected group of animals (n=16). The latter included the classification and count of the entire preantral follicle population, and the morphometric analysis of the vascular bed extension and connective stroma in the cortical region. Finally, the expression of growth and differentiation factor-9 (GDF-9) was studied. The number of follicles with diameters ranging from 1 to 3 mm and 4 to 7 mm was not affected by the degree of infection. A significant effect of the degree of udder infection was observed on the number of follicles with a diameter larger than 8 mm. Furthermore, the intensity of mastitis had no effect on the number of primordial and primary follicles, but severely affected cows showed a lower number of secondary follicles (0.5±0.1 vs. 0.2±0.03). Quantitative analysis demonstrated a decrease in the density of blood vessels (6.30±1.08 vs. 4.68±0.28) expressed as ratio of vascular bed/total area) and a higher incidence of fibrous stroma (1.60±0.99 vs. 6.04±3.08 expressed as ratio of connective tissue/total area) in the cortical area of the most affected animals. Finally, the level of GDF-9 protein within the oocytes of different follicle size was lower in the animals with the severe form of chronic mastitis (1.34±0.05 vs. 0.78±0.21 expressed as arbitrary units). In conclusion, decreased fertility of cows with chronic mastitis takes place through an effect on the ovary altering the dynamics of folliculogenesis. Within the ovary, this implies a reduction of the vascular bed and an increase in the fibrotic tissue together with a direct effect on oocyte-specific factors as GDF-9, all of which are essential regulatory elements of folliculogenesis.

Experimental small bowel transplantation from non-heart-beating donors: a large-animal study.

INTRODUCTION: The shortage of organs in the last 20 years is stimulating the development of new strategies to expand the pool of donors. The harvesting of a graft from non-heart-beating donors (NHBDs) has been successfully proposed for kidney and liver transplantation. To our knowledge, no studies are available for small bowel transplantation using NHBDs. In an experimental setting of small bowel transplantation, we studied the feasibility of using intestinal grafts retrieved from NHBDs. MATERIALS AND METHODS: Twenty five Large White piglets underwent total orthotopic small bowel transplantation and were randomly divided as follow: NHBD group (n = 15) received grafts from NHBDs; heart-beating donor (HBD) group (n = 10) received grafts from HBDs. The NHBD pigs were sacrificed inducing the cardiac arrest by a lethal potassium injection. After 20 minutes (no touch period = warm ischemia), they underwent cardiac massage, laparotomy, and aorta cannulation for flushing and cooling the abdominal organs. In HBDs, the cardiac arrest was induced at the time of organ cold perfusion. In both groups, immunosuppression was based on tacrolimus oral monotherapy. The animals were observed for 30 days. The graft absorptive function was studied at day 30 using the D-xylose absorption test. Histological investigation included HE (Hematoxilin and Eosin) microscopical analysis and immunohistological staining. RESULTS: Animals in the NHBD group died due to infection (n = 3), acute cellular rejection (n = 2), technical complications (n = 2), and intestinal failure (n = 8). In the HBD group, all animals but two were alive at the end of the study. The D-xylose absorption was significantly lower among the NHBD compared with the HBD group (P < .05). CONCLUSIONS: This study confirmed that intestinal mucosa is sensitive to ischemic injury. When the intestinal graft is harvested from NHBDs, the infectious-related mortality was higher and the absorptive function lower. Histological examination confirmed a higher grade of ischemic injury in the NHBD grafts that correlated with the clinical data. Therefore, this experimental study suggested that non-heart-beating donation may not be indicated for small bowel transplantation.

Aberrant phenotypes and quantitative antigen expression in different subtypes of canine lymphoma by flow cytometry.

Flow cytometry may be a useful tool to analyze lymphoma samples that are obtained from fine needle aspirations (FNA). This study aimed to determine if flow cytometric analysis add more objective and standardized information on the cellularity and morphology of lymphoma cells to conventional cytology. The typical immunophenotype of different lymphoma subtypes was assessed and leukocyte marker expression was evaluated to determine which antigens were more frequently over- or under-expressed in these lymphoma subtypes. Fifty FNA lymph node samples were evaluated from canine lymphomas. Thirty-one samples were identified to be of B-cell origin, sixteen were identified to be of T/NK-cell origin and three cases were classified as acute lymphoblastic leukaemia with lymph nodes involvement. The most common B-cell lymphoma subtypes were centroblastic lymphomas, whereas three cases were atypical and classified as B-large cell pleomorphic lymphomas. Among the T/NK lymphomas, small clear cells, large and small pleomorphic mixed cells, large granular lymphocytic cells and small pleomorphic cells were identified. Aberrant phenotypes and/or antigen under/over regulation was identified in thirty out of forty-seven lymphoma cases (64%; 18/31 B-cell=58% and 12/16 T-cell=75%). In B-cell lymphomas the most frequent finding was the diminished expression of CD79a (45%). CD34 expression was also observed in four cases (13%). Among T-cell lymphomas the prevalent unusual phenotype was the under-expression or absence of CD45 (25%). These findings reveal flow cytometry may be useful in confirming the diagnosis of lymphoma, as the technique allows one to add useful information about morphology of the neoplastic cells and identify antigenic markers and aberrant phenotypes.

Elemental enteral nutrition preserves the mucosal barrier and improves the trophism of the villi after small bowel transplantation in piglets.

The main goals for a successful small bowel transplantation (SBTx) are the control of acute rejection and maintenance of the mucosal barrier, which plays a key role in preventing bacterial translocation and preserving absorptive capacity. According to recent evidence that sustaining enteral nutrition (EN) as rehabilitative therapy improves the integrity of the mucosal barrier after SBTx, we studied the trophic effect of a new elemental enteral solution whose proteinic supply is represented by oligomeric-aminoacidic chains. In a swine SBTx model we studied three groups, divided by the different postoperative feeding: group 1 (n = 5): standard swine chow, group 2 (n = 5): polymeric enteral solution, group 3 (n = 5): elemental enteral solution (Peptamen, Nestlè Corp). All animals were immunosuppressed with a tacrolimus/FK778 combined oral therapy. The nutritional indices evaluated were: body weight, episodes of diarrhea, D-xylose absorption test, and histopatological and villi morphometric analysis. Three pigs died before the end of the study, two in group 1 (pneumonia and sepsis), one in group 2 (pneumonia). Mean days of diarrhea were 15, 10, and 3 in groups 1, 2, and 3, respectively (P < .05). The final/starting weight ratio was 1.08 for group 3 and 0.92 for group 2 (P < .05); the D-xylose curves showed a statistically significant difference for group 3 versus the groups 2 and 1 (P < .05), as well as for the villi height (P < .01) and width (P < .05). In conclusion, elemental enteral solution, with its basic protein supply, does not require a very complex enzymatic system to be metabolized. Thus, it may contribute to a faster recovery of the mucosal barrier and to limit the hypercatabolic state.

FK778 and tacrolimus combination therapy to control acute rejection after pig intestinal transplantation.

Malononitrilamide 715 (FK778) is a new class of low molecular weight immunosuppressant. Experimental studies in heart, liver, and kidney transplantation have shown a strong synergism when FK778 is used in combination with tacrolimus and when its administration is delayed by 7 days after the transplant. Following this indication, in a swine model of orthotopic small bowel transplantation (SBT), we assessed the efficacy of combined low dose tacrolimus and FK778 administered from day 0 or day 7. The entire small bowel was replaced in 16 piglets: group 1 (n = 5), no immunosuppression; group 2 (n = 6) oral tacrolimus to maintain whole blood trough levels between 5 and 15 ng/mL plus FK778 4 mg/kg per day; group 3 (n = 6) oral tacrolimus as in group 2 plus FK778 4 mg/kg per day administered after a 7-day delay posttransplant. The median survival was 8 days in group 1, 60 days in group 2, and 13 days in group 3. The differences between group 2 and 1 and between group 2 and 3 are statistically significant. Three episodes of major bacterial infection were detected in both group 2 and 3 (0.5 episode/animal). The infectious-related mortality was 0% in group 2 and 50% in group 3 (P < .05). Acute cellular rejection was absent or mild in all group 2 and 3 stomal biopsies. In conclusion, combining tacrolimus and FK778 allowed prolonged survival after SBT in swine when FK778 was started at the time of SBT. The delayed administration of FK778 resulted in a high incidence of lethal infectious complications.

Early outcome of different steroid-free regimens in small bowel transplantation: a large-animal study.

The intestine is a highly immunogenic organ that requires heavy immunosuppression (IS); therefore corticosteroid withdrawal after clinical small bowel transplantation (SBT) has not been standardized. In this study, we compared different immunosuppressive regimens (none with steroid or induction treatment) in a SBT pig model. Large White unrelated piglets were transplanted and divided into four groups as follow: group 1 (n = 3): no IS; group 2 (n = 10): IS with tacrolimus only; group 3 (n = 10): IS with tacrolimus and mycophenolate mofetil; group 4 (n = 5): IS with tacrolimus and rapamycin. Follow-up time was 30 days. All IS drugs were given orally; tacrolimus whole blood levels ranged between 5 and 15 ng/mL in all groups except for group 2 whose tacrolimus whole blood levels ranged between 15 and 25 ng/mL. Group 1 pigs died of graft acute rejection (ACR) after a median of 12 days. Overall survival in groups 2, 3, and 4 at day 30 was 40%, 80%, and 60%, respectively. Biochemical parameters, including glycemia and cholesterol, were into the normal range with no significant differences between groups. At the end of the study, one animal in group 2 and another one in group 4 showed histological signs of moderate to severe ACR. The incidence of infection was higher in group 2 (2.1 episodes/pig) compared to group 3 (1.25) and group 4 (1.6). This large-animal study demonstrates that tacrolimus-based IS without corticosteroids allows, in the early postoperative period (30 days) after SBT, good survival rates without an increased risk in the incidence of rejection.

Efficacy of malononitrilamide FK778 in a preclinical model of small bowel transplantation.

In a swine model of orthotopic small bowel transplantation, we assessed the efficacy of combined immunosuppressive therapy with low-dose tacrolimus plus FK778, compared with high-dose tacrolimus monotherapy. The small bowel was replaced in 23 piglets: group 1 (n = 5), no immunosuppression; group 2 (n = 12), oral tacrolimus to maintain whole blood trough levels between 15 and 25 ng/mL; and group 3 (n = 6), oral FK778 4 mg/kg/d, plus oral tacrolimus to maintain whole blood trough levels between 5 and 15 ng/mL. Follow-up time was limited to 60 days. Overall survival rates at 30 and 60 days were 0% and 0% in group 1, 30% and 0% in group 2, and 66% and 66% in group 3, respectively. The median survival time was 11 days in group 1, 28 days in group 2, and more than 60 days in group 3. The differences between groups 3 and 1 and between groups 3 and 2 were statistically significant. The numbers of major bacterial infections were 19 in group 2 (1.9 episodes per animal) and 3 in group 3 (0.75 episodes per animal). The infectious-related mortality rate was 70% in group 2 (7 cases) and 0% in group 3 (P < .05). Acute cellular rejection was absent or mild in 85% of group 2 stomal biopsy specimens and in 100% of group 3 biopsy specimens. In conclusion, combination therapy of low-dose tacrolimus is potentiated by FK778 to prevent acute cellular rejection and prolong small bowel transplant survival in pigs.

Different dose effect of HLA-DQ alpha beta heterodimers in insulin-dependent diabetes mellitus and celiac disease susceptibility.

To compare the quantitative effect of the DQ alpha beta heterodimers DQ alpha 52 Arg+, beta 57 Asp- and DQ alpha 1*0501, beta 1*0201 on susceptibility to IDDM and CD, we characterized, at the genomic level, the DQ alpha 52 and DQ beta 57 residues of 50 IDDM Italian patients observed in Rome. The results were compared with those of a previous study concerning the oligotyping of DQ dimers in a group of CD children belonging to the same population. Our data confirm that both diseases are primarily associated with HLA-DQ alpha beta heterodimers, but the distributions of the respective susceptible DQA1 and DQB1 alleles in the two diseases were different. In fact, the highest risk of IDDM is for subjects alpha SS, beta SS that could express, by either cis- or trans-association, four susceptible heterodimers and decreases in proportion to the number of these; in regard to CD, the highest risk was found for individuals who carried only one predisposing heterodimer.

A study of Italian pediatric celiac disease patients confirms that the primary HLA association is to the DQ(alpha 1*0501, beta 1*0201) heterodimer.

Celiac disease (CD) has been recently reported to be primarily associated with the DQ(alpha 1*0501, beta 1*0201) heterodimer encoded in cis on DR3 haplotype and in trans in DR5,7 heterozygous individuals. The high incidence of DR5,7 heterozygotes, reflecting the high frequency of the DR5 allele in Italy, makes the analysis of the Italian CD patients critical. Polymerase chain reaction-amplified DNA from 50 CD patients and 50 controls, serologically typed for DR and DQw antigens, was hybridized with five DQA1-specific oligonucleotide probes detecting DQA1*0101 + 0102 + 0103, DQA1*0201, DQA1*0301 + 0302, DQA1*0401 + 0501 + 0601, and DQA1*0501 and a DQB1-sequence-specific oligonucleotide probe recognizing DQB1*0201 allele. As expected by the DR-DQ disequilibria, DQA1*0201 [62% in patients versus 26% in controls, relative risk (RR) = 5] and DQA1*0501 (96% versus 56%, RR = 19) show positive association with the disease. Of CD patients, 92% (50% DR3 and 42% DR5,7) compared to 18% of the controls carry both DQA1*0501 and DQB1*0201 alleles, so that the combination confers an RR of 52, higher than both the risks of the single alleles (DQA1*0501 RR = 19, DQB1*0201 RR = 30), confirming the primary role of the dimer in determining genetic predisposition to CD both in DR3 and in DR5,7 subjects.

Different binding of NF-Y transcriptional factor to DQA1 promoter variants.

Polymorphism in the HLA-DQA1 promoter (QAP) sequences could influence the gene expression through a differential binding of transcriptional factors. Considering the main role played by the Y-box in the transcription, we focused on the QAP4 variants differing for a G vs A transition from the QAP Y-box consensus sequence. Electrophoretic Mobility Shift Assay using the two Y-box sequences was performed to determine whether this mutation could be reflected in an allele-specific binding of transcriptional factors. Indeed, the NF-Y specific band, recognised by supershift experiments, was clearly observed using the Y-box consensus probe but it was barely detectable with the QAP4 one. On the contrary, two other complexes were found to more strongly interact with QAP4 Y-box in comparison to the consensus sequence. The analysis of a selected panel of HLA homozygous lymphoblastoid cell lines by competitive RT-PCR and by Northern blotting revealed that the DQA1 *0401, *0501,*0601 alleles regulated by the QAP4 promoters were less expressed at the mRNA level than the DQA1* 0201 allele regulated by the QAP2.1 variant. In conclusion, these results show an evident reduction of NF-Y binding to the mutated QAP4 Y-box and a decreased mRNA accumulation of the DQA1 alleles regulated by these variants.

Absence of in vivo DNA-protein interactions in the DQA2 and DQB2 promoter regions.

The DQ subregion of the human major histocompatibility complex (HLA) contains two pairs of loci: the DQA1/B1 genes (hereafter called DQ1), coding for the DQ molecules, and the DQA2/B2 pseudogenes (hereafter called DQ2). These pseudogenes are highly homologous to the functional DQ1 genes and they have no apparent abnormal features in their sequences that could prevent their activity. Only recently a low expression of the DQA2 gene has been observed whereas the DQB2 transcript was never found. The comparison between the DQ1 and DQ2 regulatory sequences revealed several differences in their W, X, and Y cis-acting elements. To examine the DNA/protein interactions in the DQ promoter regions, we performed in vivo footprinting experiments. Whereas the functional DQ1 loci showed a series of DNA-protein contact points in the X and Y boxes, the promoters of the DQ2 pseudogenes displayed an unoccupied phenotype. These findings suggest that the very low level of DQA2 expression and the apparent lack of DQB2 activity are caused by the reduced binding affinity of specific transcription factors.

Distinctive features of the alpha 1-domain alpha helix of HLA-C heavy chains free of beta 2-microglobulin.

Only a few monoclonal antibodies are available with a restricted specificity to HLA-C products. In the present report, we demonstrate that antibody L31, previously shown to react with beta 2m-less (free) class I MHC heavy chains, binds to an epitope (residues 66-68 of the alpha 1 domain alpha helix) present on all the HLA-C alleles corresponding to the accepted (CW1 through CW8) serologic specificities, and on a few HLA-B heavy chains sharing with HLA-C an aromatic residue at position 67. Extensive IEF blot testing of HLA homozygous, EBV-transformed B-lymphoid cells indicates that HLA-C molecules are present at significantly lower levels than HLA-B polypeptides not only at cell surface, as previously demonstrated, but also in total cellular extracts. Testing of metabolically labeled HLA-CW1, -CW5, and -CW6 transfectants and HLA homozygous lymphoid cells, particularly HLA-CW1-expressing cells, demonstrates that the L31 epitope is present on a subpopulation of naturally occurring HLA-C molecules distinct from that identified by antibody W6/32 to beta 2m-associated heavy chains. Pulse-chase experiments demonstrate that this epitope is transiently made available to antibody binding at early biosynthetic stages, but becomes hidden upon assembly with beta 2m. Thus, free HLA-C and other Y/F67+ heavy chains are characterized by distinctive antibody binding features in a region (residues 66-68) included in a previously identified HLA-C restricted motif, which has been suggested to be the primary cause of distinctive features of the antigen-binding groove, low affinity for endogenous peptide antigens and beta 2m, and preferential uptake of exogenous peptides, possibly of viral origin. We also show that HLA-CW1 heavy chains, both free and beta 2m associated, acquire sialilation. Free HLA-CW1 heavy chains are expressed at the cell surface even when unsialilated, albeit at low levels.

A mouse monoclonal antibody against a polymorphic determinant in a defined subset of DR molecules.

The hybridoma technique was used to produce a mouse monoclonal antibody, designated as XI 21.4, which belongs to the IgG2a class. It is active in complement-dependent cytotoxicity and detects a B-cell antigenic determinant associated with DR1, DR4, DRw10, and, possibly, DRw9. Microfingerprinting of the immunoprecipitate from a homozygous DR4 cell line shows a typical alpha DR pattern and a beta pattern coinciding with that of DR4 molecules.

A study of HLA class II antigens in an Italian paediatric population with coeliac disease.

One hundred and twenty-one Italian children with coeliac disease (CD) have been compared with a control population from the same geographical area for the distribution of HLA-DR and DQ antigens. The pattern of an increase in DR3, DR7, and of heterozygotes DR5/7 was associated with an excess of heterozygotes DQw2/DQw3 in the CD population. These findings suggest that epitopes determined by specific combinations of DQ alpha and beta chains (combinatorial determinants) predispose to the disease.

HLA antigens and adult rheumatoid arthritis: a study with a monoclonal antibody.

Adult rheumatoid arthritis (RA) is a very heterogeneous disease that is associated with HLA-antigens, although no absolute association has been found with any particular HLA type. Forty-one seropositive RA patients have been studied with a local monoclonal antibody named X1 21.4 (9w940), strongly associated with HLA-DRI, DR4, Drw10 antigens, to verify a possible correlation with the disease. The results obtained have also been compared with the data reported on MC1, a serologically defined determinant correlated with RA. X1 21.4 monoclonal antibody appears to be associated with the disease and it could identify one epitope involved in the susceptibility to RA.

A nonneutralizing human IgM monoclonal antibody inhibiting hemagglutination of H3N2 influenza A strains.

A mouse-human hybridoma has been produced by fusing human splenocytes from a Cooley's anemia patient with the murine myeloma P3-NS1/1-Ag 4-1. The hybridoma is stable after 18 months and secretes human IgM. The antibody reacts with some H3N2 influenza A strains and detects an epitope that is part of the hemagglutinin antigen, but does not affect virus infectivity.

Polymorphism in the upstream regulatory region of DQA1 gene in the Italian population.

Polymorphism in the 5'-upstream regulatory region of the DQA1 gene has been recently described. Using PCR-SSO method and SSCP analysis we have investigated this polymorphism in a group of 111 Italian blood donors which had been oligotyped for DRB1, DQA1 and DQB1 genes. Eight allelic variants were detected. Looking at the relationships among QAP sequences and DQA1 and DRB1 genes, three alternative situations were found: 1. a one-to-one relation between QAP and DQA1 alleles, independently of the other class II genes; 2. the same QAP allele in association with different DQA1-DRB1 haplotypes; 3. the same DQA1 allele with different QAP sequences according to the DRB1 specificity. No unexpected associations with DQB1 gene were found. These results must be interpreted considering that DQA1 and DRB1 genes are transcribed in opposite directions so that the promoter region of DQA1 gene lies between DQA1 and DRB1, close to the former but several hundreds kb away from the latter.

[Comparison between phantoms for quality control in xeromammography]. / Confronto tra i fantocci per i controlli di qualità in xeromammografia.

An indicator of operating technique (phantom) is very useful for quality control in diagnostic radiology. We tested experimentally that the "Random Phantom" is the most suitable between those commercially available for xeromammography. In fact this indicator points out image quality alterations caused even by very small variations of the physical parameters affecting image quality. We did not test the phantoms for mammographic technique but we believe that the "Random" is the most suitable in this field too.