Thyroid Hormones and the Developing Brain

Thyroid hormones are critical for the development of the brain during pregnancy and in the early postnatal period. Environmental factors (such as iodine deficiency), maternal thyroid dysfunction, and neonatal thyroid malformations may cause permanent neurological deficits with severe mental retardation and cretinism.

Timing and genetic factors contribute to the expression of neurological deficits.The importance of timing may be illustrated by differences in neurological outcome in endemic cretinism and in congenital hypothyroidism. In endemic cretinism, the resulting maternal iodine deficiency causes fetal brain hypothyroidism throughout gestation. Early fetal hypothyroidism, before the fetal thyroid gland is active, results in profound and irreversible mental retardation, deaf–mutism, spasticity, and ataxia, though thyroid function may be normal after birth. Endemic cretinism may be prevented by administration of iodine starting in the first trimester of pregnancy. Despite knowledge of this simple remedy, iodine deficiency remains the most prevalent preventable cause of mental retardation worldwide.

In congenital hypothyroidism, fetal thyroid gland hypofunction is compensated by maternal thyroid hormone secretion. Untreated neonates with congenital hypothyroidism demonstrate symptoms of hypothyroidism and growth retardation together with mental retardation, spasticity, and language deficits. However, neurological deficits in congenital hypothyroidism are less severe than in endemic cretinism. Early thyroid hormone replacement prevents most neurological symptoms in neonatal hypothyroidism, though mild deficits in cognitive functioning may persist.

The importance of genetic factors may be illustrated by a study performed in iodine-deficient areas in China. This study found an association between DIO2 polymorphisms and mental retardation in children. Individual differences in tolerance to low thyroid hormone concentrations during brain development may be explained byDIO2 variations that affect conversion of T₄ to T₃ in the brain.

Genetic factors, such as mutation in the MCT8 gene, may cause selective neuronal hypothyroidism with normal or even increased serum T₃ concentrations. Intraneuronal hypothyroidism, similar to systemic hypothyroidism during fetal life, results in severe mental retardation, hypotonia, and absence of speech. This mutation provides a molecular basis for the Allan–Herndon–Dudley syndrome.

Source :

Thyroid Disease and Mental Disorders: Cause and Effect or Only comorbidity?

Robertas Bunevièius; Arthur J. Prange Jr- Current Opinion Psychiatry.

Galectin-3 and HBME-1 expression in well-differentiated thyroid tumors with follicular architecture of uncertain malignant potential

Well-differentiated encapsulated tumors of the thyroid gland with a follicular architecture may cause diagnostic difficulties due to the presence of incomplete or equivocal capsular penetration—which may induce the suspicion of follicular carcinoma—or the occurrence of focal nuclear changes such as clearing, overlapping, grooves and pseudoinclusions, which may raise the possibility of the follicular variant of papillary carcinoma. Depending on the severity of these changes and the bias of the observer, terms such as atypical adenoma, 'hybrid' carcinoma, and—more recently—well-differentiated carcinoma not otherwise specified were proposed. In a recent editorial from the Chernobyl Pathologists Group the suggestion was made to label tumors with 'borderline' features as well-differentiated tumor of uncertain malignant potential (WDT-UMP) in the presence of questionable papillary carcinoma-type nuclear changes with or without questionable capsular penetration, or follicular tumor of uncertain malignant potential (FT-UMP) in the presence of questionable capsular penetration without nuclear changes.

Several immunocytochemical markers of malignancy have been claimed to be useful to distinguish follicular adenoma from carcinoma and also to identify papillary carcinoma and its variants, both in surgical and cytological specimens. These include Galectin-3,HBME-1,cytokeratin 19, thyroperoxidase, and high mobility group (HMG)-Y proteins.

Galectin-3 is a member of a family of -galactoside binding animal lectins. This protein is expressed in many tissues and cells at both nuclear and cytoplasmic levels and has multiple functions, including cell–cell and cell–matrix adhesion, cell growth, neoplastic transformation and spread, cell cycle regulation and apoptosis, and cell repair processes. Galectin-3 has been found to be increased in several human malignant tumors, including well-differentiated follicular-derived thyroid carcinomas. HBME-1 was originally described as a marker of normal and malignant mesothelial cells, since it recognizes a currently unknown antigen expressed by those cells. It was later shown to also stain most papillary thyroid carcinomas, and also a fraction of follicular carcinomas, while adenomas are generally negative. In a recent study on oxyphilic tumors of the thyroid,Galectin-3 and HBME-1 expression were found to be related to molecular alterations such as PAX8-PPARgamma translocations and ras oncogene mutations.

The aim of the present study was to investigate the expression and the possible diagnostic role of these two markers in a series of well-differentiated thyroid neoplasms with a follicular architecture that fulfilled the criteria for WDT-UMP or FT-UMP, as proposed by Williams et al.

Tissues

The diagnosis was based on the proposed criteria for these tumors. In particular, WDT-UMP was represented by an encapsulated tumor composed of follicular cells having incompletely developed papillary carcinoma-type nuclear changes. In these tumors, no blood vessel invasion was present, while capsular penetration was either absent or questionable. FT-UMP was defined as an encapsulated tumor with follicular architecture, composed of conventional or oxyphilic cells, and having incomplete or questionable capsular penetration, but neither vascular invasion nor papillary carcinoma-type nuclear changes. Based on these criteria, 13 cases qualified as WDT-UMP (one of these was a small nodule in the context of Hashimoto's thyroiditis) and eight as FT-UMP. In all, 14 cases of follicular variant of papillary carcinoma and 15 follicular adenomas were collected from the same Institutions and served as control groups. All cases had hematoxylin & eosin (H&E) stains available for review and paraffin blocks for immunohistochemical stainings.

Immunohistochemistry

A purified monoclonal antibody to Galectin-3 was used at the dilution of 1/200, as previously described. This monoclonal is now commercially available (Mabtech, Naka, Sweden). HBME-1 antibody was purchased from DakoCytomation (Glostrup, Denmark) and used at the dilution of 1/50. Both markers were revealed with a biotin-free immunoperoxidase procedure (EnVision, DakoCytomation), preceded by heat-induced antigen retrieval (three 3-min microwave oven passages at 750 W in citrate buffer). Positive controls for immunohistochemistry were a papillary thyroid carcinoma for Galectin-3 and a pleural mesothelioma for HBME-1. Macrophages at the periphery of goiter nodules and endothelial cells served as positive internal controls in most cases. The immunoreactivity was scored as negative, focally positive (+: less than 25%), positive (++: 25–50%) or diffusely positive (+++: more than 75%), based on the extent of the reaction. A case was scored as positive only when strong signals in the cytoplasm or along the cell membrane were detected for Galectin-3 and HBME-1, respectively.

Source :

Modern Pathology (2005) 18, advance online publication.

Highlights in Gynecology-Cervical Cancer Screening and the Role of HPV-DNA Testing

Dr. Alan G. Waxman MD, MPH (University of New Mexico), and Dr. Kenneth Noller, MD (New England Medical Center Boston, Massachusetts), both dealt with the issue of changes in cytology screening and the role of human papillomavirus (HPV)-DNA testing. Invasive cervical cancer has been reduced by 70% in the United States through the use of cervical cytology. There has never been a national screening program here; rather, patients are screened opportunistically. Thus, the affluent are often over-screened and the indigent are under-screened. Most women with cervical cancer either have never been screened or have not had a Pap test in at least 5 years. Although we will never be able to detect every case of cervical cancer, the majority would be identified if there were universal testing.

Variations in Cervical Screening Guidelines -- When Should Screening Commence?

Guidelines for cervical cancer screening from The American College of Obstetricians and Gynecologists (ACOG), The American Cancer Society (ACS), and the U.S. Preventive Services Task Force (USPSTF) have all been recently published. Dr. Waxman noted that the different agencies recommend much that is similar. ACOG Practice Bulletin #45 recommends beginning screening no later than age 21 or approximately 3 years after onset of vaginal intercourse. However, he stressed the importance of providing appropriate preventive healthcare to adolescents not yet requiring a Pap test, such as screening for sexually transmitted infections and contraceptive counseling.

The reason why the recommendation to initiate cervical screening with onset of sexual activity was changed to initiation 3 years after onset of sexual activity is because although the cervix is extremely vulnerable to HPV infection in adolescence, this infection is usually transient and usually clears in 1 to 2 years. In addition, most dysplasias in the adolescent regress spontaneously. Most importantly, cancer develops over years and is rare in the first 2 decades of life or within 3 years of onset of intercourse. Lastly, by limiting the screening in these patients who are highly susceptible yet for the most part who only have transient dysplasia will reduce the anxiety and morbidity from unnecessary follow-up procedures.

Why even designate an age limit of 21? The ACS rationale is that providers may not get an adequate sexual history. Adolescents also may be unwilling to disclose prior consensual or nonconsensual intercourse. Dr. Waxman made sure to point out that there is still room for individualization of screening. ACOG's practice bulletin acknowledges the "unpredictable nature of follow-up in younger women," and therefore states that screening may be started earlier at the physician's discretion. The ACS also states that "Provider discretion and patient choice should guide screening in women ≥21 who have never had vaginal intercourse."

When Is it Appropriate to Cease Cervical Screening?

Differences in opinion exist, however, with regard to when to stop screening women. The USPSTF recommends against routine screening after age 65 if recent screening has been adequate, normal Pap test results were obtained, and the patient is not otherwise at high risk for cervical cancer. The ACS recommends that women with an intact cervix may elect to discontinue screening at age 70 or older after they have had 3 or more documented consecutive satisfactory negative Pap test results and no abnormal Paps within the 10 years before age 70 and if they are at low risk and in good health. ACOG states that evidence is too inconclusive to set an upper age limit for cervical cancer screening. However, if screening is discontinued, it is recommended that risk factors be assessed during annual exam to determine whether restarting screening is appropriate.

The logic for setting upper limits for age for cervical cancer screening is that the incidence of cervical cancer plateaus at about age 65 for most US women and that new cervical cancers in older women are mostly seen in unscreened and under-screened women. By way of example, in a cohort of 2561 postmenopausal women who participated in the Heart Estrogen/progestin Replacement Study (HERS) and who had gone 1 to 2 years without screening after having a negative Pap, 110 required diagnostic work-ups for abnormal Paps. This led to 231 interventions all to find a single case of mild-to-moderate dysplasia. We must consider the morbidities associated with false-positive tests, which include anxiety, discomfort, and increased health costs. Notably, in a survey of ACOG physicians conducted by Dr. Noller, 74% never stop doing Paps.

Screening vaginal cytology after hysterectomy, as per the ACOG recommendations, may be discontinued in women who have had a total hysterectomy for benign indications. However, if a woman has had prior cervical intraepithelial neoplasia (CIN) 2 or 3, annual screening should be continued until 3 consecutive negative smears are obtained. These recommendations are based on the fact that vaginal cancer is extremely rare. A study by Pearce found no significant pathology in 9610 patients post vaginal hysterectomy even though 1.1% had abnormal cytology; almost all were false positives.

What Is the Appropriate Screening Interval?

The next issue broached was the safety of increasing the intervals between Pap smears. It seems that previously well-screened women are at little risk of developing squamous cancer within 3 years of their last negative Pap. The incidence of CIN 2+ in women with at least 3 prior negative screening tests according to the National Breast and Cervical Cancer Program is extremely low, with no cancers identified and only 16 cases of CIN 2+ out of 32,230 women screened (0.05%). Numerous studies have shown little difference in detection of new cancers with 1-, 2-, or 3-year screening intervals. This along with the potential harm, inconvenience, and cost from over-screening have made this issue quite pertinent. Sawaya and colleagues have calculated that 209,000 Paps and 11,502 colposcopies would need to be done in women aged 45 to 60 with 3 negative prior Paps to prevent a single case of cervical cancer.ACOG Practice Bulletin #45 still allows room to individualize screening interval, depending on the weight of risk factors, ability to determine past screening history, and ability to monitor the patient in the future. Yearly testing may be warranted in individual cases and remains acceptable. Most agree that women younger than 30 years of age should be screened yearly. It would be reasonable to extend the screening interval to 2 to 3 years for a woman older than 30 if she has had 3 consecutive negative Paps. High-risk populations (eg, HIV positive, immunosuppressed, DES exposed, high-grade dysplasia in the past) should be closely followed.

Primary Screening With Pap and HPV-DNA Testing

Primary screening with Pap and HPV has recently been approved by the US Food and Drug Administration (FDA). Primary testing with high-risk HPV DNA plus Pap is appropriate for primary screening in women older than age 30 at a frequency of no more than every 3 years. The negative predictive value approaches 100%. In an interesting study by the Kaiser group,it was determined that if both Pap and high-risk HPV-DNA results for a given woman were negative, the negative predictive value for her not having CIN 3 at 5 years was 99.84%. If HPV causes cervical cancer, one might wonder, why not just screen everyone with HPV DNA? The answer is that HPV is a young person's infection affecting many women younger than 30. As mentioned earlier, most HPV infections do not translate into high rates of severe dysplasia and most are spontaneously cleared. Cervical cancer affects older women who have persistent infection. Although HPV-DNA testing is very sensitive for identifying patients who will have CIN 2 or greater, its specificity is not as good as Pap alone, and its positive predictive value is poor.In patients older than 30, however, the sensitivity (86%) and specificity (83%) of HPV testing are much more acceptable.

Summary

• Although invasive cervical cancer has been reduced by 70% in the US through the use of cervical cytology, there has never been a national screening program here; rather, patients are screened opportunistically. Thus, the affluent are often over-screened and the indigent are under-screened.
• Guidelines for cervical cancer screening from ACOG, ACS, and the USPSTF have all been recently published. The guidelines vary slightly with respect to when to commence screening and when to cease.
• Generally, patients younger than 30 years of age should be screened yearly, but it would be reasonable to extend the screening interval to 2 to 3 years for a woman older than 30 if she has had 3 consecutive negative Paps. Primary testing with high-risk HPV DNA plus Pap is appropriate for primary screening in women older than age 30 at a frequency of no more than every 3 years.

References :

1. ACOG Practice Bulletin: Clinical management guidelines for obstetrician-gynecologists.

2. Saslow D, Runowicz CD, Solomon D, Moscicki AB, Smith RA, Eyre HJ, Cohen C; American Cancer Society. American Cancer Society guideline for the early detection of cervical neoplasia and cancer.

3.Pearce KF, Haefner HK, Sarwar SF, Nolan TE. Cytopathological findings on vaginal Papanicolaou smears after hysterectomy for benign gynecologic disease. N Engl J Med.

Childhood Pets Linked to Lower Allergy Risk

NEW YORK (Reuters Health) Jul 22 - Good news for families that would love to have a furry dog or cat but hesitate for fear the kids might become allergic: Fido or Kitty might actually be good for children's health, scientists say.

They found that children who were exposed to animals at a young age had lower rates of nasal allergies as adolescents.

"Family pets, in particular dogs...need not be removed to prevent allergies, and in fact may protect against them," said Melanie Matheson of the University of Melbourne, lead author of the study.

Looking at survey responses from nearly 8,500 adults from Europe and Australia, Matheson and colleagues focused on those who grew up around house pets or farm animals, and those who had the troublesome runny noses, itchy eyes, and sore throats that plague nasal allergy sufferers.

Growing up with pets has already been linked to a lower risk of other types of allergies. A 2010 study from the University of Cincinnati showed than owning a dog may decrease the risk of childhood eczema, a skin condition. Similarly, a 2011 study from Henry Ford Hospital in Detroit found that growing up with pets cut kids' risk of developing pet allergies by half.

In the new study, published online July 13th in the Journal of Allergy and Clinical Immunology, more than one in four respondents said they had nasal allergies. In most cases, people said their allergies started when they were adolescents.

A number of factors were linked to a higher risk of nasal allergies in the study. Some, like a family history of allergies and the mother smoking while pregnant, are well documented risk factors.

But the research team also found that small children who had lots of exposure to other little kids - because they had young siblings, for instance, or attended day care - had lower risks of nasal allergy. And the more siblings a child had, the lower the odds that the child would have nasal allergies later in life.

The scientists saw a similar pattern among people who grew up on a farm or had pets before age five. Compared to rates in people who didn't have those experiences in early childhood, the odds of having nasal allergies in adolescence were 30% lower in people who grew up on a farm, while having a dog and cat were each associated with a 15% reduction.

Furthermore, people who'd had siblings and animal exposure had lower rates of nasal allergies compared to those who'd had only one or the other experience.

These results were consistent in the 13 countries surveyed, "despite the differences in pet ownership and farming between countries," Matheson told Reuters Health.

The study design cannot prove that exposure to pets or other children are the cause of the lower risk of nasal allergies. Although the authors accounted for several factors, including education and family history of allergies, there may be another cause of the reduced nasal allergy risk that is associated with pets and siblings.

Also, the researchers only had information on exposure to animals before age five, so they don't know whether being around animals at an older age would have any effect on allergy risk.

While the results of the study are promising, it would be premature to suggest that parents buy pets or have more children, said Dr. Jonathan Bernstein, professor of medicine of University of Cincinnati College of Medicine and a co-author of an earlier report on the same topic.

Still, he said, the results provide further evidence that avoiding exposures may not be the best way to protect children against allergies.

Antigen Retrieval

One of the earliest methods of antigen retrieval was proteolytic digestion of tissue sections employed prior to the application of the primary antibodies. A number of proteolytic enzymes served this purpose, including trypsin, proteinase K, pronase, pepsin, ficin, DNase, and others. Not only are the enzymes different but there is also variation in concentration, duration, and temperature of digestion.

Furthermore, not all antigens benefit from proteolytic digestion, and some show deleterious effects with loss of staining. In addition, inappropriate protocols result in tissue breakdown and loss of morphology with high levels of background and falsepositive staining.

The introduction of heat-induced antigen retrieval signified a major milestone in immunohistology, greatly enhancing our ability to demonstrate antigens in FFPT with greater consistency.

The initial technique was achieved with MWs, which has remained the most convenient method for antigen retrieval, but a variety of other methods of generating heat have since been spawned including water baths, hot plates, wet autoclaves, pressure cookers, and vegetable steamers. Shi et al (1991) described MW heating of FFPT in the presence of heavy metal solutions, such as lead thiocyanate, up to temperatures of 100o C to “unmask” a wide variety of antigens for immunostaining. It was subsequently shown that MW-irradiation of deparaffinised-rehydrated sections in 10 mmol citrate buffer at pH 6.0 produced, with few exceptions, increased intensity and extent of immunostaining of a wide variety of tissue antigens. The use of citrate buffer eleminated the need to employ heavy metal solutions which, ehen heated, generate toxic fumes. Several commercial antigen retrieval reagents are available but they mostly do not produce any better results than that obtained with citrate buffer.

MWs are a form of non-ionizing radiation with a typically standard frequency of 2.45 GHz, a wavelength of 12.2 cm and photon energy of 10-5 electron volts. When dipolar molecules such as water or the polar side chains of proteins are exposed to the rapidly alternating electromagnetic fields, they oscillate through 180o at the rate of 2.45 billion cycles per second. The molecular movement or kinetics so induced results in the generation of instantaneous heat that is proportional to the energy flux and continues until radiation ceases.

It was only recently recognized that molecules other than water and the polar side chains of proteins may oscillate in the electromagnetic field generated by MWs. Molecules with an uneven distribution of electrical charge such as inorganic material and copper oxides can also be rotated.

All methods of heat generation listed above suffer from problems with accurate temperature and time control. These two variables have been shown to be critical to the process of heat-induced antigen retrieval. They are inversely related so that antigen retrieval at lower temperatures requires longer durations to achieve the same results as that obtained with higher temperatures. The time taken to attain the desired temperature from variable starting temperatures, time required to cool to room temperature, and actual temperature attained are variables that cannot be controlled with most methods of heating. Furthermore, there is the problem of unevenness of heating within microwave cavities, making the entire process impossible to standardize with resulting inconsistencies in methodology. Computerized control of time and temperature that is available with some commercial MW instruments takes the guesswork out of heat-induced antigen retrieval.

Accurate time and temperature control not only produces superior antigen retrieval across the spectrum of diagnostic antigens but accurate heating to 120oC or “superheating” has proven to produce notably better antigen staining.

Our understanding of the effects of formaldehyde on proteins dates back to work done in the 1940s (Fraenkel-Conrat et al, 1947; Fraenkel-Conrat and Olcott, 1948a, 1948b). A recent comprehensive review of the fixative action of formaldehyde and antigen masking is available. The amino acid side chain of proteins includes many groups that may react with aldehydes that contribute to the stabilization of proteins. However, despite the vast

literature on the subject of protein modification by formaldehyde, there is no clear consensus as to which are the predominant molecular species resulting from this method of fixation.

There is no doubt that some of the cross-linked adducts are very stable and remain irreversibly changed even after extensive washing, while others revert under varying conditions to free formaldehyde and the amino acid. Without a complete understanding of the actions of formaldehyde on proteins, it is not surprising that we do not fully understand the mechanisms of antigen retrieval.

Heat appears to be a common denominator in antigen retrieval produced by a variety of methods including MWs. Heat is hypothesized to cause protein denaturation based on the observation that some antigens or endogenous enzymatic activities may be lost after heat treatment and heat induces reversal of various chemical modifications of the protein structure that result from formalin fixation. Other actions that produce antigen retrieval include the loosening or breakage of the cross-linkages caused by formalin fixation, hydrolysis of schiff bases, and multiple pathways including extraction of diffusible blocking proteins, precipitation of proteins and dehydration of the tissue sections to allow better penetration of antibody and increased accesibility to epitopes, all or some of which may be achieved by other methods of retrieval including enzyme digestion and change in pH. MW energy may itself mobilize the last traces of paraffin that may not have been extractable by standard techniques, there by improving antibody penetration.

It was proposed that the calcium complex formation that occurs with formalin fixation may mask antigens and that the release of calcium from this cage-like complex may require a considerable amount of energy such as high temperature heating and calcium chelation by citrate. However, it has been argued that while this calcium effect may be acting with some antigens it may not be sufficient to explain the loss of immunoreactivity for many other antigens and is unlikely to represent the general mechanism of antigen retrieval.

The role of kinetics in antigen retrieval is also not known. While the focus has been on heat as the responsible factor in MW retrieval, the rapidly oscillating electromagnetic field of MWs may itself have an effect on chemical reactions and proteins. While heat or thermal energy will increase molecular kinetics and hasten chemical reactions, the rapid rotation of molecules directly induced by the MWs will give rise to greatly increased collision of molecules, which will in turn accelerate chemical reactions. The heat generated may represent only an epiphenomenon secondary only to the kinetics. One study that examined MW stimulation of CEA/anti-CEA reaction in an enzyme-linked immunosorbent assay system found that despite continuous cooling by ice MW stimulation increased reaction rates by a factor of 1000, allowing the investigators to conclude that such rate increases were far too large to be explained solely by the modest increase in temperature.

Another study went further to elucidate the existence of a “microwave effect” (Choi et al,1997) The authors showed that the rate of droplet temperature increase obtained in a thermal cycler was similar to that achieved by MW irradiation. However, the immunostaining obtained from a 3-minute incubation at 37oC in the thermal cycler followed by 2-minute incubation without heating was much weaker than that seen with MWs. Similarly it was demonstrated that 7-s MW irradiation followed by 5-min room temperature incubation for each step of the avidin-biotin peroxidase complex procedure produced good immunolabelling.

The droplet temperature raised no more than 5oC following 7-s irradiation at 100% power in a 850-watt oven so that temperature was not a significant component of the accelerated reaction. Others have argued that there is no significant MW effect and the accelerated reactions are a function of heat. It has been concluded that MW irradiation did not produce cleavage or polymerisation of proteins and irradiation resulted an electrophoretic pattern that was similar to that obtained when lysozyme and hemoglobin was heated in formaldehyde to 60o C for 30 min. Interestingly, results to the contrary have been shown in a study of S-adenosylhomocysteine hydrolase and 5’-methylthioadenosine phosphorylase, two thermophilic and thermostable enzymes, where exposure to MWs caused a non-thermal, irreversible and time-dependent inactivation of both enzymes, In a model immunostaining system using short synthetic peptides to mimic the antibodybinding site of common diagnostic protein targets, Sompuran et al (2006), found that not all of the peptides studies exhibited the formalin-fixation and antigen retrieval phenomenon. One group of peptides was recognized by antibody even after prolonged exposure to formalin while another group exhibited the formalin-fixation and antigen-retrieval phenomenon only after another irrelevant protein was mixed with the peptide before fixation. Amino acid sequence analysis indicated that fixation and antigen retrieval were associated with a tyrosine in or near the antibody-binding site bound covalently to a nearby arginine implicating the Mannich reaction as an important factor in the process.

The Mannich reaction is a complicated, multistep interaction, which firstly involves a reaction between formaldehyde and an amine to produce an iminium ion. The iminium ion may then react with another carbonyl-containing molecule to form an intermediate product and for this to occur, the carbonyl-containing molecule must be in an enol configuration. In the final step of the reaction, the iminium ion and the enol react together to form a stable product. The findings of Sompuran et al (2004) concurred with those of Fraenkel-Conrat et al (1947, 1948) who had indicated that of all the protein cross-linking reactions that occur as a result of formalin fixation, the Mannich reaction is different, in that the cross-linkages can be hydrolysed with heat or alkaline treatment. Antibodies appear to recognize linear protein epitopes in FFPT and antigen retrieval may simply remove cross-linked proteins that are sterically interfering with antibody binding. The recent demonstration that antigen retrieval produces immunohistological staining results in FFPT that are comparable or better than that in acetone-fixed fresh frozen section and that heat-induced antigen retrieval enhances immunostaining in unfixed fresh frozen sections and dot-blot protein extracts is further support of the concept that intrinsic natural steric barriers exist and interfere with antibody binding. The demonstration that MWs can also be employed to enhance the demonstration of HER2/neu gene in chromogenic in-situ hybridization (CISH) is particularly interesting and it suggests that similar mechanisms may be operative in the ‘masking’ of DNA.

However, it would seem that these observations provide some insights into the action of antigen retrieval with some peptides but answers to the majority still remain unknown.

The demonstration that exposure to ultrasound can significantly increase antibody-antigen reaction in immunostaining lends further support to the relevance of molecular movement as an important factor in the acceleration of the chemical reaction as the amount of heat generated by this physical modality is negligible. A number of other hypothetical physical mechanisms may also play a role in the actions of MWs.

Although the proton energy generated in MW fields is too small to alter covalent bonding, they may readily affect the integrity of non-covalent secondary bonding, including hydrophobic interactions, hydrogen bonds and van der Waal’s interactions that make up the precise steric interactions at the cell membrane.

The combination of heat retrieval with enzymatic digestion has allowed enhanced demonstration and localization of a number of antigens including the immunoglobulins. Proteolytic digestion can be performed with a number of enzymes, and at varying concentrations and for varying durations. It can precede or follow heat induced antigen retrieval with different results. For optimal outcomes, it is necessary to explore all possible combinations and permutations of these variables with the realization that excesses can result in loss of antigen and cell morphology.

The chemical composition of the retrieval solution may affect the efficacy of the process and a wide variety of solutions have been advocated including citrate buffer, Tris buffer, glycine-hydrochloric acid, EDTA, urea, heavy metal solutions, and other proprietary reagents.

The molarity of the solution may also significantly influence immunostaining. The pH of the retrieval solution has been shown to be one of the most important factors in antigen retrieval. Three patterns of staining reflect the influence of pH.

Some antigens (CD20, AE1, EMA, NSE and PCNA) showed no variation at pH values ranging from 1.0 to 10.0, other antigens (MIB1, ER) displayed a dramatic decrease in staining intensity at middle pH values (pH 3.0-6.0) with strong staining above and below the range, and a third pattern was demonstrated by other antigens (CD43, HMB45) which were weakly stained at low pH (1.0-2.0) and displayed a sharp rise in intensity with increasing pH.

Use of MWs to accelerate antibody-antigen reactions in the staining of labile lymphocyte membrane antigens in cryostat sections and the exposure of cryostat sections briefly to MWs before the commencement of immunolabelling produced better quality cytomorphology and staining. A similar procedure has been adopted for freshly frozen brain sections with notable enhancement of immunostaining, without affecting the integrity of cytomorphology. MWs have been successfully used to accelerate immunolabelling in paraffin-embedded sections and the same technique has been applied for immunofluorescence labeling.

MWs have been applied between sequential rounds of a three-layer immunoenzyme staining (mouse Mab, goat anti-mouse IgG and mouse PAP or mouse APAAP) and color development technique for multiple antigen detection. The MWs denatured bound antibody molecules resulting in the blocking of cross reactivity between the sequential staining steps, allowing the use of primary and other antibodies raised in the same species.

Besides serving a role in antigen retrieval, MWs also inactivated peroxidase and alkaline phosphatase enzymes present in PAP and APAAP complexes, which would otherwise have led to inappropriate color development.

Source :

Biochemistry and Hystocytochemistry Research Developments : Editor by Stefan Fuchs and Max Auer.

Higher Vitamin D Levels Linked to Lower Diabetes Risk

(San Diego, California) — Higher levels of vitamin D in the blood appear to be associated with a reduced risk for incident diabetes among people at high risk for the disease, according to a new report.

Anastassios G. Pittas, MD, from the division of endocrinology, diabetes, and metabolism at the Tufts New England Medical Center in Boston, Massachusetts, and colleagues presented the findings here at the American Diabetes Association 71st Scientific Sessions.

According to Dr. Pittas, vitamin D might play a role in diabetes by improving insulin secretion and insulin sensitivity. "Most of the evidence focuses on a favorable effect in pancreatic beta cells," he told.

To determine the relation between vitamin D status and risk for incident diabetes, the researchers analyzed data from the Diabetes Prevention Program (DPP), a 3-group trial comparing intensive lifestyle modification or metformin with placebo for the prevention of diabetes in patients with prediabetes.

The mean follow-up of the 2039-person cohort was 3.2 years. Plasma vitamin D levels were measured at yearly intervals, and subjects were assessed for incident diabetes. For this analysis, only participants in the intensive lifestyle and placebo groups of the DPP were considered.

Participants with vitamin D levels in the highest tertile (median concentration, 30.1 ng/mL) had a hazard ratio of 0.74 (95% confidence interval [CI], 0.59 to 0.93) for developing diabetes, compared with those with vitamin D levels in the lowest tertile (median concentration, 12.8 ng/mL).

The findings also suggest a dose-dependent effect for vitamin D levels; the hazard ratio for incident diabetes was lowest (0.46; 95% CI, 0.23 to 0.90) in the people with the highest vitamin D levels (50 ng/mL or higher), compared with those with the lowest levels (below 12 ng/mL).

In a subgroup analysis by tertiles of vitamin D, the association was similar in the placebo group (0.72; 95% CI, 0.53 to 0.96) and the lifestyle group (0.80; 95% CI, 0.54 to 1.14).

According to Dr. Pittas, "this study offers several methodological advantages over previous studies." Vitamin D status was assessed multiple times during follow-up, not just once at baseline, which might not reflect long-term vitamin D status.

"Our study also includes a large clinically relevant population at high risk for diabetes, with a substantial proportion of nonwhite participants, which improves the external validity of the results," he said. However, he added, "this is an observational study and therefore confounding cannot be excluded. It would be premature to recommend vitamin D specifically for prevention of diabetes."

"This prospective study confirms that there is an association between levels of vitamin D and risk of diabetes, even when correcting for body weight, with no absolute threshold of serum 25-hydroxy vitamin D," said independent commentator Clifford Rosen, MD, from the Jackson Laboratory in Bar Harbor, Maine. Dr. Rosen is a vitamin D researcher and member of the Institute of Medicine Committee that reviewed the evidence on calcium and vitamin D.

"The implications of this study relate to the importance of performing a randomized placebo-controlled trial of vitamin D for the prevention of type 2 diabetes in those at high risk," he told Medscape Medical News. "In the interim, clinicians should at least focus on maintaining vitamin D levels in high-risk individuals at or around 20 ng/mL," he added.

Source :

American Diabetes Association (ADA) 71st Scientific Sessions: Abstract 0117-OR. Presented June 25, 2011.

2010 AHA Guidelines: The ABCs of CPR Rearranged to "CAB"

Chest compressions should be the first step in addressing cardiac arrest. Therefore, the American Heart Association (AHA) now recommends that the A-B-Cs (Airway-Breathing-Compressions) of cardiopulmonary resuscitation (CPR) be changed to C-A-B (Compressions-Airway-Breathing).

The changes were documented in the 2010 American Heart Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care, published in the November 2 supplemental issue of Circulation: Journal of the American Heart Association, and represent an update to previous guidelines issued in 2005.

"The 2010 AHA Guidelines for CPR and ECC [Emergency Cardiovascular Care] are based on the most current and comprehensive review of resuscitation literature ever published," note the authors in the executive summary. The new research includes information from "356 resuscitation experts from 29 countries who reviewed, analyzed, evaluated, debated, and discussed research and hypotheses through in-person meetings, teleconferences, and online sessions ('webinars') during the 36-month period before the 2010 Consensus Conference."

According to the AHA, chest compressions should be started immediately on anyone who is unresponsive and is not breathing normally. Oxygen will be present in the lungs and bloodstream within the first few minutes, so initiating chest compressions first will facilitate distribution of that oxygen into the brain and heart sooner. Previously, starting with "A" (airway) rather than "C" (compressions) caused significant delays of approximately 30 seconds.

"For more than 40 years, CPR training has emphasized the ABCs of CPR, which instructed people to open a victim's airway by tilting their head back, pinching the nose and breathing into the victim's mouth, and only then giving chest compressions," noted Michael R. Sayre, MD, coauthor and chairman of the AHA's Emergency Cardiovascular Care Committee, in an AHA written release. "This approach was causing significant delays in starting chest compressions, which are essential for keeping oxygen-rich blood circulating through the body," he added.

The new guidelines also recommend that during CPR, rescuers increase the speed of chest compressions to a rate of at least 100 times a minute. In addition, compressions should be made more deeply into the chest, to a depth of at least 2 inches in adults and children and 1.5 inches in infants.

Persons performing CPR should also avoid leaning on the chest so that it can return to its starting position, and compression should be continued as long as possible without the use of excessive ventilation.

9-1-1 centers are now directed to deliver instructions assertively so that chest compressions can be started when cardiac arrest is suspected.

The new guidelines also recommend more strongly that dispatchers instruct untrained lay rescuers to provide Hands-Only CPR (chest compression only) for adults who are unresponsive, with no breathing or no normal breathing.

Other Key Recommendations

Other key recommendations for healthcare professionals performing CPR include the following:

• Effective teamwork techniques should be learned and practiced regularly.
• Quantitative waveform capnography, used to measure carbon dioxide output, should be used to confirm intubation and monitor CPR quality.
• Therapeutic hypothermia should be part of an overall interdisciplinary system of care after resuscitation from cardiac arrest.
• Atropine is no longer recommended for routine use in managing and treating pulseless electrical activity or asystole.

Pediatric advanced life support guidelines emphasize organizing care around 2-minute periods of continuous CPR. The new guidelines also discuss resuscitation of infants and children with various congenital heart diseases and pulmonary hypertension.

Source :
- 4th EIDCP National Symposium
- Medscape news