Effects of lesions of the histaminergic tuberomammillary nucleus on spontaneous sleep in rats

STUDY OBJECTIVES: Extensive evidence suggests that histaminergic neurons promote wakefulness. Histaminergic neurons are found exclusively in the tuberomammillary nucleus (TMN), and electrolytic lesions of the posterior hypothalamus, where the TMN resides, produce intense hypersomnolence. However, electrolytic lesions disrupt fibers of passage, and the effects of fiber-sparing, cell-specific TMN lesions on sleep and wakefulness are unknown. Hence, we placed cell-specific lesions in the TMN to determine its role in spontaneous wakefulness. DESIGN: TMN neurons in rats are relatively resistant to excitotoxins. Hence, we ablated them using saporin conjugated to hypocretin 2, which ablates hypocretin receptor-bearing neurons such as TMN neurons. One to 2 weeks after bilateral injections of Hcrt2-SAP into Sprague-Dawley rats, we correlated loss of TMN neurons with changes in sleep. SETTING: N/A PARTICIPANTS: N/A INTERVENTIONS: N/A MEASUREMENTS AND RESULTS: Four days after injections with hypocretin-2-saporin, the number of TMN neurons was markedly decreased, and most were lost after 12 days, as determined by immunohistochemistry for adenosine deaminase, a marker of TMN neurons. Nearby nonhistaminergic neurons were similarly ablated. Rats with an average 82.5% loss of TMN cells (determined 2 weeks after injection) did not have marked changes in total sleep amounts compared to saline-treated rats 1 or 2 weeks following the injection, except for a slight decrease in rapid eye movement sleep during the lights-on period for the first week only. The percentage of remaining TMN neurons positively correlated with the average duration of wake bouts during the lights-off period. CONCLUSION: The absence of gross changes in sleep after extensive loss of histaminergic neurons suggests that this system is not critical for spontaneous wakefulness.     

Gelatin-chondroitin-hyaluronan tri-copolymer scaffold for cartilage tissue engineering

The mechanism by which the cell synthesizes and secretes extracellular matrix (ECM) and is, in turn, regulated by the ECM is termed dynamic reciprocity. The aim of the present work was to produce a gelatin/chondoitin-6-sulfate/hyaluronan tri-copolymer to mimic natural cartilage matrix for use as a scaffold for cartilage tissue engineering. The scaffold produced had a uniform pore size of about 180 microm and adequate porosity of 75%. Porcine chondrocytes were seeded onto the tri-copolymer scaffold and cultured in Petri dishes or spinner flasks for 2, 3, 4, or 5 weeks. Chondrocytes were uniformly distributed in the scaffold in the spinner flask cultures, but less so in the Petri dish cultures. Secretion of ECM was found under histology examination. In spinner flask cultures, chondrocytes retained their phenotype for at least 5 weeks, as shown immunohistochemically, and synthesized type II collagen. These results show that gelatin/chondroitin sulfate/hyaluronan tri-copolymer has potential for use as a cartilage tissue engineering scaffold.     

The effect of antigen and mode of immunization on the allotypic distribution of enhanceable plaque-forming antibody

The relation between the genotypic control and phenotypic expression of antibody formation and between gene dose and product concentration depends on and is modified by antigen-mediated selection and by antigen-induced cell division. This dependence was studied in terms of allotypic specificities of rabbit light chain as markers and with different antigens and. regimens of immunization. The number of enhanceable plaque-forming cells of the same allotype was measured in the spleens of rabbits, homozygous and heterozygous at the A_b locus. A significant departure from a simple gene-dose relationship was observed. The regular preponderance of one allotype over another (“pecking order”: A4>A6>A5>A9) was confirmed. The preponderance in A⁴/A⁵ animals was found to persist whether the allotype ratio of enhanceable plaques was tested after one, two or ten injections with SRBC, after immunization with chemically modified SRBC or with DNP-HGG. There was no significant difference in the cross-reactivity of A4 and AS plaque-forming SRBC antibody. The distribution of allotype ratios of responding A⁴/A⁵ animals changed with prolonged immunization from trimodal to bimodal and finally approached unimodality. After two injections with SRBC, the incidence of individual heterozygotes, making a monoallotypic enhanceable plaque response, was greatest among animals with allotypes from the extremes of the pecking order (i.e. A⁴/A⁹). We can account for our observations and for the distribution of allotype ratios in the response to strong and to weak antigen on the basis of the following assumptions:

• I a small number of receptor-carrying precursors of antibody-forming cells is involved in the response to one cr two injections with antigen;
• II the specificity spectrum for antigens which evoke a heterogeneous response is the same for plaque-forming antibodies of different allotypic specificity (A4 or A5);
• III in heterozygotes, the ratio of receptors with different allotypic specificities is different from 1 and is related to the distance in the pecking order; the greater the distance, the greater the difference from 1;
• IV there is continuous recruitment from precursor cells during prolonged immunization.

     

Human papillomavirus typing with a polymerase chain reaction-based genotyping array compared with type-specific PCR

BACKGROUND: Type-specific persistence of human papillomavirus (HPV) infection can cause invasive cervical cancer. OBJECTIVES: To evaluate the efficacy of HPV detection and typing with a general polymerase chain reaction (PCR)-based genotyping array and to compare it with a type-specific PCR assay. STUDY DESIGN: Four hundred and thirty-three cervical samples were tested with a modified MY11/GP6+ PCR-based reverse-blot assay (EasyChip HPV Blot; King Car, Taiwan [hereafter HPV Blot]) and with 20 genotypes of L1-type-specific PCR (HPV-6, -11, -16, -18, -31, -33, -35, -39, -45, -51, -52, -53, -56, -58, -59, -62, -66, -68, -70, and -71 [CP8061]). RESULTS: The concordance of the two tests in determining HPV positivity was 96.8% (419/433), with a Cohen's kappa=0.93 (95% CI: 0.90-0.97) and McNemar's test of P=1.0, which indicates excellent agreement. The overall concordance of the two tests in the identification of type-specific HPV was 91.0% (394/433). Sensitivity (90-100%), specificity (99.2-100%), and accuracy (98.6-100%) rates of HPV Blot against the gold standard were satisfactory for HPV-16, -18, -58, -33, -52, -39, -45, -31, -51, -70 while HPV-71 (63.6%) had suboptimal sensitivity. Though the kappa values between the two tests for many individual genotypes could not be reliably calculated because of low positivity, the kappa values for HPV-16, -52, and -58 were excellent (0.93, 0.96, and 0.95, respectively). CONCLUSION: The modified MY11/GP6+ PCR-based HPV Blot assay is accurate and sensitive for detection and genotyping of HPV in cervical swab samples.     

Determination of biogenic amines in fish implicated in food poisoning by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatography (MECC) method for the simultaneous determination of seven biogenic amines in fish was developed. The peaks of all components were successfully separated within 11.5 min. MECC was performed with 0.06 M sodium deoxycholate in 0.02 M borate buffer (pH 9.2)-methanol (95:5, v/v) solvent. The average recoveries for all components ranged from 84.4 to 100.3%. The application of this method to detect amines in fried marlin fillet implicated in a food poisoning incident indicated that a high level (56.24 mg/100 g) of histamine was present in the sample. Another 10 fish samples collected from markets were also analyzed and did not contain detectable levels of histamine (<2.5 mg/100 g).     

Antibody against Epstein-Barr virus DNA polymerase activity in sera of patients with nasopharyngeal carcinoma

A salt-dependent DNA polymerase activity was demonstrated in the culture of an EBV-producing, lymphoblastoid cell line (NPC-204 cells) treated with 5-iodo-2'-deoxyuridine (IUdR). There was a high frequency of levels of antibody to this enzyme in sera of patients with nasopharyngeal carcinoma (NPC). In contrast, sera from healthy subjects had little or no neutralizing activity. The high antibody level appeared as early as stage 1 of the disease in many NPC patients. The levels of the antibody increased with the progression of the disease and declined in treated patients. The results strongly suggest that tests measuring serum antibody against EBV DNA polymerase activity can be used for early diagnosis and prognosis of NPC.     

Differential susceptibility of human T(h)1 versus T(h) 2 cells to induction of anergy and apoptosis by ECDI/antigen-coupled antigen-presenting cells

Antigen-coupled antigen-presenting cells (APC) serve as potent tolerogens for inhibiting immune responses in vivo and in vitro, apparently by providing an antigen-specific signal through the TCR in the absence of co-stimulation. Although this approach has been well studied in rodents, little is known about its effects on human T cells. We evaluated the specificity and mechanisms of tolerization of human T cells in vitro using monocyte-enriched adherent cells that were pulsed with antigen and treated with the cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI). Autologous antigen-coupled APC selectively tolerized T cells of the T(h)1 but not T(h)2 lineage through a mechanism that involved both antigen-specific and antigen-non-specific elements. The tolerization process was dependent on the ECDI and antigen concentration, and the coupling time, and was reflected by initial up-regulation of CD25. However, upon re-stimulation with fresh APC and antigen, tolerized T(h)1 cells failed to proliferate or to produce T(h)1 cytokine message or secreted protein, had decreased expression of CD25, CD28 and B7 and increased expression of MHC class II molecules, and demonstrated an enhanced commitment to apoptosis. T(h)1 cell tolerization could be prevented by adding anti-CD28 antibody, IL-2 or untreated APC at the same time as the ECDI/antigen-coupled APC, or reversed by adding anti-CD28 antibody or IL-2 upon re-stimulation with fresh APC plus antigen. Thus, the tolerizing effect of ECDI/antigen-coupled APC on human T(h)1 cells appears to involve a reversible anergy mechanism leading to apoptosis, whereby the targeted T cells receive full or partial activation through the TCR, without coordinate co-stimulation. These data suggest dichotomous signaling requirements for inactivating cells of the T(h)1 and T(h)2 lineages that may have important implications for treatment of T(h)1-mediated autoimmune or inflammatory diseases.