Heat shock proteins: A dual carrier-adjuvant for an anti-drug vaccine against heroin

Heroin is a highly abused opioid that has reached epidemic status within the United States. Yet, existing therapies to treat addiction are inadequate and frequently result into rates of high recidivism. Vaccination against heroin offers a promising alternative therapeutic option but requires further development to enhance the vaccine’s performance. Hsp70 is a conserved protein with known immunomodulatory properties and is considered an excellent immunodominant antigen. Within an antidrug vaccine context, we envisioned Hsp70 as a potential dual carrier-adjuvant, wherein immunogenicity would be increased by co-localization of adjuvant and antigenic drug hapten. Recombinant Mycobacterium tuberculosis Hsp70 was appended with heroin haptens and the resulting immunoconjugate granted anti-heroin antibody production and blunted heroin-induced antinociception. Moreover, Hsp70 as a carrier protein surpassed our benchmark Her-KLH cocktail through antibody-mediated blockade of 6-acetylmorphine, the main mediator of heroin’s psychoactivity. The work presents a new avenue for exploration in the use of hapten-Hsp70 conjugates to elicit anti-drug immune responses.     

Deep subcutaneous adipose tissue: a distinct abdominal adipose depot

Objective: Abdominal visceral (VAT) and subcutaneous adipose tissue (SAT) display significant metabolic differences, with VAT showing a functional association to metabolic/cardiovascular disorders. A third abdominal adipose layer, derived by the division of SAT and identified as deep subcutaneous adipose tissue (dSAT), may play a significant and independent metabolic role. The aim of this study was to evaluate depot‐specific differences in the expression of proteins key to adipocyte metabolism in a lean population to establish a potential physiologic role for dSAT. Research Methods and Procedures: Adipocytes and preadipocytes were isolated from whole biopsies taken from superficial SAT (sSAT), dSAT, and VAT samples obtained from 10 healthy normal weight patients (7 women and 3 men), with a mean age of 56.4 ± 4.04 years and a mean BMI of 23.1 ± 0.5 kg/m². Samples were evaluated for depot‐specific differences in insulin sensitivity using adiponectin, glucose transport protein 4 (GLUT4), and resistin mRNA and protein expression, glucocorticoid metabolism by 11β‐hydroxysteroid dehydrogenase type‐1 (11β‐HSD1) expression, and alterations in the adipokines leptin and tumor necrosis factor‐α (TNF‐α). Results: Although no regional differences in expression were observed for adiponectin or TNF‐α, dSAT whole biopsies and adipocytes, while intermediary to both sSAT and VAT, reflected more of the VAT expression profile of 11β‐HSD1, leptin, and resistin. Only in the case of the intracellular pool of GLUT4 proteins in whole biopsies was an independent pattern of expression observed for dSAT. In an evaluation of the homeostatic model, dSAT 11β‐HSD1 protein (r = 0.9573, p = 0.0002) and TNF‐α mRNA (r = 0.8210, p = 0.0236) correlated positively to the homeostatic model. Discussion: Overall, dSAT seems to be a distinct abdominal adipose depot supporting an independent metabolic function that may have a potential role in the development of obesity‐associated complications.     

Ultrasound evaluation of visceral fat and metabolic risk factors during early pregnancy

Objective: The objective was to study the relationships between ultrasound estimated visceral fat and metabolic risk factors during early pregnancy. Research Methods and Procedures: Thirty consecutive healthy pregnant women at 11 to 14 weeks of gestation were studied. Maximum subcutaneous fat thickness (SFT) and visceral fat thickness (VFT) were successfully measured by ultrasound. Fasting plasma glucose, insulin, triglycerides, total cholesterol, high‐density lipoprotein cholesterol (HDL‐C), and blood pressure were measured. Insulin resistance was calculated by using the homeostasis model assessment (HOMA). Results: VFT significantly correlated with diastolic blood pressure (r = 0.37, p = 0.04), glycemia (r = 0.37, p = 0.04), insulinemia (r = 0.59, p = 0.001) insulin sensitivity (HOMA; r = 0.59, p = 0.001), triglycerides (r = 0.58, p = 0.03), HDL‐C (r = ?0.39, p = 0.03), and total cholesterol/HDL‐C ratio (p = 0.002), whereas SFT was significantly correlated with only diastolic blood pressure (p = 0.03). VFT better significantly correlated with the metabolic risk factors than pre‐gestational BMI [r = 0.39, p = 0.03 for insulinemia, r = 0.42, p = 0.02 for insulin sensitivity (HOMA), and r = 0.49, p = 0.01 for triglycerides and not significant for the rest]. Discussion: Visceral fat thickness can be easily measured by ultrasound at early pregnancy and correlates better than BMI with metabolic risk factors.     

Genetic variation in the β, β‐carotene‐9′, 10′‐dioxygenase gene and association with fat colour in bovine adipose tissue and milk

β, β‐carotene‐9′, 10′‐dioxygenase (BCO2) plays a role in cleaving β‐carotene eccentrically, and may be involved in the control of adipose and milk colour in cattle. The bovine BCO2 gene was sequenced as a potential candidate gene for a beef fat colour QTL on chromosome (BTA) 15. A single nucleotide base change located in exon 3 causes the substitution of a stop codon (encoded by the A allele) for tryptophan⁸⁰ (encoded by the G allele) (c. 240G>A, p.Trp80stop, referred to herein as SNP W80X). Association analysis showed significant differences in subcutaneous fat colour and beta‐carotene concentration amongst cattle with different BCO2 genotypes. Animals with the BCO2 AA genotype had more yellow beef fat and a higher beta‐carotene concentration in adipose tissues than those with the GA or GG genotype. QTL mapping analysis with the BCO2 SNP W80X fitted as a fixed effect confirmed that this SNP is likely to represent the quantitative trait nucleotide (QTN) for the fat colour‐related traits on BTA 15. Moreover, animals with the AA genotype had yellower milk colour and a higher concentration of beta‐carotene in the milk.     

Primary subcutaneous Nocardia asteroides infection in a renal allograft recipient

Nocardia asteroides is an important opportunistic pathogen in immunocompromised hosts. The primary infection is usually in the lungs and is followed by dissemination to other parts of the body. Primary subcutaneous infection with Nocardia asteroides has been reported rarely (three reports) and no such case has been reported in a renal transplant recipient. We describe here a case of renal transplant recipient who developed primary subcutaneous infection with Nocardia asteroides within one and half years of the transplantation. This revised version was published online in June 2006 with corrections to the Cover Date.     

First finding of Dirofilaria repens in a natural population of Aedes albopictus

The invasive mosquito Aedes albopictus (Skuse) (Diptera: Culicidae) has become widespread in Italy during the past decade. Also Italy has foci of canine filariasis caused by Dirofilaria (Spirurida: Onchocercidae), due to subcutaneous D. repens Railliet & Henry as well as the dog heartworm D. immitis (Leidy) transmitted by various vector mosquitoes (Diptera: Culicidae). In 2002, at Fiumicino, west of Rome (Lazio Region), 17% of dogs were found to have D. repens microfilariae in peripheral blood. To evaluate the role of Ae. albopictus as a vector of Dirofilaria in this area, female mosquitoes were collected daily, June-October 2002, landing on dog or human bait in a rural house at Focene. Mosquitoes were maintained at 27 degrees C and 70% RH for 6 days, to allow development or purging of filaria larvae, then identified and frozen for subsequent molecular assay with filaria-specific ribosomal S2-S16 primers. To distinguish specimens harbouring infective L3 Dirofilaria larvae, DNA was extracted separately from the mosquito abdomen and head-thorax. Dirofilaria species were identified by sequencing, confirmed by polymerase chain reaction of positive specimens using primers specific for D. immitis and D. repens. Dirofilaria DNA was detected in 3/154 (2%) of Ae. albopictus females examined: D. repens DNA in head-thorax and abdomen of one collected 27th July; D. immitis in the abdomen of one collected 24th September; DNA of both D. immitis and D. repens in the head-thorax of one collected 11th October 2002. Thus Ae. albopictus is a potential vector of both Dirofilarias in Italy, representing risks for veterinary and human health.     

Effectiveness of weekly subcutaneous recombinant human erythropoietin administration for chemotherapy-induced anemia

The effects of weekly subcutaneousrecombinant human erythropoietin (r-hEPO)administration on anemia during chemotherapy includingcisplatin and 5-fluorouracil in patients with head andneck carcinomas were examined. Weekly subcutaneousr-hEPO administration in cancer patients has not beeninvestigated previously. Patients were treated withr-hEPO 100 IU/kg (2 patients), 200 IU/kg (6 patients),or 400 IU/kg (5 patients), or placebo, andeffectiveness was evaluated by monitoring hemoglobinconcentration changes after administration for 8weeks. Hemoglobin concentrations in all 3 r-hEPOdosage groups were higher than that in the controlgroup during chemotherapy. All r-hEPO doses producedimprovements in the anemia induced by chemotherapy;however, the 400 IU/kg dose was most effective. Therequirement for blood transfusions decreased inpatients receiving r-hEPO therapy, and no significantside-effects were associated with r-hEPOadministration. These results suggest thatchemotherapy-induced anemia can be prevented by weeklysubcutaneous r-hEPO administration.     

CDKN2B expression and subcutaneous adipose tissue expandability: Possible influence of the 9p21 atherosclerosis locus

Risk alleles within a gene desert at the 9p21 locus constitute the most prevalent genetic determinant of cardiovascular disease. Previous research has demonstrated that 9p21 risk variants influence gene expression in vascular tissues, yet the biological mechanisms by which this would mediate atherosclerosis merits further investigation. To investigate possible influences of this locus on other tissues, we explored expression patterns of 9p21-regulated genes in a panel of multiple human tissues and found that the tumor suppressor CDKN2B was highly expressed in subcutaneous adipose tissue (SAT). CDKN2B expression was regulated by obesity status, and this effect was stronger in carriers of 9p21 risk alleles. Covariation between expression of CDKN2B and genes implemented in adipogenesis was consistent with an inhibitory effect of CDKN2B on SAT proliferation. Moreover, studies of postprandial triacylglycerol clearance indicated that CDKN2B is involved in down-regulation of SAT fatty acid trafficking. CDKN2B expression in SAT correlated with indicators of ectopic fat accumulation, including markers of hepatic steatosis. Among genes regulated by 9p21 risk variants, CDKN2B appears to play a significant role in the regulation of SAT expandability, which is a strong determinant of lipotoxicity and therefore might contribute to the development of atherosclerosis.     

CLA differently regulates adipogenesis in stromal vascular cells from porcine subcutaneous adipose and skeletal muscle

Conjugated linoleic acid (CLA), a mixture of isomers of linoleic acid, has previously been shown to be able to decrease porcine subcutaneous (SC) adipose tissue levels while increasing the count of intramuscular (IM) adipose tissue in vivo. However, the underlying mechanisms through which it acts are poorly understood. The objective of this study was to investigate the different effects of CLA on adipogenesis in cultured SC adipose tissue and IM stromal vascular cells obtained from neonatal pigs. As shown here, trans-10, cis-12 CLA decreased the expression of adipocyte-specific genes as well as adipose precursor cell numbers and the accumulation of lipid in cultured SC adipose tissue stromal vascular cells. However, the cis-9, trans-11 CLA did not alter adipogenesis in SC cultures. On the other hand, both CLA isomers increased the expression of adipocyte-specific genes in IM cultures, together with the increasing accumulation of lipid and Oil Red O-stained cells. Collectively, these data show that CLA decreases SC adipose tissue but increases IM adipose tissue by different regulation of adipocyte-specific gene expression. These results suggest that adipogenesis in IM adipocytes differs from that in SC adipocytes.     

Salmonella Typhimurium strain expressing OprF‐OprI protects mice against fatal infection by Pseudomonas aeruginosa

Pseudomonas aeruginosa poses a major threat to human health and to the mink industry. Thus, development of vaccines that elicit robust humoral and cellular immunity against P. aeruginosa is greatly needed. In this study, a recombinant attenuated Salmonella vaccine (RASV) that expresses the outer membrane proteins fusion OprF_190–342‐OprI_21–83 (F1I2) from P. aeruginosa was constructed and the potency of this vaccine candidate assessed by measuring F1I2‐specific humoral immune responses upon vaccination through s.c. or oral routes. S.C. administration achieved higher serum IgG titers and IgA titers in the intestine and induced stronger F1I2‐specific IgG and IgA titers in lung homogenate than did oral administration, which resulted in low IgG titers and no local IgA production. High titers of IFN‐γ, IL‐4, and T‐lymphocyte subsets induced a mixed Th1/Th2 response in mice immunized s.c., indicating elicitation of cellular immunity. Importantly, when immunized mice were challenged with P. aeruginosa by the intranasal route 30 days after the initial immunization, s.c. vaccination achieved 77.78% protection, in contrast to 41.18% via oral administration and 66.67% via Escherichia coli‐expressed F1I2 (His‐F1I2) vaccination. These results indicate that s.c. vaccination provides a better protective response against P. aeruginosa infection than do oral administration and the His‐F1I2 vaccine.