Unrestricted recognition of class I neodeterminants generated by exon shuffling

We and others have previously reported that homologous exchange of alpha 1 or alpha 2 domains between different alleles led to loss of most determinants recognized by cytotoxic T lymphocytes (CTL) raised against the parental H-2 class I antigens. Here we demonstrate that exchange of alpha 1 or alpha 2 domains between the Kk and Kd allele results in the formation of neodeterminants against which allospecific CTL can be generated in responder mice of various related and unrelated H-2 haplotypes. In fact, only CTL against the neodeterminants on the hybrid molecules were found and none against the parental Kk and Kd determinants. In addition our data show that recognition of the neodeterminants is H-2 unrestricted. These findings suggest that in the hybrid molecules the basic structure of a major histocompatibility complex molecule has been preserved although most parental allodeterminants on the alpha 1 and alpha 2 domains have been modified.     

Neutralizing antibodies in gene-defective hosts

In a host with a normal immune system and a complete gene defect, the nondefective gene product will be immunogenic. Consequently, neutralizing antibodies against the respective protein can arise either 'spontaneously' or after immunization, as shown in patients and in animal models, such as knockout mice. Accordingly, patients with X-linked or homozygous autosomal gene defects are at risk of developing neutralizing antibodies, in particular after protein substitution or gene therapy. This Review compares and exemplifies the various genetic and immunological contexts that lead to 'neutralizing and generated by gene defect' or 'nagged' antibodies, and outlines implications and solutions for therapeutic strategies.     

The nonselective cation channel in the basolateral membrane of rat exocrine pancreas

Nonselective Ca²⁺-sensitive cation channels in the basolateral membrane of isolated cells of the rat exocrine pancreas were investigated with the patch clamp technique. With 1.3 mmol/l Ca²⁺ on the cytosolic side, the mean openstate probabilityP _o of one channel was about 0.5. In insideout oriented cell-excised membrane patches the substances diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 3,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) were applied to the cytosolic side. These compounds inhibited the nonselective cation channels by increasing the mean channel closed time (slow block). 100 mol/l of NPPB or DPC decreasedP _o from 0.5 (control conditions) to 0.2 and 0.04, respectively, whereas 100 mol/l of DCDPC blocked the channel completely. All effects were reversible. 1 mmol/l quinine also reducedP _o, but in contrast to the abov mentioned substances, it induced fast flickering. Ba²⁺ (70 mmol/l) and tetraethylammonium (TEA⁺; 20 mmol/l) had no effects. We investigated also the stilbene disulfonates 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and 4,4-dinitro-2,2-stilbenedisulfonate (DNDS). 10 mol/l SITS applied to the cytosolic side increasedP _o from 0.5 to 0.7 and with 100 mol/l SITS the channels remained nearly permanently in its open state (P _o1). A similar activation of the channels was also observed with DIDS and DNDS. These effects were poorly reversible. The stilbene disulfonates acted by increasing the channel mean open time. When the channel was inactivated by decreasing bath Ca²⁺ concentration to 0.1 mol/l, addition of 100 mol/l of SITS had no effect. Similarly, reducing bath Ca²⁺ concentration from 1.3 mmol/l in presence of 100 mol/l SITS (channels are maximally activated) to 0.1 mol/l, inactivated the channels completely. These results demonstrate, that SITS can only activate the channels in the presence of Ca²⁺. SITS had no effects, when applied to the extracellular side in outside out patches. In summary, the substances DPC, NPPB and DCDPC inhibit nonselective cation channels, where DCDPC has the most potent and NPPB the smallest effect; whereas SITS, DIDS and DNDS activate the channel when applied from the cytosolic side in the presence of Ca²⁺ ions.     

The carboxyl terminus of the epithelial Ca(2+) channel ECaC1 is involved in Ca(2+)-dependent inactivation

The family of epithelial Ca(2+) channels (ECaC) is a unique group of highly Ca(2+)-selective channels consisting of two members, ECaC1 and ECaC2. We used carboxyl terminal truncations and mutants to delineate the molecular determinants of the Ca(2+)-dependent inhibition of ECaC. To this end, rabbit ECaC1 was expressed heterologously with green fluorescent protein (GFP) in human embryonic kidney 293 (HEK293) cells using a bicistronic vector. Deletion of the last 30 amino acids of the carboxyl terminus of ECaC1 (G701X) decreased the Ca(2+) sensitivity significantly. Another critical sequence for Ca(2+)-dependent inactivation of ECaC1 was found upstream in the carboxyl terminus. Analysis of truncations at amino acid 635, 639, 646, 649 and 653 disclosed a critical sequence involved in Ca(2+)-dependent inactivation at positions 650-653. C653X showed decreased Ca(2+) sensitivity, comparable to G701X, while E649X lacked Ca(2+)-dependent inactivation. Interestingly, the number of green fluorescent cells, which is an index of the number of transfected cells, was significantly smaller for cells transfected with truncations shorter than E649 than for cells transfected with wild-type ECaC. However, the expression level of GFP was restored in the presence of the ECaC blocker ruthenium red, suggesting that these truncations resulted in deleterious Ca(2+) influx. In conclusion, we have identified two domains in the carboxyl terminus of ECaC1 that control Ca(2+)-dependent inactivation.     

Fertilization and early embryology: Effects of persistent chlorinated hydrocarbons on fertility and embryonic development in the rabbit

The commercial polychlorinated biphenyl (PCB) formulation Aroclor1260 (4 mg/kg body weight), technical grade dichlorodiphenyltrichloroethane(DDT; 3 mg) and Lindane (-hexachlorocyclohexane; 0.8 mg) wereadministered orally, either separately or in combination, tosexually mature female rabbits three times per week for 12–15weeks. Oviductal and uterine luminal fluid, cleavage stage embryos(day 1 post coitum), blastocysts (day 6), fetuses, exocoelicfluid and placentae (day 11) were analysed, firstly for chlorinatedhydrocarbon residues, and secondly for embryonic and fetal development.The doses applied were well tolerated by the treated animals.PCB and DDT accumulated in uterine secretions (day 6) but notin oviductal luminal fluid (day 1). Both chlorinated hydrocarbonswere found in preimplantation blastocysts. Residues in day 11fetuses were 16- (DDT) or 18-fold (PCB) higher than in day 6blastocysts. Significant amounts were also detected in placentaltissue and in exocoelic fluid. A specific accumulation of thehighly chlorinated biphenyl congener no. 180 was noted in fetuses,placentae and exocoelic fluid. The clear accumulation of thechlorinated hydrocarbon compounds in luminal fluid and embryonictissue is contrasted by rather weak effects on fertility. Nostatistically significant differences between treated animalsand controls were observed for fertilization rate and pre- andpost-implantation (up to day 11 post coitum) losses. However,in females exposed to PCB, a 20% higher loss of blastocystswas noticed, as compared with controls (P > 0.05). This effectwas shown on day 6 of embryonic development and may be due tothe embryotoxic activities of PCB.     

Comparison of GB virus C, HIV, and HCV infection markers in hemophiliacs exposed to non-inactivated or inactivated factor concentrates.

BACKGROUND: Until the mandatory introduction of viral inactivation techniques of blood plasma products in the early 1980s many recipients of these products were infected with various viral pathogens. OBJECTIVES: To determine the rate of transmission of GB virus C/hepatitis G virus (GBV-C/HGV) HCV, and HIV through non-virus-inactivated clotting factor concentrates in hemophiliacs, as well as the relation between amount of administered clotting factor and risk for GBV-C/HGV infection. STUDY DESIGN: In this cross-sectional study, we determined retrospectively the rates of infection markers for GBV-C/HGV, HCV, and HIV in a German cohort of hemophiliacs treated with documented amounts of non-virus-inactivated clotting factor concentrates (group A) and in a second group of hemophiliacs who were treated exclusively with virus-inactivated clotting factor (group B). The presence of anti-virus antibodies was determined by ELISA. Viral RNA was detected by RT-PCR. Markers for viral infections were compared to amounts of administered non-virus-inactivated clotting factor. RESULTS: Among hemophiliacs treated with documented amounts of non-virus-inactivated clotting factor the prevalence for GBV-C/HGV, HCV, and HIV was 40.3%, 98.6%, and 56.3%, respectively. In contrast to HIV, the rate of GBV-C/HGV infections did not increase with increasing amounts of consumed non-inactivated clotting factor. Even in the subgroup of heavily treated hemophiliacs the rate of GBV-C/HGV infection markers did not exceed 45%. CONCLUSIONS: The amount of non-virus-inactivated clotting factor is not predictive for the risk of GBV-C/HGV infection in hemophiliacs. Despite repeated parenteral exposure more than 55% of hemophiliacs were not infected with GBV-C/HGV. Our findings indicate a high frequency of host factors preventing parenteral transmission of GBV-C/HGV.     

Prognostic value of MMP-2, -9 and TIMP-1,-2 immunoreactive protein at the invasive front in advanced head and neck squamous cell carcinomas

In head and neck cancer as well as in other carcinomas, tumor expansion and spread to distant sites require the secretion of destructive enzymes that degrade the extracellular matrix. A variety of proteases contribute to matrix destruction. Characteristics of the invasive tumor front may reflect tumor prognosis better than do other parts of the tumor. Therefore, it was the aim of the present study to (i) compare central and peripheral tumor zones for differences in the expression of matrix-metalloproteinases (MMP) -2 and -9 and their naturally occurring inhibitors (tissue inhibitor of matrix-metalloproteinases (TIMP) -1 and -2), (ii) examine the morphological potential of malignancy, and (iii) correlate these findings with clinicopathological parameters. The study population consisted of 106 surgical specimens of advanced head and neck squamous cell carcinomas. The invasive front was graded for malignancy, and immunohistochemical staining with MMP-2, MMP-9, TIMP-1 and TIMP-2 antibodies was performed. Both MMP-2 and MMP-9 were found to be significantly overexpressed at the tumor front. The MMP-2-positive invasive front exhibited diminished overall survival times. In multivariate analysis, MMP-2 expression retained its correlation with overall survival in addition to nodal status and total malignancy score. Expression of TIMP-2 correlated with local tumor invasion. We conclude that the expression of MMP-2 at the invasive front is a marker of poor survival and appears to be associated with early recurrence in initially lymph node-negative patients.