Dry period length affects rumen adaptation in dairy cattle precalving and during the first weeks after calving.

Omitting or shortening the dry period may result in a fairly constant ration throughout the transition period of dairy cows, reducing the need for adaptation of cow metabolism and rumen function to a new lactation. The objective of this study was to determine the effect of dry period length on rumen adaptation and cow metabolic state during the transition period. Twelve pregnant, rumen-cannulated Holstein Friesian dairy cows at the end of their first lactation were assigned to one of 3 treatments: a conventional (60 d), short (30 d) or no dry period (0 d). At dry-off, cows received a dry cow ration until calving. Lactating cows received a lactation ration. Cows were monitored from 8 wk before calving until 8 wk after calving for milk yield and dry matter intake (DMI). Rumen biopsies were taken from 3 locations in the rumen at 60, 40 and 10 d before calving and 3, 7, 14, 28 and 56 d after calving to assess papillae dimensions. Blood was sampled weekly from 3 wk before until 8 wk after calving, and liver biopsies were taken at wk -2, wk 2 and wk 4 relative to calving. Prepartum, DMI and milk yield were greater for cows with a short or no dry period, compared with cows with a conventional dry period. Postpartum, DMI was greater for cows with a short dry period compared with cows with a conventional dry period. Plasma glucose concentration was greater for cows without a dry period, compared with the other dry period lengths postpartum. Plasma concentrations of nonesterified fatty acids and ß-hydroxybutyrate, and liver triglyceride content, did not differ among dry period. Rumen papillae differed in size based on biopsy location, but there was no interaction between biopsy location and the effect of dry period length. Rumen papillae surface area for cows managed for a 30 d or 60 d dry period decreased toward calving. At 40 d prepartum, papillae surface area was greater for short and no dry period treatment compared with a conventional dry period. At 10 d prepartum, papillae surface area was greater for the no dry period treatment compared with both other treatments, and this difference was still present 3 d postpartum. Cows managed for a short dry period showed faster increase in papillae dimensions after calving compared with cows managed for a conventional dry period. From d 28 onwards, no differences in papillae surface area were observed. The faster rumen adaptation postpartum may be related to the increased DMI during the first weeks postpartum for cows managed for a short dry period. However, this did not result in improved metabolic status or milk yield. The results from the present study demonstrate that the dietary changes related to a conventional dry period length affected rumen papillae development, not only prepartum but also early postpartum. Further optimization of dry period length as well as dietary composition throughout the transition period may support cows in their adaptation to a new lactation.

Evaluation of 3 northwest European seaweed species on enteric methane production and lactational performance of Holstein-Friesian dairy cows.

Seaweeds have been studied for their ability to reduce enteric methane emissions of ruminants when fed as a feed supplement. In vivo research with dairy cattle is mainly limited to the seaweed species Ascophyllum nodosum and Asparagopsis taxiformis, whereas in vitro gas production research covers a broader range of brown, red, and green seaweed species from different regions. The objective of the present study was to determine the effect of Chondrus crispus (Rhodophyta), Saccharina latissima (Phaeophyta), and Fucus serratus (Phaeophyta), 3 common northwest European seaweeds, on enteric methane production and lactational performance of dairy cattle. Sixty-four Holstein-Friesian dairy cattle (16 primiparous, 48 multiparous) averaging (mean ± standard deviation) 91 ± 22.6 d in milk and 35.4 ± 8.13 kg/d fat- and protein-corrected milk yield (FPCM) were randomly assigned to 1 of 4 treatments in a randomized complete block design. Cows were fed a partial mixed ration [54.2% grass silage, 20.8% corn silage, and 25.0% concentrate; dry matter (DM) basis] with additional concentrate bait in the milking parlor and the GreenFeed system (C-Lock Inc.). The 4 treatments consisted of a control diet without seaweed supplement (CON), or CON supplemented with 150 g/d (fresh weight of dried seaweed) of either C. crispus (CC), S. latissima (SL), or a 50/50 mix (DM basis) of F. serratus and S. latissima. Milk yield (28.7 vs. 27.5 kg/d, respectively), fat- and protein-corrected milk (FPCM) yield (31.4 vs. 30.2 kg/d, respectively), milk lactose content (4.57 vs. 4.52%, respectively), and lactose yield (1,308 vs. 1,246 g/d, respectively) increased for SL compared with CON. Milk protein content was lower for SL compared with the other treatments. Milk fat and protein contents; yields of fat, protein, lactose, and FPCM; feed efficiency; milk nitrogen efficiency; and somatic cell count did not differ between CON and the other treatments. Depending on week of experiment, milk urea content was higher for SL compared with CON and CC. No effects were observed of the treatments compared with CON for DM intake, number of visits to the GreenFeed, or gas emission (production, yield, or intensity) of CO2, CH4, and H2. In conclusion, the seaweeds evaluated did not decrease enteric CH4 emissions and did not negatively affect feed intake and lactational performance of dairy cattle. Milk yield, FPCM yield, milk lactose content, and lactose yield increased, and milk protein content decreased, with S. latissima.

Comparison of fractionation methods for nitrogen and starch in maize and grass silages.

In in situ nylon bag technique, many feed evaluation systems use a washing machine method (WMM) to determine the washout (W) fraction and to wash the rumen incubated nylon bags. As this method has some disadvantages, an alternate modified method (MM) was recently introduced. The aim of this study was to determine and compare the W and non-washout (D+U) fractions of nitrogen (N) and/or starch of maize and grass silages, using the WMM and the MM. Ninety-nine maize silage and 99 grass silage samples were selected with a broad range in chemical composition. The results showed a large range in the W, soluble (S) and D+U fractions of N of maize and grass silages and the W, insoluble washout (W-S) and D+U fractions of starch of maize silages, determined by both methods, due to variation in their chemical composition. The values for N fractions of maize and grass silages obtained with both methods were found different (p < 0.001). Large differences (p < 0.001) were found in the D+U fraction of starch of maize silages which might be due to different methodological approaches, such as different rinsing procedures (washing vs. shaking), duration of rinsing (40 min vs. 60 min) and different solvents (water vs. buffer solution). The large differences (p < 0.001) in the W-S and D+U fractions of starch determined with both methods can led to different predicted values for the effective rumen starch degradability. In conclusion, the MM with one recommended shaking procedure, performed under identical and controlled experimental conditions, can give more reliable results compared to the WMM, using different washing programs and procedures.

Prediction of urinary nitrogen and urinary urea nitrogen excretion by lactating dairy cattle in northwestern Europe and North America: a meta-analysis.

A meta-analysis was conducted on the effect of dietary and animal factors on the excretion of total urinary nitrogen (UN) and urinary urea nitrogen (UUN) in lactating dairy cattle in North America (NA) and northwestern Europe (EU). Mean treatment data were used from 47 trials carried out in NA and EU. Mixed model analysis was used with experiment included as a random effect and all other factors, consisting of dietary and animal characteristics, included as fixed effects. Fixed factors were nested within continent (EU or NA). A distinction was made between urinary excretions based on either urine spot samples or calculated assuming a zero N balance, and excretions that were determined by total collection of urine only. Moreover, with the subset of data based on total collection of urine, a new data set was created by calculating urinary N excretion assuming a zero N balance. Comparison with the original subset of data allowed for examining the effect of such an assumption on the relationship established between milk urea N (MUN) concentration and UN. Of all single dietary and animal factors evaluated to predict N excretion in urine, MUN and dietary crude protein (CP) concentration were by far the best predictors. Urinary N excretion was best predicted by the combination of MUN, CP, and dry matter intake, whereas UUN was best predicted by the combination of MUN and CP. All other factors did not improve or only marginally improved the prediction of UN or UUN. The relationship between UN and MUN differed between NA and EU, with higher estimated regression coefficients for MUN for the NA data set. Precision of UN and UUN prediction improved substantially when only UN or UUN data based on total collection of urine were used. The relationship between UN and MUN for the NA data set, but not for the EU data set, was substantially altered when UN was calculated assuming a zero N balance instead of being based on the total collection of urine. According to results of the present meta-analysis, UN and UUN are best predicted by the combination of MUN and CP and that, in regard to precision and accuracy, prediction equations for UN and UUN should be derived from the total collection of urine.

Immune responses against islet allografts during tapering of immunosuppression--a pilot study in 5 subjects.

Transplantation of isolated islet of Langerhans cells has great potential as a cure for type 1 diabetes but continuous immune suppressive therapy often causes considerable side effects. Tapering of immunosuppression in successfully transplanted patients would lower patients' health risk. To identify immune biomarkers that may prove informative in monitoring tapering, we studied the effect of tapering on islet auto- and alloimmune reactivity in a pilot study in five transplant recipients in vitro. Cytokine responses to the graft were measured using Luminex technology. Avidity of alloreactive cytotoxic T Lymphocytes (CTL) was determined by CD8 blockade. The influence of immunosuppression was mimicked by in vitro replenishment of tacrolimus and MPA, the active metabolite of mycophenolate mofetil. Tapering of tacrolimus was generally followed by decreased C-peptide production. T-cell autoreactivity increased in four out of five patients during tapering. Overall alloreactive CTL precursor frequencies did not change, but their avidity to donor mismatches increased significantly after tapering (P = 0·035). In vitro addition of tacrolimus but not MPA strongly inhibited CTL alloreactivity during tapering and led to a significant shift to anti-inflammatory graft-specific cytokine production. Tapering of immunosuppression is characterized by diverse immune profiles that appear to relate inversely to plasma C-peptide levels. Highly avid allospecific CTLs that are known to associate with rejection increased during tapering, but could be countered by restoring immune suppression in vitro. Immune monitoring studies may help guiding tapering of immunosuppression after islet cell transplantation, even though we do not have formal prove yet that the observed changes reflect direct effects of immune suppression on immunity.

Functional consequences of HLA-DQ8 homozygosity versus heterozygosity for islet autoimmunity in type 1 diabetes.

Human leukocyte antigen (HLA) class II haplotypes are established risk factors in type 1 diabetes (T1D). The heterozygous DQ2/8 genotype confers the highest risk, whereas the DQ6/8 genotype is protective. We hypothesized that DQ2/8 trans-molecules composed of α and ß chains from DQ2 and DQ8 express unique ß-cell epitopes, whereas DQ6 may interfere with peptide binding to DQ8. Here we show that a single insulin epitope (InsB13-21) within the T1D prototype antigenic InsB6-22 peptide can bind to both cis- and trans-dimers, although these molecules display different peptide binding patterns. DQ6 binds a distinct insulin epitope (InsB6-14). The phenotype of DQ8-restricted T cells from a T1D patient changed from proinflammatory to anti-inflammatory in the presence of DQ6. Our data provide new insights into both susceptible and protective mechanism of DQ, where protecting HLA molecules bind autoantigens in a different (competing) binding register leading to 'epitope stealing', thereby inducing a regulatory, rather than a pathogenic immune response.

Milk urea concentration as an indicator of ammonia emission from dairy cow barn under restricted grazing.

Bulk milk urea concentration was evaluated to assess its potential as an indicator of ammonia emission from a dairy cow barn in a situation with restricted grazing. An experiment was carried out with a herd of, on average, 52 Holstein-Friesian dairy cows. The cows were housed in a naturally ventilated barn with cubicles and a slatted floor, were fed ensiled forages and feed supplements, and each day were allowed 8.5 h of grazing. The experiment was a balanced randomized block design, replicated 3 times. The experimental factor was the bulk milk urea level, which was adjusted to levels of 15, 35, and 55 mg of urea per 100 g of milk, respectively, by changing the level of nitrogen fertilization of the pasture, the herbage mass and grass regrowth age, and the level and type of feed supplement. Ammonia emission from the barn was measured using sulfur hexafluoride as the tracer gas. Ammonia emission generally increased upon an increase in adjusted milk urea levels. A dynamic regression model was used to predict ammonia emission from bulk milk urea concentration, temperature, and a slurry mixing index. This model accounted for 66% of the total variance in ammonia emission and showed that emission increases exponentially with increasing milk urea concentration. At levels of 20 and 30 mg of urea per 100 g of milk, ammonia emission increased by about 2.5 and 3.5%, respectively, when milk urea concentration increased by 1 mg/100 g. Furthermore, emissions from the barn increased 2.6% when temperature increased by 1°C. The study showed that bulk milk urea concentration is a useful indicator for ammonia emissions from a dairy cow barn in a situation with restricted grazing.

Accumulation of autoreactive effector T cells and allo-specific regulatory T cells in the pancreas allograft of a type 1 diabetic recipient.

AIMS/HYPOTHESIS: Simultaneous kidney-pancreas transplantation is an established treatment for patients with type 1 diabetes and end-stage renal failure, even though restored beta cell function may become affected by recurrent islet autoimmunity or graft rejection. We characterised infiltrating lymphocytes isolated from a pancreatic graft with normal endocrine function in a kidney-pancreas recipient with type 1 diabetes. METHODS: The pancreas graft was removed due to recurrent graft pancreatitis of unknown cause. Pancreas-infiltrating lymphocytes and peripheral blood mononuclear cells (PBMC) were isolated and characterised phenotypically and functionally. RESULTS: Compared with PBMC, pancreas-infiltrating lymphocytes exhibited a distinct activation/memory phenotype and T cell receptor profile that were indicative of selective infiltration of the pancreas. Islet autoreactive CD8(+) T cells could be detected in the pancreas and were increased in frequency compared with PBMC. Additionally, an augmentation of CD8(+) CD28(-) regulatory T cells was observed in the pancreas; these induced expression of the inhibitory receptor immunoglobulin-like transcript-3 on antigen-presenting cells in a donor HLA class I-specific manner. CONCLUSIONS/INTERPRETATION: These data demonstrate the simultaneous presence of regulatory and effector T cells in the pancreas allograft of a recipient with type 1 diabetes. They also indicate that circulating islet autoreactive T cells may reflect immunological processes in pancreatic tissue, even though their frequency in the periphery may lead to underestimation of their presence in the pancreas. Additional specificities were also present in the pancreas that were undetectable in the circulation.

Allograft-specific cytokine profiles associate with clinical outcome after islet cell transplantation.

Islet cell transplantation can cure type 1 diabetes, but allograft rejection and recurrent autoimmunity may contribute to decreasing insulin independence over time. In this study we report the association of allograft-specific proliferative and cytokine profiles with clinical outcome. Peripheral blood mononuclear cells were obtained of 20 islet recipients. Cytokine values in mixed lymphocyte cultures (MLC) were determined using stimulator cells with graft-specific HLA class II. Qualitative and quantitative cytokine profiles were determined before and after islet transplantation, blinded from clinical outcome. Cytotoxic T Lymphocyte precursor (CTLp) assays were performed to determine HLA class I alloreactivity. Allograft-specific cytokine profiles were skewed toward a Th2 or regulatory (Treg) phenotype after transplantation in insulin-independent, but not in insulin-requiring recipients. IFNgamma/IL10 ratio and MLC proliferation decreased after transplantation in insulin-independent recipients (p = 0.006 and p = 0.01, respectively). Production of the Treg cytokine IL10 inversely correlated with proliferation in alloreactive MLC (p = 0.008) and CTLp (p = 0.005). Production of IL10 combined with low-MLC reactivity associated significantly with insulin independence. The significant correlation between allograft-specific cytokine profiles and clinical outcome may reflect the induction of immune regulation in successfully transplanted recipients. Islet donor-specific IL10 production correlates with low alloreactivity and superior islet function.

Relevance of cytotoxic alloreactivity under different immunosuppressive regimens in clinical islet cell transplantation.

Islet or beta cell transplantation provides a promising cure for type 1 diabetes patients, but insulin-independency decreases frequently over time. Immunosuppressive regimens are implemented attempting to cope with both auto- and alloimmunity after transplantation. We analysed the influence of different immunotherapies on autoreactive and alloreactive T cell patterns and transplant outcome. Patients receiving three different immunosuppressive regimens were analysed. All patients received anti-thymocyte globulin induction therapy. Twenty-one patients received tacrolimus-mycophenolate mofetil maintenance immunosuppression, whereas the other patients received tacrolimus-sirolimus (SIR, n = 5) or SIR only (n = 5). Cellular autoreactivity and alloreactivity (CTL precursor frequency) were measured ex vivo. Clinical outcome in the first 6 months after transplantation was correlated with immunological parameters. C-peptide levels were significantly different between the three groups studied (P = 0.01). We confirm that C-peptide production was correlated negatively with pretransplant cellular autoreactivity and low graft size (P = 0.001, P = 0.007 respectively). Combining all three therapies, cellular autoimmunity after transplantation was not associated with delayed insulin-independence or C-peptide production. In combined tacrolimus-SIR and SIR-treated patients, CTL alloreactivity was associated with less insulin independence and C-peptide production (P = 0.03). The percentage of donors to whom high CTLp frequencies were measured was lower in insulin-independent recipients (P = 0.03). In this cohort of islet cell graft recipients, clinical outcome in the first 6 months after transplantation correlates with the applied immunosuppressive regimen. An association exists between insulin-independence and lower incidence of CTL alloreactivity towards donor human leucocyte antigen. This observational study demonstrates the usefulness of monitoring T cell reactivity against islet allografts to correlate immune function with graft survival.

Immunological efficacy of heat shock protein 60 peptide DiaPep277 therapy in clinical type I diabetes.

An immunogenic peptide (p277) from the 60-kDa heat shock protein (hsp60) arrested beta-cell destruction in non-obese diabetic mice. A randomized, double-blind, phase Ib/II clinical trial of DiaPep277 peptide treatment was performed in recent-onset type 1 diabetes patients with remaining insulin production. We studied the immunological efficacy of this peptide therapy and correlated this with clinical outcome. Forty-eight C-peptide-positive patients were assigned subcutaneous injections of 0.2, 1.0 or 2.5 mg p277 (n = 12 per dosage) at entry, and 1, 6 and 12 months, or four placebo injections (n = 12). T cell autoimmunity to hsp60, DiaPep277, glutamic acid decarboxylase and tetanus toxoid (recall response control) were assayed by proliferation and cytokine secretion assays (enzyme-linked immunospot) at regular intervals until 18 months after the first injection. All treated patients at each dosage of peptide demonstrated an altered immune response to DiaPep277, while the majority of placebo-treated patients remained non-responsive to treatment (P = 0.00001), indicating a 100% efficacy of immunization. Cytokine production in response to therapy was dominated by interleukin (IL)-10. IL-10 production before therapy and decreasing autoantigen-specific T cell proliferation were associated with beta-cell preservation. Third-party control immune responses were unaffected by therapy. No potentially adverse immunological side effects were noted. DiaPep277 is immunogenic in type 1 diabetic subjects and has immune modulating properties. Immunological monitoring distinguished therapy from placebo treatment and could determine immunological efficacy. Declining or temporary proliferative responses to peptide DiaPep277 treatment may serve as an immunological biomarker for clinical efficacy.

Review: Converting nutritional knowledge into feeding practices: a case study comparing different protein feeding systems for dairy cows.

Improving milk nitrogen efficiency through a reduction of CP supply without detrimental effect on productivity requires usage of feeding systems estimating both the flows of digestible protein, the exported true proteins and from these predict milk protein yield (MPY). Five feeding systems were compared in their ability to predict MPY v. observed MPY in two studies where either protein supply or protein and energy supply were changed. The five feedings systems were: Cornell Net Carbohydrate and Protein System (v6.5.5), Dutch protein evaluation system (1991 and 2007), Institut National de la Recherche Agronomique in France (INRA), National Research Council and NorFor. The key characteristic of the systems with the best predicted MPY was the inclusion of a variable efficiency of utilisation of protein supply taking into account the supply of both protein and energy. The systems still using a fixed efficiency had the highest slope bias in their prediction of MPY. Therefore, the development of new feeding systems or improvement of existing systems should include a variable efficiency of utilisation of the protein related to both the protein and energy supply. The limitation of the current comparison did not allow determining if additional factors, as used in INRA, were beneficial. This concept should also probably be transferred to essential amino acids.

Imogen 38: a novel 38-kD islet mitochondrial autoantigen recognized by T cells from a newly diagnosed type 1 diabetic patient.

Cell-mediated autoimmune attack directed against islet proteins of approximately 38 kD in size has been associated with type 1 diabetes. A novel murine cDNA encoding an antigen of this size was cloned using a screening procedure based on the proliferative response of a human diabetic T cell clone (1C6) to a recombinant antigen epitope library. Membrane preparations from COS 7 cells transfected with the full-length 1,267-bp cDNA elicited a proliferative response from the reporter T cells comparable to that of the defined peptide epitope and native insulinoma antigen. In vitro translation and transfection experiments suggested that the protein is initially synthesized as a 44-kD protein and then processed to the native 38-kD form through the proteolytic removal of a 54-aa NH2-terminal mitochondrial targeting sequence. Differential centrifugation, Percoll density gradient centrifugation, and immunofluorescence studies confirmed localization of the antigen to mitochondria. Northern blot, Western blot, and 1C6 T cell proliferation assays showed that, although imogen 38 was more highly expressed in beta cell than alpha cell lines, it was also present in other tissues. It is concluded that imogen 38 may be a target for bystander autoimmune attack in diabetes rather than a primary autoantigen.

T-cell reactivity to beta-cell membrane antigens associated with beta-cell destruction in IDDM.

Insulin-dependent diabetes mellitus (IDDM) results from a T-cell-mediated destruction of the insulin-producing beta-cells. In this study, we designed a sensitive assay to detect and identify islet cell-reactive T-cells in patients with newly diagnosed IDDM. The relation between T-cell recognition of beta-cell antigens with IDDM and the pathogenesis of the disease (the beta-cell destruction process) was tested in a large group of IDDM patients and compared with T-cell responses in nondiabetic children with other chronic inflammations and in immunologically normal, age-matched control subjects. The results demonstrate that peripheral blood T-cells reacting with a beta-cell membrane preparation enriched for insulin-secretory granule antigen were detectable in the majority of newly diagnosed IDDM patients (27 of 40 [67%]; mean stimulation index [SI] 37.0). Such reactivity was reduced postonset in IDDM patients proportionally to the duration of the disease (11 of 30 [37%]; mean SI 8.7). Nondiabetic age-matched control subjects showed no responses or moderate responses to the granule preparation (4 of 48 [8%]; mean SI 3.4). The magnitude of the T-cell response was significantly greater in newly diagnosed IDDM patients than in IDDM patients tested at least 2 years postonset (P < 0.001). Two children in remission for insulin dependency (so-called honeymoon period) displayed exceptionally high proliferative responses to insulin-secretory granules (mean SI 86.7). These results imply that T-cell recognition of insulin-secretory granule antigens is associated with IDDM and in particular with the immune-mediated process of beta-cell destruction.

Auto- and alloimmune reactivity to human islet allografts transplanted into type 1 diabetic patients.

Allogeneic islet transplantation can restore an insulin-independent state in C-peptide-negative type 1 diabetic patients. We recently reported three cases of surviving islet allografts that were implanted in type 1 diabetic patients under maintenance immune suppression for a previous kidney graft. The present study compares islet graft-specific cellular auto- and alloreactivity in peripheral blood from those three recipients and from four patients with failing islet allografts measured over a period of 6 months after portal islet implantation. The three cases that remained C-peptide-positive for >1 year exhibited no signs of alloreactivity, and their autoreactivity to islet autoantigens was only marginally increased. In contrast, rapid failure (<3 weeks) in three other cases was accompanied by increases in precursor frequencies of graft-specific alloreactive T-cells; in one of them, the alloreactivity was preceded by a sharply increased autoreactivity to several islet autoantigens. One recipient had a delayed loss of islet graft function (33 weeks); he did not exhibit signs of graft-specific alloimmunity, but developed a delayed increase in autoreactivity. The parallel between metabolic outcome of human beta-cell allografts and cellular auto- and alloreactivity in peripheral blood suggests a causal relationship. The present study therefore demonstrates that T-cell reactivities in peripheral blood can be used to monitor immune mechanisms, which influence survival of beta-cell allografts in diabetic patients.

Molecular mimicry in type 1 diabetes mellitus revisited: T-cell clones to GAD65 peptides with sequence homology to Coxsackie or proinsulin peptides do not crossreact with homologous counterpart.

Type 1 diabetes mellitus is a T-cell mediated autoimmune disease in which the insulin-producing pancreatic beta cells are selectively destroyed. Molecular mimicry and T-cell crossreactivity to beta-cell autoantigens and environmental agents with sequence similarities have been a proposed mechanism underlying the pathogenesis of type 1 diabetes, but actual crossreactivity has not yet been demonstrated. We isolated and investigated T cells reactive to GAD65 peptides and homologous peptides of the Coxsackie virus protein P2C and proinsulin from recent onset type 1 diabetes patients, and tested their fine specificity and cytokine production profile. Six T-cell lines specific for GAD65 peptides (amino acids 491-530) with homology to proinsulin (B20-C14) were isolated from six newly diagnosed patients with type 1 diabetes, but none of the stable T-cell lines crossreacted to the homologous proinsulin peptides. Similarly, none of four T-cell lines reactive to GAD65 peptides (amino acids 247-280) with sequence homology to Coxsackie P2C (amino acids 30-50) crossreacted to the homologous viral peptide. Two T-cell lines corecognized a GAD65 peptide and a Coxsackie P2C peptide. However, the antigen-specific T-cell clones from these T-cell lines were reacting either with the GAD65 peptide or the Coxsackie P2C peptide using different restriction elements without crossreacting to the homologous peptide. Our data demonstrate that homologous peptides previously proposed to serve as targets for crossreactivity indeed are immunogenic. Yet, T-cell clones did not crossreact with linear sequence homologies, despite strong T-cell responses to individual peptides.

Relationship between chemical composition and in situ rumen degradation characteristics of maize silages in dairy cows.

Several in situ studies have been conducted on maize silages to determine the effect of individual factors such as maturity stage, chop length and ensiling of maize crop on the rumen degradation but the information on the relationship between chemical composition and in situ rumen degradation characteristics remains scarce. The objectives of this study were to determine and describe relationships between the chemical composition and the rumen degradation characteristics of dry matter (DM), organic matter (OM), CP, starch and aNDFom (NDF assayed with a heat stable amylase and expressed exclusive of residual ash) of maize silages. In all, 75 maize silage samples were selected, with a broad range in chemical composition and quality parameters. The samples were incubated in the rumen for 2, 4, 8, 16, 32, 72 and 336 h, using the nylon bag technique. Large range was found in the rumen degradable fractions of DM, OM, CP, starch and aNDFom because of the broad range in chemical composition and quality parameters. The new database with in situ rumen degradation characteristics of DM, OM, CP, starch and aNDFom of the maize silages was obtained under uniform experimental conditions; same cows, same incubation protocol and same chemical analysis procedures. Regression equations were developed with significant predictors (P<0.05) describing moderate and weak relationships between the chemical composition and the washout fraction, rumen undegradable fraction, potentially rumen degradable fraction, fractional degradation rate and effective rumen degradable fraction of DM, OM, CP, starch and aNDFom.

Effect of rumen-degradable protein balance and forage type on bulk milk urea concentration and emission of ammonia from dairy cow houses.

As the Dutch government and dairy farming sector have given priority to reducing ammonia emission, the effect of diet on the ammonia emission from dairy cow barns was studied. In addition, the usefulness of milk urea content as an indicator of emission reduction was evaluated. An experiment was carried out with a herd of 55 to 57 Holstein-Friesian dairy cows housed in a naturally ventilated barn with cubicles and a slatted floor. The experiment was designed as a 3 x 3 factorial trial and repeated 3 times. During the experiment, cows were confined to the barn (no grazing) and were fed ensiled forages and additional concentrates. The default forage was grass silage. The nutritional experimental factors were: (1) rumen-degradable protein balance of the ration for lactating cows with 3 levels (0, 500, and 1000 g/cow per d), and (2) proportion of corn silage in the forage ration for lactating cows with 3 levels (0, 50, and 100%) of forage dry matter intake. Several series of dynamic regression models were fitted. One of these models explained emission of ammonia by the nutritional factors and the temperature; another model explained ammonia emission by the bulk milk urea content and the temperature. The ammonia emission from the barn increased when levels of rumen-degradable protein balance increased. Furthermore, at a given level of rumen-degradable protein balance, the emission of ammonia correlated positively with the corn silage content in the forage ration. However, this correlation was not causal, but was the result of interaction between corn silage proportion and intake of ileal digestible protein. The bulk milk urea content and the temperature correlated strongly with the ammonia emission from the barn; the selected model accounted for 76% of the variance in emission. It was concluded that the emission of ammonia from naturally ventilated dairy cow barns was strongly influenced by diet. The emission can be reduced approximately 50% by reducing the rumen-degradable protein balance of the ration from 1000 to 0 g/cow per d. The milk urea content is a good indicator of emission reduction.

Cloned T cells from a recent onset IDDM patient reactive with insulin B-chain.

Insulin-dependent diabetes mellitus (IDDM) results from selective autoimmune destruction of insulin producing beta-cells. T-cell reactivity and autoantibodies to several islet proteins such as insulin, GAD and IA-2 are associated with IDDM in mice and men. In NOD mice, the majority of T cells from insulitis specifically recognize the insulin B-chain peptide amino acid 9-22, in contrast to the periphery where the precursor frequency is much lower. It is important to note that these cells are diabetogenic. Surprisingly, the same insulin B-chain region contains epitopes recognized by protective T cells. In fact, autoimmune diabetes in NOD mice could be prevented by prophylactic treatment with this immunodominant T-cell epitope. In humans, however, no immunodominant regions of insulin have yet been defined. We have isolated and characterized a human insulin-specific T-cell clone that was derived from peripheral blood of a newly diagnosed IDDM patient. This patient displayed weakly positive primary T-cell responses to insulin. The peptide recognized by the clone was mapped to the insulin B chain (B:11-27). Functionally, the human insulin-specific CD4+ T cells displayed a Th1/0 like cytokine profile and were restricted by HLA-DR. The previously proposed alternative superantigen-like binding of insulin-B chain peptide outside of the peptide binding groove of HLA-DR could not be confirmed, since T-cell recognition was inhibited in competition experiments of insulin-B chain peptide with HLA-DR16 binding influenza peptide HA307-319. Our results indicate that human clonal T cells isolated from a recent onset IDDM patient recognize an epitope overlapping with the insulin B-chain region that is immunodominant and potentially therapeutic in NOD mice. This observation may be useful in studying the role of insulin-specific T cells in IDDM, and may eventually help to establish peptide-based immunotherapies in IDDM.

HLA-associated inverse correlation between T cell and antibody responsiveness to islet autoantigen in recent-onset insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is a T cell-dependent immune-mediated disease. Recently, a novel islet cell antigen (ICA69) recognized by autoantibodies was described. We tested T cell responsiveness to ICA69 in peripheral blood of patients with recent onset IDDM (n = 46), patients with long-standing IDDM (n = 44), non-diabetic age-matched, islet cell autoantibody- and glutamic acid decarboxylase (GAD)65 antibody-negative first-degree relatives of IDDM patients (n = 15) and rheumatoid arthritis patients (n = 22). T cell responsiveness was significantly higher in recent onset IDDM patients, compared to IDDM patients post-disease onset, non-diabetic first degree relatives and rheumatoid arthritis patients (p < 0.001). In responding IDDM patients a significant inverse correlation between T cell and autoantibody responsiveness to ICA69 was observed (p < 0.0005). Immunogenetic evaluation revealed an association of HLA-DR3 with T cell responsiveness to ICA69 (p < 0.02) and absence of ICA69-reactive autoantibodies (p < 0.04). The increased T cell reactivity to ICA69 in the absence of antibody reactivity at onset of IDMM is associated with an HLA class II immune response gene, and therefore suggestive of a genetically controlled selective activation of T helper subsets to a specific autoantigen in humans.