A randomised phase IIb trial to assess the efficacy of ReCharge ice cream in preventing chemotherapy-induced diarrhoea.

PURPOSE: Chemotherapy-induced diarrhoea (CID) has a significant impact. A medicinal food product (ReCharge) containing iron-saturated lactoferrin and anhydrous milk fat reduces the detrimental effects of chemotherapy on the gut in animals. We report results of a randomised blinded placebo-controlled phase IIb trial investigating the efficacy and safety of ReCharge in preventing CID. METHODS: Eligible patients were adults due to start the first cycle of a 2- or 3-week-cycle chemotherapy regimen, had an Eastern Cooperative Oncology Group (ECOG) status of 3 or less, had adequate haematological, liver and renal function and provided written informed consent. Patients (197) were randomised to ReCharge or placebo. They consumed 100-g study product for 2 weeks before and 6 weeks after starting chemotherapy, completed daily diaries for 8 weeks and attended clinic visits until 12 weeks (2-week cycles) or 14 weeks (3-week cycles). The primary outcome was days with CID. RESULTS: The mean number of days with diary-recorded CID was marginally but not statistically significantly lower on ReCharge than placebo (-2.0, 95 % CI (-4.7 to 0.7), p = 0.2). The proportion reporting diarrhoea in the previous cycle at the clinic visit was 30 % lower (p = 0.012) on ReCharge. Missing diary data may have contributed to the discrepancy. No significant differences were found in quality of life or other adverse events. CONCLUSIONS: We found no clear evidence that ReCharge reduced CID as measured by patient self-report diary. The converse finding of benefit as recorded at clinic visits and incomplete adherence to diary completion indicates that further research is required into methods for measuring CID.

TCRBV14S1 and rheumatoid arthritis revisited: abnormalities in the percentage of transcribed TCRBV14S1 family genes in PBMC from rheumatoid arthritis patients.

The purpose of this study was to identify Vbeta gene families that are associated with rheumatoid arthritis (RA). A PCR-based assay was used to compare the Vbeta repertoire of unstimulated PBMC from 18 RA patients and 18 matched controls. The influence of an HLA-DRB1-binding peptide (HA307-319) on the Vbeta repertoire of PBMC in culture was compared in 11 RA patients and 10 controls. There was a larger variance in the percentage of BV14S1 transcripts in unstimulated PBMC from RA patients than from controls (p = 0.0003). The mean percentage of BV14S1 transcripts was higher in RA patients when prednisone-treated RA patients were excluded from the analysis (p = 0.0006). In vitro stimulation with the HA307-319 peptide increased the percentage of BV14S1 transcripts in PBMC from RA patients (+ 1.5 +/- 0.4%, p < 0.005) but not controls (+ 0.3 +/- 0.2%, ns), and the difference between RA patients and controls was significant (p = 0.03). In conclusion, there is an association between RA and the BV14S1 gene family in New Zealand patients.

Repertoire of transcribed peripheral blood T-cell receptor beta chain variable-region genes in acute rheumatic fever.

Patients with severe group A streptococcal infections have abnormalities in the Vbeta repertoire of peripheral blood T cells that are consistent with superantigen stimulation by cytoplasmic membrane proteins. The purpose of this study was to determine whether similar changes in Vbeta repertoire could be found for patients with acute rheumatic fever (ARF). The mean Vbeta repertoire of peripheral blood T cells in nine hospitalized ARF patients was similar to that of 34 controls and did not change during 6 months of follow-up in 6 of the ARF subjects. We were unable to detect changes in the Vbeta repertoire of peripheral blood T cells from patients with ARF that could be attributed to the influence of a superantigen.

The influence of a recombinant-HBsAg vaccine on the repertoire of transcribed T-cell receptor beta chain variable region genes in peripheral blood in humans.

The purpose of this study was to determine whether in vivo changes in the repertoire of Tcr beta chain variable region (V beta) genes could be detected in peripheral blood mononuclear cells after immunization of humans with recombinant hepatitis B virus envelope protein (rHBsAg). We measured the percentage of Tcr RNA transcripts carrying each of 20 V beta genes in human PBMC before and after immunization with rHBsAg in Polynesians (8 non-immunized controls, 26 immunized subjects) and Europeans (9 non-immunized controls, 11 immunized subjects). The per cent of RNA transcripts containing V beta 7.4 family genes was increased in immunized vs control Polynesian (+ 1.6 +/- 0.5%) vs -1.1 +/- 0.3%, P = 0.0002) and European (+1.6 +/- 0.6% vs -0.1 +/- 0.5%, P = 0.05) subjects at 48 h and 28 h post-immunization, respectively. No changes in V beta repertoire were found after 48 h in either race. Thus, there is a transient increase in frequency of T cells with Tcr containing V beta 7.4 family genes within 48 h of an immunization containing rHBsAg in humans. There are a number of explanations for this finding, including the possibility that V beta 7.4 gene family products may be preferentially involved in the primary immune response to HBsAg.

The influence of a maternal chronic hepatitis B virus infection on the repertoire of transcribed T-cell receptor beta chain variable region genes in human cord blood.

We used an anchor polymerase chain reaction method to compare the repertoires of transcribed T-cell receptor beta chain variable region (V beta) genes in cord blood T cells from neonates of hepatitis B surface antigen (HBsAg) positive (n = 40) and HBsAg negative (n = 40) women. Fifteen of the HBsAg positive women were hepatitis B e antigen (HBeAg) positive, and 25 were HBeAg negative. The percentage of V beta 7.4 transcripts was lower in cord blood T cells from neonates of HBsAg-positive relative to HBsAg-negative women (9.7% +/- 0.5% vs. 12.7% +/- 0.6%, P = .002). The percent of V beta 5.1 transcripts was higher in cord blood T cells from neonates of HBeAg-positive relative to HBeAg-negative women (9.3% +/- 0.7% vs. 7.0% +/- 0.3%, P < .001). There were no correlations between neonatal maturity at birth and V beta repertoire. In summary, a maternal chronic hepatitis B virus (HBV) infection is associated with changes in the repertoire of transcribed T-cell receptor genes in neonatal cord blood T cells. It is possible that the T-cell response to the HBV is associated with a limited repertoire of V beta genes. The mechanism of vertical chronic HBV infection in human neonates may involve changes in the T-cell response to the virus that are induced in utero.

Peripheral blood T lymphocytes in systemic vasculitis: increased T cell receptor V beta 2 gene usage in microscopic polyarteritis.

Antigen recognition by T lymphocytes is mediated by cell surface receptors T cell specificity depends on the variable, diversity and junctional (VDJ) regions of the alpha and beta polypeptide chains of the T cell receptor (TCR). The expression of the variable region genes of the beta chain (V beta) has been analysed to study the involvement of peripheral blood T cells in systemic vasculitis. RNA was extracted from peripheral blood lymphocytes of 12 patients with microscopic polyarteritis, 10 with Wegener's granulomatosis, six with unclassified vasculitis, and 28 healthy age- and sex-matched individuals. Complementary DNA was made from RNA and amplified by the anchored polymerase chain reaction (PCR) using redundant oligonucleotide primers for the TCR V beta genes. To determine if the dominant usage of a V beta gene family reflected the presence of particular T cell clones, cDNA was amplified with primers for the specific V beta gene family. The product was screened for sequence homogeneity by single-stranded conformational polymorphism (SSCP) and cloned to sequence the adjoining TCR (D beta) J beta region. A significant increase in the mean percentage expression of the V beta 2.1 gene was seen in vasculitis patients (11.4 + 1.0% (mean + s.e.m.)) compared with controls (6.6 + 0.6%; P < 0.003). The most marked increase was seen in microscopic polyarteritis (13.9 + 1.7%; P < 0.0001). There were also increases in the expression of V beta 3, 13 and 14 in peripheral blood of vasculitis patients compared with controls. SSCP analysis of V beta 2.1 amplified products indicated the presence of oligoclonal bands in a smaller proportion of patients (8/27) than controls (12/28). There was no strong evidence for the conservation of the TCR V beta 2.1 junctional region sequence data from a sample group of three patients with oligoclonal bands. Thus, a subset of patients with systemic vasculitis, particularly those with microscopic polyarteritis, have increased TCR V beta 2.1 gene expression in their peripheral blood T cell repertoire. As superantigens binding V beta 2.1 are postulated to activate T cells with diverse CDR3 sequences, it is proposed that a superantigen is involved in the immunopathogenesis of vasculitis.

Search for linkage disequilibrium between alleles in the T cell receptor alpha and beta chain loci and susceptibility to rheumatic fever.

A population-based linkage disequilibrium study was conducted to search for associations between alleles in T cell receptor alpha and beta chain polymorphic loci and susceptibility to rheumatic fever. The allele frequencies of four T cell receptor locus restriction fragment length polymorphisms were measured in 47 European and 51 Maori subjects with a history of rheumatic fever. These allele frequencies were compared to the allele frequencies in three or four independently recruited, race-matched control groups totalling 125 Europeans and 117 Maoris with no history of rheumatic fever. The polymorphisms studied were (locus/enzyme/probe) C alpha/Taq1/Y14, V alpha/Taq1/Y14, V beta 7/BAMHI/V beta 7.4 and V beta 8/BAMHI/V beta 8.1. There was no evidence for linkage disequilibrium between rheumatic fever and these Tcr alleles in either the Maori or European subjects.

Lymphocyte responses to DR1/4 restricted peptides in rheumatoid arthritis.

OBJECTIVE: To determine whether analog and unrelated DR1/4 binding peptides alter DR1/4 restricted responses of peripheral blood lymphocytes (PBL) from patients with rheumatoid arthritis (RA). METHODS: PBL from 25 patients with RA and 12 healthy controls were cultured with DR1/4 restricted peptides of the influenza haemagglutinin, amino acids 307-319 (HA) and matrix proteins, amino acids 17-29 (IM). Responses were determined by 3H-thymidine uptake proliferation assays and limiting dilution analysis. Competitor peptides were analogs HA-R312 and HA-K313 differing from HA by one amino acid at the 312 or 313 position respectively or unrelated peptides which bind to DR1/4. RESULTS: The responses of eight patients with RA to the two stimulatory influenza peptides did not differ significantly from controls and this was confirmed by the frequency estimate of T cells in PBL which responded to HA (mean frequency: 1 in 9.0 x 10(4), n = 5, in DR1/4+ RA patients, 1 in 7.6 x 10(4), n = 5, in DR1/4+ healthy controls). DR1/4 binding analogs of the HA peptide inhibited HA specific peptide responses of PBL from patients with RA and controls. Inhibition was also detected with unrelated peptides which bind to DR1/4 but to which the individual did not respond. CONCLUSION: Similar responses to two DR1/4 restricted peptides were observed in patients with RA and controls. Both antigen analog- and unrelated peptide-major histocompatibility complexes (MHC) can result in the inhibition of antigen specific responses in multi-clonal human lymphocyte populations. However, an analog peptide may be stimulatory in some individuals. These results provide some initial data for the development of a rational approach to MHC-specific immunomodulation in rheumatoid arthritis.

T cell receptor polymorphisms in Caucasians and Polynesians.

The purpose of this study was to find genetic polymorphisms that might be useful in studies of Polynesian-Caucasian racial admixture and Polynesian disease susceptibility. The allele frequencies of six T cell receptor locus RFLP were measured in 73 Caucasians and two Polynesian ethnic groups comprising 86 Maoris and 95 Samoans. The RFLP studied were (locus/enzyme/probe): C alpha/Taq1/Y14, V alpha/Taq1/Y14, C beta/BglII/Y35, C gamma/Pvu II/HGP02, V beta 7/BamHI/V beta 7.4 and V beta 8/Bam HI/V beta 8.1. Racial differences in allele frequency were present with all six RFLP (P < 0.001). The allele frequencies of the V alpha/Taq1/Y14 and the V beta 7/BamHI/7.4 RFLP were similar in the two Polynesian groups, both of which differed from the Caucasians. The 1.4 kb allele of the V alpha/Taq1/Y14 RFLP and the 8.0 kb allele of the V beta 7/BamHI/7.4 RFLP were present in low frequency in both Polynesian groups compared to the Caucasian group, consistent with a gene flow effect. These alleles may be useful in studies of Caucasian-Polynesian racial admixture.

Population study of T cell receptor V beta gene usage in peripheral blood lymphocytes: differences in ethnic groups.

The T cell receptor (TCR) V beta repertoire in peripheral blood lymphocytes (PBL) of a large number of healthy individuals was analysed by quantifying V beta-specific mRNA using the method of anchored multiprimer DNA amplification and a reverse dot blot assay. Among 16 V beta gene families examined, particular V beta genes were noted to be unequally expressed in the PBL of 70 healthy donors. The frequently used genes belong to the V beta 4, 5, 6, 8 and 13 (12) families, while V beta 1, 9 and 15 were the least frequently used gene families. This bias in gene usage was observed in all individuals. Marked deviation from the mean percentage usage was noted for some V beta genes in individuals when their PBL were examined serially, but the common pattern of biased usage was not grossly distorted. When the TCR repertoire of different ethnic groups was examined, a lower mean frequency of V beta 3.2 was seen in the repertoire of 19 Caucasians compared with 25 age-matched Samoans (P < 0.003). Conversely, the expression of V beta 5.1 and V beta 5.3 was higher in Caucasians than in 51 age-matched Polynesians (Maoris and Samoans, P < 0.003). Considering the 20% co-efficient of variation in the estimate of V beta gene usage, our data from 70 unrelated individuals suggest that in PBL, individual variations in the TCR repertoire were superimposed upon a common biased usage of V beta genes in the general population.

HLA-DR4 subtypes in New Zealand Polynesians. Predominance of Dw13 in the healthy population and association of Dw15 with rheumatoid arthritis.

OBJECTIVE: To analyze HLA-DR4 alleles in New Zealand Polynesians with rheumatoid arthritis (RA). METHODS: Thirty Polynesians and 30 Caucasians with RA, as well as 65 Polynesian and 60 Caucasian healthy blood donors, were DR4 subtyped using the polymerase chain reaction and sequence-specific oligonucleotide probes. RESULTS: The frequency of DR4 (DRB1*04) was increased in both Polynesian (P < 0.001) and Caucasian (P < 0.005) RA patients compared with race-matched controls. Dw4 (DRB1*0401) was detected in 15 of 30 Caucasian patients but only 2 of 30 Polynesian patients (P < 0.001). In Polynesians, RA was associated with Dw15 (DRB1*0405), which was present in 11 of 30 patients and 3 of 65 controls (P < 0.001). Dw13 (DRB1*0403) was the most frequent DR4 allele in healthy Polynesians, but was not significantly associated with RA. CONCLUSION: The predominance of the Dw13 subtype in Polynesians may explain in part the low prevalence of RA in this population. The association of Dw15 with RA in Polynesians supports the hypothesis that the third hypervariable region of DR beta determines susceptibility to RA.

T cell receptor alpha-chain polymorphic allele frequencies in Caucasians and Polynesians.

Restriction length polymorphisms in the variable and constant regions of the T cell receptor alpha-chain were examined in 42 Caucasians, 29 Maoris and 27 Pacific Islanders. Southern blots of Taq I digested DNA were hybridized with the T cell receptor alpha-chain probe pY14. Our results confirm that a 1.4 kb T cell receptor alpha chain-Taq 1 band is allelic to a 0.5 kb band. A significant difference in the frequency of the 1.4 and 0.5 kb alleles of the variable region of the alpha-chain was detected in Caucasians when compared with Maoris or Pacific Islanders (P < 0.0001). No differences in the frequency of the 2.0 and 7.0 kb alleles of the constant region gene were detected between any of the racial groups. These data may be relevant to ethnic differences in susceptibility to immune disorders.

Effect of extended lactation and diet on transferrin concentrations in rat milk.

Milk transferrin in the rat is immunochemically identical to serum transferrin. Its concentration in milk during normal lactation (10 pups for 21 d) varies biphasically, decreasing from a value of 1.5 mg/ml in colostrum to barely detectable values at d 4 and 8 of lactation, and thereafter increasing to reach values of 4 mg/ml at d 21. The effect of extended lactation on transferrin in milk was investigated in two experiments in which litters were replaced by 4-d-old litters at d 8 and 12 of lactation or at d 20 of lactation. Transferrin concentrations in milk in both experiments increased in a similar manner to reach values of 10 mg/ml at d 28 through d 36 of lactation. Serum transferrin and serum insulin and prolactin concentrations were not significantly altered in these experiments. Premature exposure of dams to older pups did not affect the pattern of milk transferrin concentrations. Milk transferrin concentrations were, however, modulated by altering the milk demand (changing litter sizes) and by restricting either the total food intake or the protein content of the diet. These restrictions led to lower transferrin concentrations.

Nutritional regulation of milk protein messenger RNA concentrations in mammary acini isolated from lactating rats.

The effect of starvation, starvation and refeeding and feeding a low-protein diet on concentrations of the mRNAs for the alpha-, beta- and gamma-caseins, alpha-lactalbumin and whey acidic protein has been determined using dot-blot hybridization analyses with total RNA extracted from mammary acini isolated from lactating rats. Starvation for 48 h decreased the concentrations of all RNA species to between 5 and 20% of those for rats feeding ad libitum when expressed on a DNA basis. Refeeding for 24h restored the concentrations to control values. Consumption of a low protein diet reduced the concentrations of each mRNA by about 50%. Only minor changes were detected in the mRNA concentrations for alpha-casein, alpha-lactalbumin and whey acidic protein in samples prepared at six hourly intervals from rats receiving a restricted intake (40% ad libitum) of the control diet.

Serum albumin secretion in rat milk.

1. The concentration of serum albumin in rat milk, rat serum, the stomach contents of suckling rats and mammary homogenates has been measured. 2. Serum albumin in expressed milk ranges in concentration between 3.5 and 6 mg/ml and amounts to an average of 4-5 mg/100 mg total protein whereas, in the stomach contents of suckling rats, serum albumin was 3.2 mg/100 mg total protein. 3. Corresponding concentrations of a second soluble milk protein, alpha-lactalbumin, were 2.9 mg/100 mg total protein in the milk and 1.2 mg/100 mg total protein in the stomach contents. 4. Serum albumin is a major protein in mammary homogenates at 10 mg/100 mg total soluble protein and the calculated total mammary pool of albumin amounts to 110 mg/rat. 5. There was a significant negative correlation between the serum albumin concentration and the ratio of potassium to sodium ions in milk samples obtained from rats milked with varying oxytocin treatments. 6. No evidence for albumin mRNA could be detected when mammary total RNA was probed with a complementary DNA (cDNA) for rat albumin. 7. The data are consistent with albumin being a major whey protein in the rat, it being synthesized at an extramammary site and transferred to the milk space by a paracellular mechanism from an extravascular mammary pool rather than directly from the serum.

Effect of dietary protein and food restriction on milk production and composition, maternal tissues and enzymes in lactating rats.

Lactating rats have been fed either a protein-restricted diet (10 vs. 20% casein in the control diet) or the control diet at 80, 60 and 40% of the voluntary intake for 7 d from d 7 of lactation. Food consumption, changes in maternal live weight, litter live weight gain and the mass of several maternal tissues were determined together with the activity of several mammary and liver enzymes, including 10 that are essential for fatty acid and complex lipid synthesis. Milk production was estimated from the litter weight gain and litter weight. Lactating rats fed the 20% protein diet ad libitum consumed three times that of nonlactating rats; their liver and kidney masses were significantly higher and their adipose mass was lower. The livers of the lactating rats were fatty, containing 118 mg lipid/g compared with 42 mg/g for the nonlactating rats. Lactating rats fed either the protein-restricted diet or the control diet at 40 and 60% of the ad libitum intake of the control diet had lower mammary, liver and kidney masses than rats consuming the control diet ad libitum. Both protein and food restriction led to lower rates of milk production than those of ad libitum-fed control rats as evidenced by the decrease in litter live weight gains. The concentrations of total lipid, total protein and lactose in milk were not affected by these dietary treatments. The concentration of alpha-lactalbumin in milk of rats fed the low protein diet was, however, lower than that in the milk of all rats receiving the control diet, irrespective of intake. Consumption of the restricted diets resulted in only small changes in specific activities (mu/mg protein) of 15 mammary enzymes. In the livers, lactation led to higher specific activities of all four soluble lipogenic enzymes examined but did not affect the particulate enzymes involved in complex lipid synthesis. The dietary restrictions resulted in lower specific activities of the soluble enzymes compared with those of the lactating rats consuming the control diet ad libitum without affecting the particulate enzymes. Total activities of these enzymes were, however, lower than those for the control rats as a result of the smaller liver mass in the rats receiving the restricted diets.

Protein synthesis in mammary acini isolated from lactating rats: effect of maternal diet.

Protein synthesis in the rat mammary gland has been studied using acini isolated from mammary tissue by collagenase digestion. When the acini were incubated with radioactively labeled amino acids, both cellular and milk proteins were synthesized and milk proteins were secreted into the incubation medium. Antisera to the lipogenic enzyme, fatty acid synthase, and the milk proteins, alpha-lactalbumin and the caseins, raised in rabbits, were shown to be specific by analyzing immunoprecipitates on sodium dodecyl sulfate--polyacrylamide gels. The rates of synthesis and secretion of each protein by acini prepared from rats during late gestation and at specific stages of lactation reflect their previously observed concentration in the mammary gland or milk of rats at the corresponding stage of gestation or lactation. Rats were treated according to one of the following regimes between d 7 and 14 of lactation: they were fed a control (20% casein) or a low protein (10% casein) diet ad libitum, they were fed the control diet restricted to 25 g/d (40% of the voluntary intake), they were fed the control diet for 5 d and starved for 48 h or they were treated as in 3 and then refed the control diet ad libitum for 24 h. Food restriction and starvation both resulted in lowered rates of synthesis of all proteins examined compared with either the control or refed animals. Starvation also lowered the rates of secretion of the milk proteins. Consumption of the low protein diet caused a specific decrease in both the rates of synthesis and secretion of alpha-lactalbumin compared with the control rats without affecting the synthesis and secretion of the caseins.

Milk composition of rats feeding restricted litters.

Milk samples were taken from rats feeding ten pups and from both the suckled and non-suckled glands of rats feeding two pups. The lipid, protein and lactose concentrations were similar in the milks from the secreting glands, but the fluid from the non-suckled glands contained less lactose and lipid but significantly higher total protein and transferrin concentrations. The fatty acid compositions of the milk from the three sources were very similar. The mammary tissue from the rats feeding ten pups had a higher DNA content/g wet wt. than either the suckled or non-suckled mammary tissue of the rats feeding two pups. The specific activities of several lipogenic enzymes were significantly lower in the non-suckled mammary tissue.

Effect of changes in litter size at mid-lactation on lactation in rats.

The effect of altering the number of pups per litter from ten to two or two to ten at day 14 of lactation in rats was investigated. Reducing litter size had no effect on the daily live weight gain whereas increasing the litter size resulted in an initial weight loss followed by an impaired weight gain. Maternal food consumption decreased to values appropriate for rats feeding two pups within 2 days after litter reduction, but required at least 5 days after the increase in litter size to increase to values appropriate for rats feeding large litters from parturition. Altering the litter size resulted in maternal serum insulin concentrations that were intermediate between those expected for rats feeding two or ten pups from parturition. Maternal serum prolactin concentrations increased after litter sizes were increased and decreased initially after litter reduction before being restored to normal levels. The activities of the lipogenic enzymes, fatty acid synthase, glucose-6-phosphate dehydrogenase and 'malic' enzyme in the mammary gland all decreased within 72 h of litter reduction to levels appropriate for rats feeding two pups. Although all three enzymes increased in activity after litter size increase, only fatty acid synthase had reached values appropriate for rats feeding ten pups by 72 h.