Assignment of weight-based immunoglobulin G1 (IgG1) and IgG2 units in antipneumococcal reference serum lot 89-S(F) for pneumococcal polysaccharide serotypes 1, 4, 5, 7F, 9V, and 18C

Weight-based assignments for immunoglobulin G1 (IgG1) and IgG2 subclass antibodies to Streptococcus pneumoniae capsular polysaccharides (PnPs) in antipneumococcal standard reference serum lot 89-S (lot 89-S), also known as lot 89-SF, have been determined for serotypes 1, 4, 5, 7F, 9V, and 18C. This extends the usefulness of lot 89-S beyond the IgG1 and IgG2 subclass assignments for serotypes 3, 6B, 14, 19F, and 23F made previously (A. Soininen, H. Kayhty, I. Seppala, and T. Wuorimaa, Clin. Diagn. Lab. Immunol. 5:561-566, 1998) to cover 11 major serotypes associated with the highest percentage of pneumococcal disease worldwide. A method of equivalence of absorbances in enzyme immunosorbent assays was used to determine the IgG1 and IgG2 antibody concentrations for the additional serotypes in lot 89-S, based on the subclass values previously assigned for PnPs serotypes 6B, 14, and 23F. This cross-standardization method assures consistency with previous antibody assignments in that reference serum. The newly assigned subclass values for serotype 9V, and previously assigned values for serotype 14, were used to quantitate PnPs antibodies in sera from adult and pediatric subjects immunized with a pneumococcal conjugate vaccine. There was a predominance of IgG1 anti-PnPs antibodies in pediatric sera and IgG2 anti-PnPs antibodies in the adult sera. The IgG1 and IgG2 subclass assignments for the 11 PnPs serotypes in antipneumococcal standard reference serum lot 89-S are useful for quantitating and characterizing immune responses to pneumococcal infection and vaccination regimens.     

ASSOCIATION OF THE C2-DEFICIENCY GENE (C2*QO) WITH THE C4A*4, C4B*2 GENES

C4 typing in thirteen homozygous C2-deficient patients and in a family with heterozygous C2 deficiency showed a strong association of the C2-deficiency gene (C2*QO) with the relatively rare C4A* C4B*2 haplotype.     

The role of C1, C1-inactivator and C4 in modulating immune precipitation

To clarify the mechanism of inhibition of immune precipitation by early components of the classical pathway of complement, aggregation of 125I-BSA-rabbit-anti-BSA antibody complexes was performed in the presence of purified C1, C1-inactivator (C1-In) and C4. C1 delayed the rate of immune precipitation in a concentration dependent manner. This phenomenon was not influenced by the presence of 0.3 mM p-nitrophenyl-p-guanidinobenzoate (NPGB) which inhibits C1 activation. The antiaggregational effect of C1 was reversed by 10 mM EDTA and by C1-In at a C1-In/C1 molar ratio of greater than or equal to 4/1. C1-In was not effective when the reaction was performed in the presence of NPGB. Thus, although the inhibitory effect of C1 on immune precipitation was not dependent upon C1 activation, the formation of C1 was required to observe the effect of C1-In. The addition of C4 to C1 did not modify the slow aggregation of complexes, even when a limiting concentration of C1 was used. C1-In and EDTA were both able to cause similar rapid precipitation of complexes prepared in the presence of C1 and C4, demonstrating that C4 did not play a significant role in delaying the precipitation reaction. However, soluble complexes prepared in the presence of C1 and C4 were specifically precipitated by the addition of excess anti-C4 antibody, attesting to the binding of C4 to immune complexes. These observations suggest that the processing of immune complexes in vivo may not be similar in different classical pathway complement deficiency states.     

Expression of claudins 1, 2, 3, 4, 5 and 7 in various types of tumours

AIMS: Claudins are adhesion molecules present in tight junctions. To evaluate their usefulness as differentiation markers claudins 1, 2, 3, 4, 5 and 7 were studied in 116 epithelial and 92 non-epithelial tumours. METHODS AND RESULTS: Immunoreactivity for all claudins could be seen in different carcinomas. There were, however, tumour type-specific differences in their expression. Lower expression of claudin 2 was seen in breast and prostatic carcinomas, while hepatocellular and renal carcinomas expressed lower levels of claudins 4 and 5. In contrast to epithelial tumours, lymphomas did not express claudins and most soft tissue tumours and naevocytic lesions were negative or showed weaker, mainly cytoplasmic positivity for some claudins. Of non-epithelial tumours, claudin 5 was found only in angiosarcomas and benign vascular tumours, which also showed reactivity for claudins 2, 3 and 7, but was not expressed in any other soft tissue lesions or lymphomas. CONCLUSIONS: The results show that claudins 1, 2, 3, 4, 5 and 7 can be used as markers for epithelial differentiation and to distinguish epithelial neoplasms from lymphomas and selectively also from soft tissue and naevocytic lesions. Since these claudins show type-specific differential expression in epithelial tumours, they may also be of some value in distinguishing different epithelial tumours from each other. Additionally, claudin 5 shows promise as a marker for endothelial lesions compared with other soft tissue lesions.     

Depth absorbed dose and LET distributions of therapeutic 1H, 4He, 7Li, and 12C beams

The depth absorbed dose and LET (linear energy transfer) distribution of different ions of clinical interest such as 1H, 4He, 7Li, and 12C ions have been investigated using the Monte Carlo code SHIELD-HIT. The energies of the projectiles correspond to ranges in water and soft tissue of approximately 260 mm. The depth dose distributions of the primary particles and their secondaries have been calculated and separated with regard to their low and high LET components. A LET value below 10 eV/nm can generally be regarded as low LET and sparsely ionizing like electrons and photons. The high LET region may be assumed to start at 20 eV/nm where on average two double-strand breaks can be formed when crossing the periphery of a nucleosome, even though strictly speaking the LET limits are not sharp and ought to vary with the charge and mass of the ion. At the Bragg peak of a monoenergetic high energy proton beam, less than 3% of the total absorbed dose is comprised of high LET components above 20 eV/nm. The high LET contribution to the total absorbed dose in the Bragg peak is significantly larger with increasing ion charge as a natural result of higher stopping power and lower range straggling. The fact that the range straggling and multiple scattering are reduced by half from hydrogen to helium increases the possibility to accurately deposit only the high LET component in the tumor with negligible dose to organs at risk. Therefore, the lateral penumbra is significantly improved and the higher dose gradients of 7Li and 12C ions both longitudinally and laterally will be of major advantage in biological optimized radiation therapy. With increasing charge of the ion, the high LET absorbed dose in the beam entrance and the plateau regions where healthy normal tissues are generally located is also increased. The dose distribution of the high LET components in the 7Li beam is only located around the Bragg peak, characterized by a Gaussian-type distribution. Furthermore, the secondary particles produced by high energy 7Li ions in tissuelike media have mainly low LET character both in front of and beyond the Bragg peak.     

Functional analysis of congenital stationary night blindness type-2 CACNA1F mutations F742C, G1007R, and R1049W

Congenital stationary night blindess-2 (incomplete congenital stationary night blindness (iCSNB) or CSNB-2) is a nonprogressive, X-linked retinal disease which can lead to clinical symptoms such as myopia, hyperopia, nystagmus, strabismus, decreased visual acuity, and impaired scotopic vision. These clinical manifestations are linked to mutations found in the CACNA1F gene which encodes for the Ca(v)1.4 voltage-gated calcium channel. To better understand the physiological effects of these mutations, three missense mutants, F742C, G1007R and R1049W, previously shown to be mutated in patients with CSNB-2, were transiently expressed in human embryonic kidney (HEK) tsA-201 cells and characterized using whole-cell patch clamp. The G1007R mutation is located in transmembrane segment 5 (S5) of domain III and R1049W is located in the extracellular linker between S5 and the P-loop of domain III. Both mutants produced full length proteins that targeted to the membrane but did not support ionic currents. In 20 mM Ba(2+), F742C (S6 domain II) produced a approximately 21 mV hyperpolarizing shift in half activation potential (V(a[1/2])) and a approximately 23 mV hyperpolarizing shift in half inactivation potential (V(h[1/2])). Additionally, F742C displayed slower inactivation kinetics and a smaller whole cell conductance (G(max)). In physiological 2 mM Ca(2+), F742C produced a approximately 19 mV hyperpolarizing shift in V(a[1/2]). These findings suggest that the pathology of CSNB-2 in patients with these missense mutations in the Ca(v)1.4 calcium channel is the result in either a gain of function (F742C) or a loss of function (G1007R, R1049W).     

Significance of System L Amino Acid Transporter 1 (LAT-1) and 4F2 Heavy Chain (4F2hc) Expression in Human Developing Intestines

To clarify the significance of expression of system L amino acid transporter 1 (LAT1) and 4F2 heavy chain (4F2hc) in the developing intestine, immunohistochemical investigation and molecular analysis were performed in the human embryonic and/or fetal intestines, ranging from 28–30 days to 34–35 weeks gestation. The molecular analysis for the expression of LAT1 and 4F2hc mRNAs was done in the pure epithelial cell samples prepared after laser assisted microdissection. The immunoreactivities against LAT1 and 4F2hc were detected along the basolateral cell membrane of the primitive gut epithelium at 28–30 days gestation. According to advance in gestational age of up to 24–25 weeks gestation, the immunoreactivity of LAT1 was predominantly observed in the supranuclear cytplasmic localization with a granular or dot-like staining pattern. Up to 8–9 weeks gestation, the immunoreactivity of 4F2hc showed almost the same as that of LAT1. However, after the age of 12–13 weeks gestation, the immunoreactivity of 4F2hc was predominantly localized along the cell membrane of apical surface of the epithelial cells. No apical and linear membranous localization of LAT1 was observed until nearly 20 weeks gestation. In the late gestational stage, both the immunoreactivities against LAT1 and 4F2hc were localized along the apical surface of the epithelial cells. In conclusion, the expression of LAT1 and 4F2hc in early developing intestine suggests they have a more important role in cell proliferation rather than functional differentiation. The predominant cytoplasmic localization of LAT1 during mid-fetal life seems to be largely inactive as amino acid transporter. On the other hand, the apical and linear membranous co-localization of LAT1 and 4F2hc in the late fetal life suggests that these molecules may play a role as a functional amino acid transporter in the fetal intestinal epithelium.     

The fluid-phase binding of human C4 and its genetic variants, C4A3 and C4B1, to immunoglobulins

Covalent binding of the fourth complement protein, C4, to immune complexes is an important first step in the complement mediated processing of the complexes. Many of the initial encounters between the proteins of the complement system and antigen and antibody occur in solution, and prior to this report, studies of the interactions between them have focused on complement binding to preformed immune precipitates that most likely are not found in vivo. We have characterized the covalent binding of C4b to immunoglobulin molecules in a fluid-phase system consisting only of antibody in solution and purified C4 and C1s. We demonstrate that human C4b binds to IgG in the fluid phase, that its covalent binding is predominantly to the heavy chain of IgG, and that the covalent linkage is by either amide or acyl ester bonds. In addition, we compare the covalent binding efficiencies of two genetic variants of C4, C4A3 and C4B1, to IgG. C4A3 binds 3-4 times more IgG than C4B1 over a range of C4 concentrations, and C4A3 has a higher binding efficiency than C4B1 for IgM, IgA, IgG2a and F(ab')2 as well as for a protein antigen, BSA. Furthermore, we found that whereas C4A3 is bound to immunoglobulins in the fluid-phase predominantly by amide linkage, C4B1 is bound by either amide or acyl ester bonds. The results presented here suggest that the covalent binding efficiency of C4A3 and C4B1 to IgG is similar to that reported for their covalent binding to small molecules.     

Analysis of Ig subclass deficiency: First reported case of IgG2, IgG4, and IgA deficiency caused by deletion of C alpha 1, psi C gamma, C gamma 2, C gamma 4, and C epsilon in a Mongoloid patient

BACKGROUND: IL-4 and IL-13 have been shown to be critical for expression of the asthma phenotype in a murine model and may modulate human fibroblast function. OBJECTIVE: We hypothesized that IL-4 and IL-13 would increase airway fibroblast proliferation and reduce the ability of dexamethasone to decrease this proliferation. METHODS: Six subjects with severe asthma, 5 subjects with mild asthma, and 5 healthy subjects underwent bronchoscopy with endobronchial biopsy. Biopsy specimens were placed in Dulbecco modified Eagle medium and cultured, and only fibro-blasts from the first and second passages were evaluated. Cells were incubated with IL-4 (50 ng/mL), IL-13 (10 ng/mL), and the combination for 48 hours in the presence and absence of dexamethasone, 10(-7) mol/L, and 10(-8) mol/L. Fibroblasts were also incubated with IFN-gamma at 50 ng/mL to assess the response of a T(H)1 cytokine on proliferation. RESULTS: Fibroblast proliferation, determined by (3)H-thymidine incorporation, was significantly increased by IL-4 in subjects with mild asthma as compared with IL-4 in subjects with severe asthma and healthy subjects (P =.003), IL-13 (P =.011), and the combination (P =.004). Dexamethasone also increased proliferation in the group with mild asthma as compared with the group with severe asthma and the healthy group (10(-7) mol/L, P =.02; 10(-8) mol/L, P =.02). IFN-gamma did not significantly alter airway fibroblast proliferation. CONCLUSION: IL-4, IL-13, and dexamethasone all significantly increased fibroblast proliferation in subjects with mild asthma.     

Level of complement activity and components C1, C4, C2, and C3 in complement response to bacterial challenge in malnourished rats.

In experimentally induced malnutrition in rats, there was no significant difference between the measured level of complement activity of the classical pathway (50% hemolytic complement [CH50]) and that of the alternative pathway (ACH50), although the levels of complement components C1, C4, C2, and C3 were depressed significantly. The complement activity showed a temporary elevation with a peak at 2 or 3 days after bacterial challenge with Staphylococcus aureus in rats, and we call this the complement response. After 3 days, CH50 and C3 in the malnourished rats and ACH50, CH50, and C3 in the well-nourished rats showed a significant increase, and C1, C4, and C2 in both groups tended to elevate. On the basis of these observations, the significance of the elevation of C3 in the complement response to bacterial infection showed a strong influence by enhancing the activation of both the classical and the alternative pathways, since C3 is known to be the junction of both complement pathways. In this way, C3 responded to an earlier stage than did the other components and may contribute to maintaining the body defense system against infection.     

Effects of H-2 haplotype and gender on the lifespan of A and C57BL/6 mice and their F1, F2, and backcross offspring

The effects of H-2 type and gender on the lifespan of A and C57BL/6 mice and their F1, F2, and backcross offspring were studied. The proportion of mice remaining alive, percent survivors, was calculated at monthly intervals for each mating group. Statistical analyses of these survival data showed that, in agreement with studies from other laboratories, C57BL/6 (H-2b) mice lived significantly longer than A (H-2a) mice. When the survival curves for A and C57BL/6 backcross and F2 offspring were analyzed to test for the presence of an H-2 effect on mouse lifespan, no statistically significant association was detected, although a trend toward increased 10th decile survivorship among female H-2a mice was noted. The mice used in this study were also evaluated for the presence of an effect of gender on lifespan. Female mice of the A strain, and F1, F2, and backcross groups, but not the C57BL/6 mice, exhibited significantly longer lifespans when compared with their male counterparts. Thus these data show a significant gender effect, but only a trend towards an H-2 effect, on the lifespan of A and C57BL/6 mice and their F1, F2, and backcross offspring.     

TRAF4通过泛素化E2F1调控乳腺癌细胞凋亡

目的 探讨肿瘤坏死因子受体相关因子4(TRAF4)是否通过E2F1转录因子(E2F1)调控乳腺癌细胞凋亡.方法 流式细胞分析检测TRAF4对乳腺癌细胞凋亡的调控情况以及E2Fl在其中的作用;Western blot检测TRAF4、精氨酸甲基转移酶5(PRMT5)和E2F1之间的调控关系;免疫荧光及免疫共沉淀检测TRAF...     

Arachidonic acid pathway members PLA2G7, HPGD, EPHX2, and CYP4F8 identified as putative novel therapeutic targets in prostate cancer

The arachidonic acid and prostaglandin pathway has been implicated in prostate carcinogenesis, but comprehensive studies of the individual members in this key pathway are lacking. Here, we first conducted a systematic bioinformatic study of the expression of 36 arachidonic acid pathway genes across 9783 human tissue samples. The results showed that the PLA2G7, HPGD, EPHX2, and CYP4F8 genes are highly expressed in prostate cancer. Functional studies using RNA interference in prostate cancer cells indicated that all four genes are also essential for cell growth and survival. Clinical validation confirmed high PLA2G7 expression, especially in ERG oncogene-positive prostate cancers, and its silencing sensitized ERG-positive prostate cancer cells to oxidative stress. HPGD was highly expressed in androgen receptor (AR)-overexpressing advanced tumors, as well as in metastatic prostate cancers. EPHX2 mRNA correlated with AR in primary prostate cancers, and its inhibition in vitro reduced AR signaling and potentiated the effect of antiandrogen flutamide in cultured prostate cancer cells. In summary, we identified four novel putative therapeutic targets with biomarker potential for different subtypes of prostate cancer. In addition, our results indicate that inhibition of these enzymes may be particularly powerful when combined with other treatments, such as androgen deprivation or induction of oxidative stress.