Healthy Early Preantral Follicle Can Be Obtained in a Culture of Frozen–Thawed Human Ovarian Tissue of 32 Weeks

The objective of this study was to report morphological and functional evidence of a well-preserved preantral follicle recovered from human frozen–thawed ovarian tissue in a long-term culture. The tissue was originally obtained from a 26-year-old woman with breast cancer. The ovarian cortex was collected by laparoscopy and frozen/thawed and cultured for 32 weeks in minimum essential medium α-MEM, supplemented with insulin transferrine selenite (ITS), human serum (HS), antibiotics, follicle-stimulating hormone (FSH). and N-acetyl cysteine (NAC). Thawed tissue samples were examined by light microscopy (LM), transmission electron microscopy (TEM), and real-time RT-PCR. LM examination of cortical pieces after 32 weeks of culture showed a healthy early preantral follicle; TEM and real-time PCR confirmed its good state of preservation. The synergy in action of NAC and FSH plays an important role in follicle growth of ovarian tissue cultures. For the first time a well-preserved preantral follicle was found in a culture of frozen–thawed human ovarian tissue.     

Sequence Analysis of the 5′-Flanking Region of the Gene Encoding Human HOXA-7

We have isolated and characterized the immediate 5-flanking region (886 bp) of the gene encoding human HOXA-7. When the total sequence was compared with those of mice, 93% of the 3 518 bp (nt 370–886) sequences were identical, in which the 245 bases just preceding theAUG initiator codon (nt 614) was as highly conserved as in the coding region (nt 614–886). Sequences further upstream (nt 1–370) by comparison were highly diverged. In the 245 bp region, 8 stop and 3 initiation (including the initiator) codons were located, and a 50-aa long presumptive polypeptide was encoded. Nucleotide sequence analysis revealed three Spl and oneAP2 binding sites, as well as one CAATbox. However, there was no consensus sequence for a TATA box in the 5 flanking region. One RARE repeat, one krox20 and three Hox-PBC binding sites were detected. Since many of the factor recognition sites were located in the immediate 5 flanking sequences of a highly-conserved region, it might be speculated that a regulatory mechanism for Hox gene expression is conserved throughout the evolution and one possible mechanism could be at the post-translational level.     

The Development of GABA-containing Neurons in the Broca''''s Area of Human Fetus

Prenatal development of GABA-containingneurons in the Broca's area of human fetusranging in age from 17 weeks to full-termnewborn infant were studied by means ofGABA immunocytochemistry.The GABA-con-taining neurons were found in marginal layer,cortical plate,intermediate zone and subventri-     

Investigation of the SV40 – Human Cancer Association: Look for the Full Signature of the Virus

In the controversy about the association of simian virus 40 with human cancers, the greatest problem is the ascertainment of SV40 exposure. This difficulty would be resolved if one were to look for all components of SV40 infection. How does SV 40 circulate in the human community? Do cancer patients with SV40-positive tumors have serological correlates of SV 40 infection and of SV40-induced cancer? SV40 association with a cancer should be studied in the context of the known risk factors for that cancer. The tumor cell-virus relationship should be characterized with respect to viral integration and viral localization to the tumor cell. Specimens should be masked and the assays should include panels of specimens to estimate analytic sensitivity and specificity. In view of the rarity of some of the tumors reported to be associated with SV40, a multi-institutional investigation initiated and coordinated by the NIH would be most effective.     

Rhythmic Photostimulation and the Number of α-Rhythm Dipoles in the Human Brain

The numbers of -rhythm equivalent current dipoles (ECD) arising in the human brain before and during rhythmic photostimulation at the -rhythm frequency was studied in six healthy adult subjects. Dipoles were calculated using a single-dipole model for the whole of the -range and three subranges by solution of inverse equations in a three-layer model of the head obtained by simultaneous use of EEG data and MRI tomograms of the subjects' heads. The number of apparent ECD was significantly associated with rhythmic photostimulation and depended on the phase of the -rhythm wave at which stimulation started and on the type of visual illusion (circle, spiral, grid) appearing during this time. The relationship between these data and the hypothetical wave process scanning the human visual cortex at the frequency of the -rhythm is discussed.     

Mechanism of Action of Prothynosin α in the Human Autologous Mixed Lymphocyte Response

Abstract

Prothymosin α(Prota), an immunologically active polypeptide derived initially from rat thymus, and now pig thymus, was tested for its effect on autoantigen-induced human T cell proliferation in vitro. Pig ProTa was found to enhance the autologous mixed lymphocyte response (auto-NLR). Optimum enhancement was achieved at doses which varied among different donors. Treatment of the stimulatory monocytes with ProTa resulted in considerably higher auto-MLR responses as compared to those with non treated monocytes. ProTa was without effect on T lymphocytes. In contrast, T lymphocytes exhibited enhanced proliferative activity when treated with ProTa in the environment of autologous monocytes. Horeover, supernatants from cultures of monocytes incubated with ProTa(ProTa-sup) were also shown to enhance the human auto-NLR either after addition in cultures or after preincubation with responder T lymphocytes. In addition, ProTa-sup did not demonstrate any detectable inter-leukin I (IL 1) or interleukin 2 (1L 2)-like activity Furthermore, ProTa-sup induced an increase in IL 2 production in auto-NLR cultures. The enhancement of T-cell proliferation and IL 2 production by ProTa-sup was maximal when this material was added at the beginning of the auto-MLR, and no effect of ProTa-sup was seen if the latter was added 3 days after initiation of the culture. Finally, Prota-sup was also shown to increase the expression of IL 2 receptors on T lymphocytes activated in the auto-MLR. These studies suggest that ProTa enhances the human auto-HLR through ProTa -sup which is released after interaction of monocytes with ProTa ProTa-sup then increases directly T lymphocyte proliferation by elevating IL 2 production and expression of IL 2 specific receptors on autoactivated T lymphocytes.     

Severity of Staphylococcus aureus Infection of the Skin Is Associated with Inducibility of Human β-Defensin 3 but Not Human β-Defensin 2

Gram-positive bacteria are the predominant cause of skin infections. Antimicrobial peptides (AMPs) are believed to be of major importance in skin''s innate defense against these pathogens. This study aimed at providing clinical evidence for the contribution of AMP inducibility to determining the severity of Gram-positive skin infection. Using real-time PCR, we determined the induction of human β-defensin 2 (HBD-2), HBD-3, and RNase 7 by comparing healthy and lesional mRNA levels in 32 patients with Gram-positive skin infection. We then examined whether AMP induction differed by disease severity, as measured by number of recurrences and need for surgical drainage in patients with Staphylococcus aureus-positive lesions. We found that HBD-2 and -3, but not RNase 7, mRNA expression was highly induced by Gram-positive bacterial infection in otherwise healthy skin. Less induction of HBD-3, but not HBD-2, was associated with more-severe S. aureus skin infection: HBD-3 mRNA levels were 11.4 times lower in patients with more than 6 recurrences (P = 0.01) and 8.8 times lower in patients reporting surgical drainage (P = 0.01) than in the respective baseline groups. This suggests that inducibility of HBD-3 influences the severity of Gram-positive skin infection in vivo. The physiological function of HBD-2 induction in this context remains unclear.Gram-positive bacteria, in particular Staphylococcus aureus and group A streptococci (GAS), are by far the most common cause of skin infections (3). Human skin expresses a variety of antimicrobial peptides (AMPs), which are believed to be of major importance in innate defense against pathogens (21, 25). These structurally diverse molecules are currently best categorized into the following two major classes: (i) constitutively expressed AMPs, such as RNase 7, which are present at high levels in healthy skin under physiological conditions; and (ii) inducible AMPs, such as human β-defensin 2 (HBD-2) and HBD-3, which are found at low concentrations in healthy skin but can be upregulated by inflammatory and bacterial stimuli.Our current understanding of the role of AMPs in preventing and limiting human Gram-positive skin infections is based largely on studies demonstrating their in vitro antibacterial activity, their content in healthy and inflamed skin, and their expression in cultured keratinocytes after exposure to bacteria, bacterial components, and proinflammatory cytokines (1, 4-8, 11, 12, 16, 18, 22, 24). Direct evidence from in vivo studies supporting the involvement of AMPs in defense of human skin against these infections is scarce. Attempts to clarify AMP function by comparing patients with atopic dermatitis (AD) and psoriasis and attributing the known higher propensity of AD patients for S. aureus colonization and infection to differences in AMP expression have led to conflicting results (2, 15). Hence, there is a need to conduct studies of individuals with otherwise healthy skin to elucidate the clinical role of AMPs in Gram-positive skin infection.The strikingly high in vitro activities of RNase 7 and HBD-3 against S. aureus and GAS (6, 8, 10) suggest a particular function of these AMPs in cutaneous defense against these clinically important pathogens. Recent work by our group provided strong evidence for a key role of constitutively expressed RNase 7 in protecting healthy skin against S. aureus infection in vivo (26). For the inducible AMPs HBD-2 and -3, there was no association of constitutive, noninduced mRNA expression with S. aureus skin infection (26). Whether induced expression of these AMPs is clinically important in defense against Gram-positive skin infections is still not known, although this has been hypothesized widely (1, 25). In a recent review, for instance, Schröder and Harder speculated that “recurrent skin infections may be associated with a dysregulation of antimicrobial peptide and protein production caused by lack of induction” (21). In particular, the high in vitro activity of HBD-3 against S. aureus suggests that interindividual differences in its inducibility may explain some of the variability in presentation and course of skin infections caused by this pathogen, which range from superficial self-limiting disease to deep-seated and recurrent furunculosis (19, 20).In this study, we investigated the induction of RNase 7, HBD-2, and HBD-3 in previously healthy individuals suffering from skin infection caused by S. aureus and GAS. Inducibility, or the skin''s capacity to increase AMP expression in response to infection, was approximated by observed AMP induction and was defined accordingly, as the difference between the lesional mRNA concentration and the baseline concentration in healthy skin. To explore the potential role of AMP inducibility in preventing and limiting S. aureus infection of the skin, we studied whether AMP induction was associated with two clinically relevant measures of disease severity, namely, the need for surgical drainage and the number of recurrences.     

Trends in Human Fecal Carriage of Extended-Spectrum β-Lactamases in the Community: Toward the Globalization of CTX-M

SUMMARY

In the last 10 years, extended-spectrum β-lactamase-producing enterobacteria (ESBL-E) have become one of the main challenges for antibiotic treatment of enterobacterial infections, largely because of the current CTX-M enzyme pandemic. However, most studies have focused on hospitalized patients, though today it appears that the community is strongly affected as well. We therefore decided to devote our investigation to trends in ESBL-E fecal carriage rates and comprehensively reviewed data from studies conducted on healthy populations in various parts of the world. We show that (i) community ESBL-E fecal carriage, which was unknown before the turn of the millennium, has since increased significantly everywhere, with developing countries being the most affected; (ii) intercontinental travel may have emphasized and globalized the issue; and (iii) CTX-M enzymes, especially CTX-M-15, are the dominant type of ESBL. Altogether, these results suggest that CTX-M carriage is evolving toward a global pandemic but is still insufficiently described. Only a better knowledge of its dynamics and biology will lead to further development of appropriate control measures.     

Characteristics of Radioimmunoassays for the α- and β-Subunits of Human Luteinizing Hormone

Abstract

The binding characteristics and specificities of the National Hormone and Pituitary Program (NHPP) kits for the radioimmunoassay of the alpha- and beta-subunits of human luteinizing hormone (hLH-α and hLH-β) were studied, as well as the specificities of the anti-hLH and anti-human follicle stimulating hormone (anti-hFSH) antisera distributed by the same organization. The affinity constants of the anti-hLH-α and anti-hLH-β antisera were calculated at 157 ± 8.4 nM^?1 and 109 ± 7.4 nM^?1, respectively. Both antisera were highly specific with regard to the other subunit. However, in the homologous hLH-α RIA, native hLH cross-reacted at 21.9%, hFSH at 17.5% and hTSH at 7.9%. The alpha-subunit of the human chorionic gonadotropin, hCG-α, was equipotent with the hLH-α standard in this assay. In the homologous hLH-β RIA, hLH showed a cross-reactivity of 14.7% while the cross-reactivities of hCG-β, hFSH and hTSH were 3.5%, 1.2% and 0.6%, respectively. The anti-hFSH antiserum was highly specific, while the anti-hLH antiserum showed non parallel competition curves. With this knowledge of the specificity of each antiserum, corrections can be properly made for the assays of hLH, hLH-α and hLH-β while the hFSH RIA can be used without correction for the presence of the three other components.     

Human equivalent of mouse disorganization: Has the case been made?

Temtamy and McKusick suggested mouse disorganization (Ds) as a model for human tibial agenesis, fibular duplication and mirror foot, but the concurrent papers by Winter and Donnai and Donnai and Winter in 1989 kindled interest and led to continued reports of patients hypothesized as human equivalent of Ds (HEDs). Subsequent reports have tended to follow one or other of the two categories outlined; (1) band/constriction with additional anomalies unexplained by bands (ABS); (2) patterns of malformation interpreted as resembling mouse Ds (non-ABS). A review of a series of cases led to a re-read of the original Ds mouse reports by Hummel in 1958 and 1959 and examination of current literature in an attempt to assess the strength of the argument that the patients might represent HEDs. Key to the approach was a paragraph in Hummel's introduction; "some of the developmental anomalies … from action of Ds are similar to those caused by other …genes…teratogens… others are unique…" The corollary is a patient is likelier to represent human DS if the anomaly(s) match these unique malformations/patterns. Presence of anomalies not specifically noted in Ds would weaken the argument for human equivalence. Reports of possible HEDs were ascertained using PubMed and literature cited by authors subsequent to the 1989 papers, up to and including January, 2010. This paper gives an overview of HEDs patients reported and concludes that the ABS type, even with non-band associated anomalies, is not likely to often represent HEDs. Many non-ABS HEDs patients had equally valid alternative hypothesis or diagnoses, malformations unreported or unusual for the Ds mouse, and/or paucity of the more unusual anomalies of the Ds mouse.     

3D CFD Simulation of Pulsatile Blood Flow in the Human Aorta

INTRODUCTION　　The human aorta is the majorblood vessel of complex geometry including curva-tures in multiple planes,branches at the apex of the arch,significant tapering andwith distensible vessel wall ( as shown in Fig.1 ) . The blood flow structures in theaorta are very complex and attribute a lot to the development of atherosclerotic le-sions,which always occur in the vicinity of arterial branches,curvatures and bifur-cations〔1～ 5〕.In order to understand the complex nature of the …     

Getting to Know the Human Brain

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Ovarian carcinoma. Do the subtypes reflect different diseases?

Lack of therapeutic options and poor reproducibility of histopathological subtypes have been the reasons that ovarian carcinomas are currently treated as monolithic entity. Histopathological grading is used to identify those patients who can be spared adjuvant therapy. With slight modifications of the WHO based subtype classification we have shown that subtypes (i.e. serous, endometrioid, clear cell, mucinous) can be reproducibly used to stratify patients according to disease-specific survival. As these pathologically identifiable subtypes have different epidemiologic and genetic risk factors, precursor lesions, molecular abnormalities and clinical behaviour, screening and management strategies have to be subtype-specific.     

Antigenic Regions on the β Chain of Human Chorionic Gonadotropin and Development of Hormone Specific Antibodies

Five peptides corresponding to four regions of the β chain of human chorionic gonadotropin (hCG), were synthesized, purified and characterized. The four regions studied were selected on the basis of sequence differences between the β chain of hCG (βhCG) and the β chains of related hormones. The peptides were found to bind rabbit and mouse anti-hCG antibodies as well as rabbit anti-β chain antibodies, but did not bind antibodies against the α chain or against other hormones. All the peptides, even in their free form, were able to elicit high titer antisera in both rabbits and mice. In all cases, anti-peptide antisera bound to the immunizing peptide as well as to the native hCG and the isolated β chain. These anti-peptide antisera did not bind to unrelated peptides, the α chain of hCG or to other hormones with very similar β chains such as human luteotropic hormone (hLH), ovine luteotropic hormone (oLH) and equine chorionic gonadotropin (eCG). Since the areas represented by these peptides elicit antibodies that are specific for human βhCG, they can formulate the basis for the development of discriminatory reagents for the β chain of hCG.     

Analysis of the CDR3 Length Repertoire and the Diversity of TCRα Chain in Human Peripheral Blood T Lymphocytes

Analysis of complementarity determining region 3 （CDR3） length of T lymphocyte receptors （TCRs） by immunoscope spectratyping technique has been used successfully to investigate the diversity of TCR in autoimmune diseases and infection diseases. In this study, we investigated the patterns of CDR3 length distribution for all 32 TCR AV gene families in human peripheral blood lymphocytes of four normal volunteers by the immunoscope spectratyping technique. It was found that PCR products exhibited an obscure band on 1.5% agarose gel electrophoresis. Each TCR AV family exhibited more than 8 bands on 6% sequencing gel electrophoresis. The CDR3 spectratyping of all TCR AV families showed a standard Gaussian distribution with different CDR3 length, and the expression frequency of CDR3 was similar among the gene families. Most of CDR3 in TCR AV family recombine in frame. However, some of the CDR3 showed out-of frame gene rearrangement. Additionally, we found that in some of TCR AV families there were 18 amino acid discrepancies between the longest CDR3 and shortest CDR3. These results may be helpful to further study the recombination mechanism of human TCR genes, the TCR CDR3 gene repertoire, and the repertoire drift in health people and disease state. Cellular ＆ Molecular Immunology.