Propagation and characterisation of dendritic cells from G-CSF mobilised peripheral blood monocytes and stem cells in common marmoset monkeys.

The common marmoset is a small New World Primate that has been used as an immunological model for a number of human diseases. Dendritic cells (DC) have not been extensively characterised in this species and in particular protocols to derive DC from living donors without the need for animal sacrifice are presently lacking. This study establishes new protocols to generate substantial numbers of marmoset DC for use in cell therapy studies. Recombinant human G-CSF was used to mobilise peripheral blood monocytes and CD34(+) stem cells in sufficient numbers for large scale in-vitro DC propagation using cytokine conditioning including IL-4, GM-CSF, FLT3-L, stem cell factor and thrombopoietin. Marmoset DC exhibited morphology similar to human DC, were capable of antigen uptake and presentation and had moderate allo-stimulatory ability. Monocyte-derived DC had a maturation-resistant immature phenotype, whereas haematopoietic precursor-derived DC were semi-mature in phenotype and function. This study confirms the feasibility of the marmoset as a unique small primate model in which to pursue DC-based immunotherapy strategies.

MHC Class II DRB genotyping is highly predictive of in-vitro alloreactivity in the common marmoset.

The common marmoset (Callithrix jacchus) is emerging as a promising alternative pre-clinical model for transplantation and immunological research. It is therefore important to establish a rapid and reliable method of confirming alloreactivity between donor-recipient pairs. In this study of a large marmoset colony (n=49), we firstly characterised MHC Class II genes (Caja-DRB*W1201, Caja-DRB1*03, Caja-DRB*W16) using, for the first time in this species, sequence-based allelic typing techniques. Exon 2 was amplified using M13-tailed PCR primers specific for known marmoset alleles, and sequenced using universal M13 sequencing primers and dye terminator cycle sequencing. Twenty-six genotypes involving monomorphic Caja-DRB*W1201, 8 Caja-DRB*W16 and 5 Caja-DRB1*03 alleles were observed. Two new DRB*W16 alleles were identified. Subsequently we investigated whether matching at MHC-DRB loci alone could accurately predict in-vitro alloreactivity as assessed by mixed lymphocyte reactions. Peripheral blood mononuclear cells (PBMC) isolated from fully and partially DRB-matched and fully mismatched animal pairs were mixed and co-cultured for T-cell proliferation. PBMC co-cultured from fully or partially mismatched pairs exhibited significant T cell proliferation above single cell controls (p<0.01). Mixed PBMC from fully DRB-matched pairs exhibited no proliferation over controls (p=0.3). Thus using Caja-DRB genotyping, suitably alloreactive donor-recipient pairs can be rapidly and accurately identified for use in further studies of cellular and solid organ transplantation.

Ethnicity and survival on dialysis in west London.

BACKGROUND: Indo-Asian and Afro-Caribbean patients have higher rates of renal failure and requirement for renal replacement therapy than the general population in the UK. Despite this, information regarding survival on dialysis is limited. METHODS: The incident hemodialysis population of a large west London renal service was reviewed from 1996 to 2001 (N = 465). RESULTS: The cohort's ethnic background was Indo-Asian (30.8%), Caucasian (49%), Afro-Caribbean (18.3%), and other (1.9%). Indo-Asians and Afro-Caribbeans were younger than Caucasian patients, with a higher rate of diabetes mellitus. Survival on hemodialysis for Indo-Asians was 97.5% and 81.6% at 1 and 3 years, respectively, compared with 92.7% and 75.2% for Caucasians, and 97.5% and 85.3% for Afro-Caribbeans (P = nonsignificant). Dialysis adequacy was observed to be associated with survival. Patients with mean single pool Kt/V of over 1.4 had survival of 90.6% and 74.8% at 2 and 5 years, respectively, compared with 74.0% and 42.9% for those with Kt/V less than 1.4 (P < 0.001). There were significantly more patients in the Indo-Asian cohort with a mean Kt/V of 1.4 or over (87.4%) compared with Caucasians (57.6%) and Afro-Caribbeans (52.4%), and the benefit of higher Kt/V was seen in all ethnic groups. In a multivariate analysis of factors including Kt/V over 1.4, age, diabetic status, gender, and ethnicity, Indo-Asian or Afro-Caribbean ethnicity did not confer a survival disadvantage. The strongest predictors of survival were age and dialysis adequacy. CONCLUSION: Indo-Asian and Afro-Caribbean hemodialysis patients have survival comparable to Caucasians despite a higher burden of diabetes.

Impact of hemodialysis on endogenous plasma and muscle carnitine levels in patients with end-stage renal disease.

BACKGROUND: End-stage renal disease (ESRD) patients undergoing hemodialysis treatment have reduced plasma L-carnitine levels; however, the relationship between dialysis age and carnitine status is poorly understood. This study examined the relationship between duration of dialysis and plasma and skeletal muscle concentrations of L-carnitine and its esters in ESRD patients. METHODS: Blood samples were collected from 21 patients at baseline and throughout the first 12 months of hemodialysis. In 5 patients, muscle samples were obtained after 0, 6, and 12 months of hemodialysis. Blood and muscle samples were collected from an additional 20 patients with a mean dialysis age of 5.10 years. L-carnitine, acetyl-L-carnitine, and total L-carnitine were measured by high-performance liquid chromatography (HPLC). RESULTS: The mean +/- SD plasma L-carnitine concentration in ESRD patients who had not yet started hemodialysis was 50.6 +/- 20.0 micromol/L. Significantly lower concentrations were observed after 12 months (29.7 +/- 10.5 micromol/L) and >12 months (22.0 +/- 5.4 micromol/L) of hemodialysis treatment. Acetyl-L-carnitine also declined with dialysis age, while plasma nonacetylated acylcarnitines continued to increase with the progression of hemodialysis therapy. An inverse relationship between dialysis age and muscle L-carnitine concentrations was observed. CONCLUSION: Long-term hemodialysis treatment is associated with a significant reduction in endogenous plasma and muscle L-carnitine levels and a significant increase in plasma acylcarnitines. The majority of the change in plasma L-carnitine concentrations occurs within the first few months of hemodialysis, while muscle levels continue to decline after 12 months of treatment.

Outcome of renal transplantation in South Asian recipients is similar to that in non-Asians.

BACKGROUND: The United Kingdom has a large South Asian population, in which there is a high rate of renal disease and which forms a significant percentage of the renal transplant waiting list. Information about short- and long-term transplant outcomes in this ethnic group is limited, although it has been suggested that graft survival is poorer in this population compared with non-Asians. METHODS: The authors examined the outcome and determinants of medium-term (5-year) survival in 245 renal transplants, 53 of which were performed in South Asian patients between 1995 and 2002. RESULTS: Three-year survival with a functioning graft was 89% for the non-Asians and 85% for the South Asians. At 5 years, this deviated to 83% and 70%, respectively, for the two groups, but this did not reach statistical significance. Acute rejection rates were similar in the two groups. South Asian ethnicity was not a significant predictor of medium-term graft loss in the authors' study. CONCLUSIONS: In this cohort of patients, South Asian ethnic background did not confer a survival disadvantage after renal transplantation.