Celiac disease

Celiac disease, or gluten-sensitive enteropathy, classically presents as diarrhea and weight loss in childhood, but it may also have protean manifestations and appear well into adult life. The increasing availability of noninvasive blood tests that are highly sensitive and specific for celiac disease enables primary care physicians to recognize the disorder in a wide variety of clinical situations. The authors believe that the disease is more common than supposed and thus offer this diagnostic review to increase awareness.     

Expression of pro form of prostate-specific antigen by mammalian cells and its conversion to mature, active form by human kallikrein 2

To study the expression, biosynthesis, and processing of prostate-specific antigen (PSA) in mammalian cells, recombinant PSA was expressed in Syrian hamster tumor cell line AV12-664 (AV12-PSA). Expression of PSA was monitored by the Tandem-MP PSA assay. PSA was secreted into the medium during the logarithmic phase of cell growth at >9 microg/ml and was stable. The PSA purified from spent medium of AV12-PSA cells did not exhibit any enzymatic activity and did not complex with the protease inhibitor, alpha-1-antichymotrypsin. These findings indicated that an inactive form of PSA was expressed by AV12-PSA cells. NH2-terminal sequencing confirmed the identity of the PSA purified from the spent medium of AV12-PSA cells to be pro-PSA. This demonstrates that PSA is expressed as pro-PSA by mammalian cells and suggests that pro-PSA may be present in biological fluids. Human kallikrein 2 (hK2), another member of the hK family, is also expressed predominantly in prostate epithelium. Although hK2 has been shown to exhibit trypsin-like activity, little is known about its natural substrates. Using purified proteins, we show that hK2 can convert pro-PSA to mature, enzymatically active PSA, thus establishing a physiological connection between hK2 and PSA. These findings imply that hK2 may be regulating PSA activity in vivo.     

Genetic construction, properties and application of a green fluorescent protein-tagged ciliary neurotrophic factor

The in vitro and in vivo actions of ciliary neurotrophic factor (CNTF) suggest that endogenous CNTF plays a role in nervous system development and maintenance. CNTF produces most, possibly all, of its effects by binding to a protein referred to as CNTF receptor alpha (CNTFRalpha). Information on CNTFRalpha tissue expression and dynamics would be advanced by the availability of reagents suitable for studying the subcellular localization and trafficking of CNTFRalpha. This paper describes the genetic construction, synthesis, purification and properties of a chimeric protein in which a highly fluorescent form of the green fluorescent protein (GFP) has been fused to human CNTF. The fusion protein, termed GFP-CNTF, was expressed in Escherichia coli. Histidine tagging of GFP-CNTF permitted ready purification by means of immobilized Ni(II) chromatography. Under non-reducing conditions GFP- CNTF migrated on SDS-PAGE with an apparent molecular mass of 50 kDa, although under reducing conditions it behaved electrophoretically as a 67 kDa species. Despite these discrepancies, the molecular mass of GFP- CNTF determined by mass spectrometry (54755) agreed well with its deduced relative molecular mass of 54536. Importantly, the absorbance profile of the GFP chromophore in GFP-CNTF was not modified by the presence of the CNTF domain. Moreover, the fluorescence emission spectrum of GFP-CNTF overlapped that of GFP, showing neither a change in absorbance shift nor a difference in the fluorescence quantum yield. Circular dichroism spectroscopy confirmed that the CNTF and GFP domains of GFP-CNTF folded independently of each other. GFP-tagged CNTF was equipotent to human CNTF in supporting the survival of cultured embryonic chicken sensory and ciliary ganglion neurons. GFP-CNTF, but not GFP, bound to immobilized CNTFRalpha and was displaced by an excess of human CNTF. GFP-CNTF specifically labeled the Purkinje cell layer in cerebellar slices from adult rat. This report is the first to describe a GFP chimera with a neurotrophic factor as the fusion partner. GFP- CNTF should provide a valuable tool for elucidating the role of CNTFRalpha in nervous system function.      

A complex nine base pair deletion in RET exon 11 common in sporadic medullary thyroid carcinoma

Genetic alteration of the RET proto-oncogene is associated with multiple endocrine neoplasia type 2A and 2B (MEN 2A and MEN 2B), familial medullary thyroid carcinoma (FMTC) and Hirschprung's disease. Oncogenically activated RET has also been demonstrated in sporadic medullary thyroid tumors, which in some cases show somatic missense mutations. We have recently described a complex 9 bp deletion in RET exon 11 in a single case of sporadic MTC. In order to determine the prevalence of this mutation among sporadic MTC tumors, we have now analysed 15 cases and five normal controls by PCR-based nonradioactive single-strand conformational polymorphism analysis (PCR-SSCP) and fragment size analysis of exon 11. DNA was extracted from microdissected tumor tissue or normal cells and subjected to nested PCR prior to analysis. A markedly divergent SSCP pattern and a PCR fragment 9 bp shorter than normal were demonstrated in 14 of the 15 MTC tumors. Sequencing revealed the deletion of nine bases encompassing a key cysteine at codon 634, often altered in MEN 2A. Four lymphocyte controls and normal thyroid tissue from one patient failed to show the deletion. Several factors in the DNA sequence environment immediately surrounding the deletions, including an extended inverted repeat, several direct repeats and a so-called symmetric element suggest that the deletional events may be non-random.     

Natural killer cell-mediated eradication of neuroblastoma metastases to bone marrow by targeted interleukin-2 therapy

Targeted interleukin-2 (IL-2) therapy with a genetically engineered antidisialoganglioside GD2 antibody-IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of neuroblastoma. The mechanism involved is exclusively natural killer (NK) cell-dependent, because NK-cell deficiency abrogated the antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell-deficient mice or immunocompetent mice depleted of CD8+ T cells in vivo. A strong stimulation of NK-cell activity was also shown in vitro. Immunohistology of the leukocytic infiltrate of livers from treated mice revealed a strong staining for NK cells but not for CD8+ T cells. The therapeutic effect of the fusion protein was increased when combined with NK-cell-stimulating agents, such as poly I:C or recombinant mouse interferon-gamma. In conclusion, these data show that targeted delivery of cytokines to the tumor microenvironment offers a new strategy to elicit an effective cellular immune response mediated by NK cells against metastatic neuroblastoma. This therapeutic effect may have general clinical implications for the treatment of patients with minimal residual disease who suffer from T-cell suppression after high-dose chemotherapy but are not deficient in NK cells.     

Rationale for en bloc vein resection in the treatment of pancreatic adenocarcinoma adherent to the superior mesenteric-portal vein confluence. Pancreatic Tumor Study Group

The enzymatic activity of protein kinase C (PKC) was measured in the cytosol and particulate fraction of parabrachial nucleus, the presumed site of conditioned taste aversion (CTA) engrams. At various time intervals after acquisition of the task (pairing saccharin consumption with subsequent LiCl poisoning) the nucleus was dissected from the frozen coronal sections. An increase (+40%) in the cytosol PKC activity was found 48 h after that pairing in comparison with controls (saline injection instead of LiCl). Particulate enzyme activity virtual did not change (-5%). Thus the total PKC activity increased significantly (21%). Qualitatively similar but less markedly expressed PKC shifts (+18% in cytosol) ere found 24 h following CTA. Twelve hours and 5 days after CTA acquisition the activity and distribution of PKC was similar to that seen in normal rats. The control experiments revealed that 6 h after LiCl injection alone (without previous saccharin consumption) translocation of PKC from the cytosol to the membrane fraction (found previously 1 h after LiCl injection alone) still persisted but did not differ from that found 6 h after its pairing with saccharin drinking (CTA). It is concluded that acquisition of conditioned taste aversion may be followed by synthesis of PKC rather than by its translocation or downregulation.     

Updated estimates of condom effectiveness

This paper reports results of a study concerning the effectiveness of condoms in preventing HIV transmission. The study expands on a 1993 meta-analysis that pooled the results of a number of studies of HIV transmission in serodiscordant couples to arrive at an overall condom effectiveness estimate of 69%. A meta-analysis of studies that compared seroconversion rates among couples who regularly use condoms and those who used them inconsistently was conducted to determine the use and/or effectiveness of condoms in preventing HIV transmission. Results of the analysis indicated that condoms are 90-95% effective when used consistently. To illustrate the impact of differential assumptions regarding the effectiveness of condoms in preventing the transmission of HIV, a community of gay men is considered in which the prevalence rate of HIV infection is 20%, supposing each man has sex once a week with a monogamous partner from the same population. The expected annual incidence of HIV infection in this community is 13% if condoms are never used, while consistent use of 95% effective condoms would reduce the incidence to about 1%. In this example, the probability of transmission for 52 acts of condom-protected intercourse is less than for a single act of unprotected intercourse. Moreover, inconsistent condom use also offers some protection against HIV in which the reduction achieved by using condoms 50% of the time is equal to almost half the reduction associated with consistent use.     

Conformationally constrained inhibitors of caspase-1 (interleukin-1 beta converting enzyme) and of the human CED-3 homologue caspase-3 (CPP32, apopain)

A systematic study of interleukin-1 beta converting enzyme (ICE, caspase-1) and caspase-3 (CPP32, apopain) inhibitors incorporating a P2-P3 conformationally constrained dipeptide mimetic is reported. Depending on the nature of the P4 substituent, highly selective inhibitors of both Csp-1 or Csp-3 were obtained.