Low incidence of human herpesvirus 8 in stem cell collections from myeloma patients.

Human herpesvirus 8 is a gammaherpesvirus which may be implicated in the pathogenesis of multiple myeloma. Viral DNA sequences have been found in the bone marrow, peripheral blood and leukapheresis products of myeloma patients. These findings have significant implications for the use of leukapheresed cells in the transplantation and immunotherapy of myeloma. The studies suggest the cell which harbours the virus may be dendritic in origin. We have previously reported that dendritic cells cultured for use in the clinical setting do not harbour HHV-8. In this study, we examined the leukapheresis products of a larger cohort of myeloma patients for the presence of HHV-8 using a highly sensitive PCR technique. A strong association between HHV-8 and myeloma was not confirmed, with only 4% of the patient samples positive for viral sequences. While further study is needed, the current use of apheresis cells and their cultured progeny in the treatment of myeloma should not be compromised.

Interleukin-1 cluster and tumor necrosis factor-alpha gene polymorphisms in polymyalgia rheumatica.

OBJECTIVE: To investigate whether polymorphisms in the interleukin (IL)-1 locus (human chrom. 2q13) and TNF-alpha gene are associated with susceptibility to or severity of polymyalgia rheumatica (PMR). METHODS: The study included 92 consecutive PMR patients diagnosed over a 5-year period who were prospectively followed-up for at least one year and 79 healthy controls over the age of 50 residing in the same area. All the patients and controls were Caucasians of Italian origin. We tested the allelic distribution of IL-1A (+4845), IL-B (-511), IL-B (+3954), IL-1RN Intron 2 VNTR and TNFA (-308). Frequencies were compared in the patient and control groups. RESULTS: A statistically significant association between PMR patients and the IL1RN*2 allele in the homozygous state was found [OR 8.46 (95% CI 1.05-68.31)]. The polymorphisms in the other genes of the IL-1 gene cluster did not reveal any association with PMR when compared with controls. A weak association between PMR patients and the TNF2 allele was also present [OR 2.09 (95% CI 1.0-4.17)]. None of the gene variants studied was associated with the disease severity of PMR. CONCLUSION: Our findings show that IL1RN*2 allele, particularly in the homozygous state, is associated with susceptibility to, but not with the severity of, PMR.

Absence of the association with CC chemokine receptor 5 polymorphism in polymyalgia rheumatica.

OBJECTIVE: Elevated RANTES serum levels are present in polymyalgia rheumatica (PMR) patients with active disease. Chemokines may contribute to the inflammatory PMR process through their binding to CC chemokine receptor 5 (CCR5). The aim of this study was to examine if the 32 base pair deletion allele in CCR5 (CCR5 delta 32 allele) might be associated with PMR susceptibility and influence the disease outcome. METHODS: We enrolled 88 consecutive patients with PMR residing in the Reggio Emilia area (Italy) who had a follow-up duration of at least one year. As a control group we used 86 healthy blood donors from the same geographic area. The CCR5 genotype of all PMR patients and controls was studied by polymerase chain reaction amplification of the region which includes the 32 deletion (CCR5 delta 32). RANTES serum levels were measured by commercial ELISA kits in CCR5 delta 32 heterozygous and CCR5 homozygous PMR patients at diagnosis before starting corticosteroid therapy and again after 6 months of therapy, as well as in 28 healthy subjects over 50 years of age. RESULTS: Frequencies of the CCR5 and CCR5 delta 32 alleles in patients and controls did not differ significantly. Homozygosity for CCR5 delta 32 was not detected in PMR patients and was detected in only one of the controls. No significant differences were observed between the patients carrying the CCR5 delta 32 allele and those homozygous for the normal CCR5 allele when we compared sex, presence of distal synovitis and systemic signs and/or symptoms, initial and cumulative prednisone dose, duration of therapy, ESR at diagnosis, frequency of relapse/recurrence and RANTES serum levels at diagnosis and after 6 months of corticosteroids. CONCLUSION: These results indicate that the frequency of the 32 deletion of the CCR5 receptor was not significantly different between PMR patients and healthy controls, and this genotype does not appear to be associated with the susceptibility to or severity of PMR.

The influence of interleukin gene polymorphism on expression of interleukin-1beta and tumor necrosis factor-alpha in periodontal tissue and gingival crevicular fluid.

BACKGROUND: A specific composite genotype of the polymorphic interleukin-1 (IL-1) gene cluster has recently been associated with severe periodontitis. One polymorphism of the composite periodontitis-associated genotype (PAG) has been functionally linked with expression of high levels of IL-1. The purpose of this study was to test whether gingival crevicular fluid (GCF) levels of IL-1beta and tumor necrosis factor-alpha (TNFalpha), and gingival tissue levels of IL-1alpha, IL-1beta, and TNFalpha correlate with PAG, and to examine the effect of conservative periodontal therapy on these levels. METHODS: Twenty-two adults with moderate to advanced periodontal disease were enrolled. Polymerase chain reaction amplification and restriction enzymes were used to identify specific polymorphisms from peripheral blood samples. GCF samples were collected at baseline and 3 weeks following conservative treatment and analyzed by ELISA for IL-1beta and TNFalpha. An interproximal gingival biopsy was collected at baseline and follow-up and analyzed for IL-1alpha, IL-1beta, and TNFalpha by ELISA. RESULTS: The genotyping identified 7 as PAG(+) and 15 as PAG(-). The 2 groups were comparable in terms of existing periodontitis and age. In shallow sites (<4 mm), total IL-1beta in GCF was 2.5 times higher for PAG(+) patients prior to treatment (P=0.03), and 2.2 times higher after treatment (P=0.04), while differences were less apparent in deeper sites. Following treatment, a reduction in IL-1beta concentration in GCF was seen for PAG(-) but not for PAG(+) patients. While not statistically significant, a trend was observed in mean tissue levels of IL-1beta which were 3.6 times higher in PAG(+) versus PAG(-) patients (P=0.09). CONCLUSIONS: These data suggest that PAG(+) patients may demonstrate phenotypic differences as indicated by elevated levels of IL-1beta in GCF.

Haplotype-specific gene expression profiles in a telomeric major histocompatibility complex gene cluster and susceptibility to autoimmune diseases.

The telomeric class III region of the major histocompatibility complex is gene dense, but apart from the three tumour necrosis factor (TNF) superfamily members (TNF, lymphotoxin alpha and lymphotoxin beta) little is known of the expression and function of the majority of the genes. Recent genetic studies in autoimmune diseases, particularly rheumatoid arthritis (RA), have suggested a human leukocyte antigen (HLA)-DR-independent disease effect in this region. To gain further insights into these associations, we used lipopolysaccharide-stimulated human macrophages to examine inducible mRNA expression and genotype-phenotype relationships for genes in this region. Following stimulation in addition to the expected induction of TNF mRNA, a 14-fold increase of ATP6V1G2 at 18 h (P<0.001) was seen, whereas B-associated transcript (BAT)2 (P<0.001) and leucocyte-specific transcript (LST)1 (P<0.001) were both downregulated. By genotyping single-nucleotide polymorphisms spanning a 70 kb interval centred on the TNF locus, we constructed haplotypes and determined associated expression profiles for 10 genes in the cluster using quantitative real-time polymerase chain reaction. Overexpression of BAT1 mRNA was associated with carriers of a haplotype containing the LST1 marker transmitted to RA cases in a family study and also DRB1(*)15 associated with susceptibility to nephritis in systemic lupus erythematosus. The implications of our findings for the understanding of genetic associations with disease susceptibility in this region are discussed.

Identification of novel single nucleotide polymorphisms within the NOTCH4 gene and determination of association with MHC alleles.

Mapping of disease susceptibility loci within the MHC has been partly hampered by the high degree of polymorphism of the HLA genes and the high level of linkage disequilibrium (LD) between markers within the MHC region. It is therefore important to identify new markers and determine the level of LD between HLA alleles and non-HLA genes. The NOTCH4 gene lies at the centromeric end of the MHC class III region, approximately 335 kb telomeric of the DRB1 locus. The encoded protein is an oncogene that is important in regulating vascular development and remodelling. A recent report has linked polymorphisms within NOTCH4 with risk of developing schizophrenia. We have investigated if coding polymorphisms exist within this gene and have identified three single nucleotide polymorphisms; a synonomous T to C transition at +1297 (HGBASE accession number SNP000064386), a synonomous A to G transition at +3061 (SNP000064387) and an A to G transition at +3063 which results in a replacement of glycine with aspartic acid at amino acid 279 (SNP000064388). The allele frequencies of +1297T, +3061A and +3063G were 0.65, 0.66 and 0.66, respectively. Linkage disequilibrium was detected both between these markers and with MHC alleles. These findings can be used in the fine mapping of disease susceptibility alleles within the MHC.

Effect of stool size and consistency on defecation.

The ability of subjects to expel from the rectum objects simulating stools of different characteristics was assessed in paired studies carried out in a total of 58 normal subjects and 25 young women with severe constipation. Our results showed that a lower percentage of normal subjects and a lower percentage of constipated patients were able to pass a 1.8 cm incompressible sphere compared with a 50 ml deformable balloon, although constipated patients found it more difficult than normal subjects to expel both types of simulated stool. It was also more difficult for normal subjects to pass a soft compressible silicon rubber simulated stool than a stool made up of a similar volume of incompressible 1 cm wooden spheres contained in a cylindrical latex envelope, but both objects were much easier to pass than the same number of 1 cm spheres placed loose within the rectum. When normal subjects were instructed to expel single incompressible spheres of different sizes placed in the rectal ampulla, the intrarectal pressure and the time needed to pass these objects varied inversely with their diameter. These results suggest that more effort is required to expel stools from the rectum if they are small and hard than if they are large and soft.

Physiological studies in young women with chronic constipation.

Manometric, radiological and neurophysiological investigations were performed on 34 women, aged between 14 and 53, who suffered with chronic constipation refractory to treatment, and on 27 age-matched normal female control subjects. The constipated patients had more difficulty in evacuating simulated stools than control subjects and 13 out of 19 patients tested obstructed defaecation by contracting the external sphincter during straining. The constipated group required a greater degree of rectal distension than control subjects to induce rectal contractions, anal relaxation and a desire to defaecate. Other modalities of rectal sensation were normal in the constipated subjects. Compared with controls, constipated patients had significantly lower anal pressures, an abnormal degree of perineal descent on straining and an obtuse anorectal angulation at rest. These results were compatible with weakness of the pelvic floor and neuropathic damage to the external sphincter. Mouth to anus transit time was abnormally prolonged in 60% of constipated patients, but was within the normal range in the remainder. Anorectal function in patients with slow transit was not significantly different from that in patients with a normal transit time. The mouth to caecum transit time of a standard meal was prolonged in constipated patients irrespective of the duration of the whole gut transit. Gastric emptying was not significantly prolonged.

Impairment of defecation in young women with severe constipation.

Anorectal manometry, radiology, and tests of simulated defecation were carried out in 14 severely constipated young women and 29 age-matched controls. The resting anal sphincter pressures were reduced in the patients, but the squeeze pressures, rectoanal inhibitory reflex, and rectal pressures upon balloon distention were all normal. At rest, the anorectal angle was more obtuse in the constipated group, but there was no overall increase in perineal descent in constipated patients compared with controls. The presence of a balloon in the rectum and the onset of pain were perceived in constipated patients at volumes that were not significantly different from those in normal volunteers. Constipated patients, however, required higher rectal volumes to induce the desire to defecate and to stimulate regular rectal contractions. Constipated patients also found it more difficult to pass simulated stools from the rectum than the normal controls and, unlike most normal controls, failed to inhibit their external anal sphincter on attempted defecation. These findings suggest that young women with severe constipation have great difficulty initiating the coordinated set of events that constitute a normal defecation response.

Dendritic cells cultured from mononuclear cells and CD34 cells in myeloma do not harbour human herpesvirus 8.

Dendritic cells (DC) are antigen-presenting cells with the potential to be a powerful adjuvant in the immunotherapy of haematological malignancy, including myeloma. Recently, human herpesvirus 8 (HHV-8) infection of dendritic cells in the long-term bone marrow stromal cultures of patients with myeloma has been reported. This finding is of great potential importance regarding oncogenesis in myeloma in addition to having significant implications for the use of DC in the immunotherapy of this disease. Therefore DC generated from mobilized blood mononuclear cells (MO-DC) and purified CD34+ cells (CD34-DC) of myeloma patients were examined for the presence of HHV-8 using a sensitive PCR technique. HHV-8 was not demonstrated in MO-DC or CD34-DC and we conclude that these cells remain a suitable vehicle for investigation in the immunotherapy of myeloma.

Genetic analysis of the interleukin-1 receptor antagonist and its homologue IL-1L1 in alopecia areata: strong severity association and possible gene interaction.

Alopecia areata is an inflammatory hair loss disease with a major genetic component. The presence of focal inflammatory lesions with perifollicular T-cell infiltrates reflects the importance of local cytokine production in the pathogenesis. In addition to its fundamental pro-inflammatory role, the interleukin-1 (IL-1) system has major effects on hair growth regulation in vitro, with the inhibitory actions of IL-1alpha and IL-1beta being opposed by the receptor antagonist IL-1ra. The novel interleukin-1 like molecule 1 (IL-1L1) which has greatest gene sequence homology with IL1RN, the gene encoding IL-1ra, is another potential IL-1 antagonist. In view of previous studies suggesting a significant role for IL1RN polymorphisms in the pathogenesis of autoimmune/inflammatory disease, we have analysed polymorphisms of IL-1ra (IL1RN+2018) and its homologue IL-1L1 (IL1L1+4734) in a case-control association study on 165 patients and a large number of matched controls. Homozygosity for the rare allele of IL1RN (IL1RN*2) was significantly associated with alopecia areata [odds ratio (OR) = 1.89, 95% CI (1.09, 3.28); P = 0.02], confirming our previous findings of significant association with the IL1RN variable number tandem repeat (VNTR). The results also revealed a novel association involving a polymorphism of the interleukin-1 receptor antagonist homologue IL1L1 at position + 4734, IL1RN+2018, and alopecia areata. The effect of a genotype combining three copies of the rare alleles at the IL1RN and IL1L1 loci conferred a more than additive increase in the risk of disease compared to IL1RN+2018 or IL1L1+4734 alone [OR 3.37 (1.60, 7.06); P = 0.002], suggesting possible synergy between the IL1RN and IL1L1 genes. This effect was stronger in patients with severe disease (alopecia totalis/universalis) [OR 4.62 (1.87, 11.40), P = 0.0022], and in those with early age at onset (< 20 years) [OR = 6.38 (2.64, 15.42), P = 0.0002]. Our results suggest that these polymorphisms within IL1RN and IL1L1 themselves or a gene in linkage disequilibrium with IL1RN and IL1L1 predispose to the more severe forms of alopecia areata.