Validation of primary CD4 gating as an affordable strategy for absolute CD4 counting in Cambodia

OBJECTIVE: To validate primary CD4 gating in lysed whole blood for absolute CD4 counts in fresh and aged blood using an affordable compact volumetric commercial flow cytometer. DESIGN: Comparison of CD4 counts between the FACSCount and the 2-parameter CyFlow SL Green. METHODS: One hundred twenty fresh blood samples from patients likely to be infected with HIV were simultaneously run on a FACSCount at the Pasteur Institute of Cambodia and on a CyFlow SL Green at the Sihanouk Hospital Center of Hope (SHCH), Phnom Penh, Cambodia. Intra- and interrun precision was assessed using 2 blood samples. Stability of CD4 counting in blood stored up to 96 hours at room temperature was assessed using 27 blood samples. RESULTS: CD4 counts on the CyFlow SL Green and on the FACSCount correlated well apart from a relative bias (R = 0.993, bias of -9.5%, 95% confidence interval [CI]: -11.8% to -7.1%, limits of agreement: -32.5% to 13.6%). Intra- and interrun variability ranged from 3% to 5% and from 5% to 6%, respectively. CD4 counts on aged blood using the CyFlow SL Green showed an interassay variability of <10%. CONCLUSIONS: Primary CD4 gating in lysed whole blood using the CyFlow SL Green is an affordable and precise method for CD4 counting. Because the fluorescence (FL) and light scatter signals have to be analyzed manually, however, intensive training of the technician and/or operator is imperative.     

Presence of a linear plasmid-like DNA molecule in the fungal pathogen Ceratocystis fimbriata Ell. & Halst.

Summary A plasmid-like molecule was detected in a strain of the ascomycete Ceratocystis fimbriata Ell. & Halst., a pathogenic fungus of Populus spp. The DNA replicon, designated pFQ501, was found to have a linear structure with a length of 6.0 kb (3.9 × 10⁶ daltons) and a density of 1.685 g/cc. This molecule was found to be associated with the mitochondria and was isolated from the gel; its restriction map was deduced from single and double digestions.     

Natural killer T cells ameliorate antibody-induced arthritis in macrophage migration inhibitory factor transgenic mice

Macrophage migration inhibitory factor (MIF) plays an important role in inflammatory diseases. It has been reported that anti-MIF treatment and mif-gene disruption ameliorate joint inflammation in a mouse model of arthritis induced by anti-type II collagen monoclonal antibodies and lipopolysaccharide (anti-IIC mAb/LPS). In the present study, using the anti-IIC mAb/LPS system, we have analyzed arthritis in MIF-transgenic (MIFTg) and wild-type C57BL/6 (WT) mice. We found that MIFTg mice developed more severe arthritis than WT mice. The histopathological scores were significantly higher in MIFTg mice and significantly increased numbers of CD69+ T cells were detected in the spleens of these arthritic MIFTg mice, compared with WT mice. Natural killer T (NKT) cells from MIFTg mice, compared with WT mice, produced reduced amounts of IL-4 upon stimulation with agr;-galactosylceramide (alpha-GalCer). Further, repeated administration of alpha-GalCer to MIFTg mice resulted in a profound reduction of both clinical and histopathological scores of arthritis, with a significant decrease in IL-6. The present findings demonstrate that overexpression of MIF exacerbates inflammation in this arthritis model and that NKT cells play an ameliorating role upon stimulation with alpha-GalCer in the inflammatory process in MIFTg mice.     

Blood lactate response to overtraining in male endurance athletes

Many physiological markers vary similarly during training and overtraining. This is the case for the blood lactate concentration ([La⁻]_b), since a right shift of the lactate curve is to be expected in both conditions. We examined the possibility of separating the changes in training from those of overtraining by dividing [La⁻]_b by the rating of perceived exertion ([La⁻]_b/RPE) or by converting [La⁻]_b into a percentage of the peak blood lactate concentration ([La⁻]_b,peak). Ten experienced endurance athletes increased their usual amount of training by 100% within 4 weeks. An incremental test and a time trial were performed before (baseline) and after this period of overtraining, and after 2 weeks of recovery (REC). The [La⁻]_b and RPE were measured during the recovery of each stage of the incremental test. We diagnosed overtraining in seven athletes, using both physiological and psychological criteria. We found a decrease in mean [La⁻]_b,peak from baseline to REC [9.64 (SD 1.17), 8.16 (SD 1.31) and 7.69 (SD 1.84) mmol · l⁻¹, for the three tests, respectively; P < 0.05] and a right shift of the lactate curve. Above 90% of maximal aerobic speed (MAS) there was a decrease of mean [La⁻]_b/RPE from baseline to REC [at 100% of MAS of 105.41 (SD 17.48), 84.61 (SD 12.56) and 81.03 (SD 22.64) arbitrary units, in the three tests, respectively; P < 0.05), but no difference in RPE, its variability accounting for less than 25% of the variability of [La⁻]_b/RPE (r=0.49). Consequently, [La⁻]_b/RPE provides little additional information compared to [La⁻]_b alone. Expressing [La⁻]_b as a %[La⁻]_b,peak resulted in a suppression of the right shift of the lactate curve, suggesting it was primarily the consequence of a decreased production of lactate by the muscle. Since the right shift of the curve induced by optimal training is a result of improved lactate utilization, the main difference between the two conditions is the decrease of [La⁻]_b,peak during overtraining. We propose retaining it as a marker of overtraining for long duration events, and repeating its measurement after a sufficient period of rest to make the distinction with overreaching. Accepted: 26 September 2000     

Presence and mechanism of macrolide-lincosamide resistance in Enterococcus columbae strains belonging to the intestinal flora of pigeons.

Faecal samples from 50 pigeons all originating from different lofts were screened for the presence of macrolide and lincosamide (ML)-resistant isolates of Streptococcus gallolyticus and Enterococcus columbae by plating the samples onto selective media. Sixty-eight ML-resistant E. columbae strains were recovered from the faecal samples of 29 animals. Two of these samples also harboured ML-resistant S. gallolyticus strains. The erm(B) gene was detected in 58 E. columbae and in five S. gallolyticus isolates. Four of these E. columbae isolates also carried the mef(A) gene. Five E. columbae strains possessed the mef(A) gene in the absence of erm(B). On the basis of the sequence of the complete erm(B) gene, 10 E. columbae isolates clustered together in six groups. In two of these isolates, the erm(B) gene sequence was identical to that of S. gallolyticus strains, indicating that exchange of resistance genes might occur between pathogenic and non-pathogenic bacterial species belonging to the pigeon's intestinal flora.     

Association of the BDNF Val66Met variation with obesity in women

Brain-derived neurotrophic factor (BDNF) has been implied in the regulation of food intake. In the present study, we genotyped the Val66Met polymorphism in a Belgian cohort of 532 obese women and 197 healthy female controls and were, for the first time, able to show an association of the 66Met allele with obesity, at least in our female cohort.     

Pain and disability in osteoarthritis: A review of biobehavioral mechanisms

Pain and disability are cardinal symptoms in osteoarthritis. The literature is reviewed in order to identify causes of these symptoms at the articular, kinesiological, and psychological level. It is concluded that pain and disability are associated with degeneration of cartilage and bone (articular level), with muscle weakness and limitations in joint motion (kinesiological level), and with anxiety, coping style, attentional focus on symptoms, and possibly depression (psychological level). Biobehavioral mechanisms of pain and disability which explain the observed associations are described and the empirical evidence for these mechanisms is evaluated. Methodological and conceptual deficiencies in the research reviewed are pointed out and suggestions for further research are given.