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Co-Director, University of Hawaii at Manoa John A. Burns School of Medicine

After development of the colored product, the slides are rinsed, counterstained, and mounted in mounting medium to be observed under a light microscope. A modification of the indirect approach includes an additional step whereby an antibody raised against the enzyme is incubated with the sample. Because this antibody is also conjugated with enzyme, its sensitivity is greatly enhanced. Avidin-biotin is another system that is widely used to increase sensitivity in such approaches. The binding reaction between these two molecules is strong, and it occurs independently of the immune reactions in the assay. In a typical avidin-biotin complex protocol, biotinconjugated primary antibodies are incubated with the samples, and after the unbound antibodies have been washed out, enzyme-conjugated avidin or streptavidin is added. The complex formed is monitored, with the final addition of substrate and mounting of the slide. In the indirect approach, an unlabeled primary antibody is first incubated with the sample, followed by secondary incubation with biotinylated antibody and the biotin-avidin enzyme complex incubation. After samples are added, unbound material is removed and enzyme-conjugated antibody is added. After removal of unbound material, enzyme substrate is added and the formation of the reaction product is measured. This measurement reflects the amount of enzyme bound to antigen that is retained in the solid phase. Control samples are always included to define the background levels of each modification. Such a membrane is attached to a plastic well, and the entire system is attached to a cassette containing a material that can absorb all waste fluid generated during the assay. The antibody and the controls can be dotted or slotted onto the membrane in separate wells. The method offers the advantages of increased sensitivity, reduced time required for its completion, and detection of many different respiratory viruses from many samples. Based on the same principle, numerous variations are used, with different sensitivity scores. Alternatively, a classic dot-blot apparatus can be used to place an antibody in dots on the membrane. Samples and controls can then be added as serial dilutions for quantitation, and waste fluids are collected from the associated vacuum system. However, although these systems are able to discriminate between serotypes of the same species. In a representative protocol, a microtiter plate or strip is coated with antibody and the remaining binding sites of the plates are blocked. The clinical sample is then added simultaneously with the Europium-conjugated antibody, and after incubation, the unbound material is washed out. This allows direct visual detection of a macromolecule that is bound onto a molecular thin film. Binding causes an increase in the thickness of the optical surface (silicon wafer film) that will alter the reflected light path and will be perceived as a color change that is observable with the naked eye. Practically, viral-specific antibodies 407 Chapter 24 408 Infections of the Respiratory Tract are immobilized on the surface and allowed to capture extracted viral antigens that are placed directly onto the surface and incubated at room temperature for a short period. After addition of a suitable substrate, a positive result appears as a color spot, whereas in the absence of antigens in the sample, the background color remains unchanged. Agglutination Assays In the latex agglutination assay, antibody-coated nanoparticles from polystyrene, polyacrylamide, and other latexes; agarose beads; or colloidal gold agglutinate in the presence of viral antigens. The method is simple, and an agglutination reaction can be observed usually after 10 to 15 minutes, but the overall sensitivity and specificity are low. Serologic Methods As indicated by the name, serologic methods attempt to detect viruses in the host by assessing the presence of specific antibodies in blood samples, but sputum and urine also may be used. Serology is a rather sensitive, specific, and relatively cheap diagnostic technique that is also used to confirm the results of other methods. IgM antibodies represent approximately one tenth of serum immunoglobulins, while secretory IgA constitutes the first line of defense against mucosal viral infections.

Choosing dams that have had at least one litter prior to the pups for treatment is also advantageous though not required. Finally, the disease model can also influence how well the neonate injection procedure is tolerated. Foust spectrum disorder had a much higher rate of pup rejection than expected based on the background strain. Should the vein not be visible or is mistargeted on initial attempts, targeting the vein on the opposite side of the head is appropriate. If the vein is punctured on both sides of the head, some leakage may occur through the first site during injection into the second site. For delivery of self-complementary adeno-associated virus, injections are routinely performed into 21-day-old mice. Adachi H, Katsuno M, Minamiyama M, Sang C, Pagoulatos G, Angelidis C, Kusakabe M, Yoshiki A, Kobayashi Y, Doyu M, Sobue G (2003) Heat shock protein 70 chaperone overexpression ameliorates phenotypes of the spinal and bulbar muscular atrophy transgenic mouse model by reducing nuclear-localized mutant androgen receptor protein. Jankowsky Abstract the rapid pace of neuroscience research demands equally efficient and flexible methods for genetically manipulating and visualizing selected neurons within the rodent brain. The use of viral vectors for gene delivery saves the time and cost of traditional germline transgenesis and offers the versatility of readily available reagents that can be easily customized to meet individual experimental needs. Here, we present a protocol for widespread neuronal transduction based on intraventricular viral injection of the neonatal mouse brain. Injections can be done either free-hand or assisted by a stereotaxic device to produce lifelong expression of virally delivered transgenes. Key words Adeno-associated virus, Neonatal brain, Intracerebroventricular injection, Viral transduction, Transgenic mouse 1 Introduction the ease of viral delivery permits genetic manipulation of the rodent brain without the time and cost of traditional germline manipulations. Rather than the spatially limited transduction obtained by stereotaxic injection of the adult, intracerebroventricular injection into the neonate achieves widespread distribution of viral particles [1­4]. Expression of virally delivered transgenes usually begins within 2­3 days after injection and persists for well over a year [1, 2]. We also introduce a complementary approach for viral injection based on stereotaxic targeting of the lateral ventricles. The only major equipment required for freehand injection is a microsyringe fitted with a fine bore needle. In contrast, stereotaxic injection requires a dedicated apparatus but can improve reproducibility of the injections for novice investigators. This benefit is worth noting, as accurate targeting of the lateral ventricles is the most critical factor for successful viral expression when using these techniques. By including a non-toxic dye (2­4 % trypan blue) in the viral solution, the injection site and ventricular spread can be visualized to provide an immediate read-out of injection accuracy. With successful targeting of the lateral ventricles, both free-hand and stereotaxic techniques provide widespread neuronal transduction throughout the brain from the olfactory bulb to the cerebellum. While free-hand and stereotaxic injection produce similar patterns of neuronal transduction, they are not identical. The angle at which the needle approaches the brain and the precise location of viral injection within the ventricle differ between the two techniques. The method we describe for free-hand injection targets the caudal portion of the ventricle and results in highest expression within the posterior and lateral regions of cortex. In contrast, the stereotaxic coordinates provided for single-site injection (as well as the alternative free-hand injection site we describe) target a more rostral location within the ventricle, leading to strongest expression in anterior regions. Both injection sites generate transgene expression throughout the cortex and beyond, but the bias of maximal expression toward rostral or caudal forebrain may influence which of the two approaches is more appropriate for a particular experiment. If desired, the stereotaxic manipulator can be used to perform two injections into each ventricle, one rostral and one caudal, to attain a more even distribution of viral transduction throughout the cortex. The density of neuronal transduction can be controlled by adjusting the viral titer, with high titer injections producing very dense viral expression and dilute injections resulting in sparse transduction of isolated cells [2]. Moreover, multiple viruses can be co-injected to express distinct proteins, using the serotype of each to bias transduction toward distinct or overlapping populations of neurons [2]. There are endless combinations of promoter, serotype, and titer than can be exploited to tailor viral expression for experimental needs [12]. This inherent flexibility has been advantageous for studies ranging from in vivo imaging to gene therapy of neurological disorders [2, 8, 13­19].

The dominant racial types as previously noted are Asian (sometimes referred to as Mongoloid or Yellow), White (sometimes referred to as Caucasoid or Indo-European), and Black (sometimes referred to as Negroid) with some authorities adding American Indian (sometimes referred to as Red) and Australian Aboriginal (sometimes referred to as Malay) types (Omi and Winant, 1994; Stringer and McKie, 1997; Thernstrom, Orlov, and Handlin 1980; Van den Berghe, 1967). Of the 2 to more than 60 arbitrary "racial" types that science has created over the centuries, the U. While virtually none of the authorities still believe in "pure" races, all concede that many groups overlap the various racial classification systems and that there are a few that cannot be classified at all. Further confusion is introduced when all attempts at definition are confounded by the belief that race is just one aspect of ethnicity. The United States is still highly stratified on the basis of race, ethnicity and class, and growing income inequality over the past decades may be accentuating these trends (Feagin and Feagin, 1999; U. Historically, some groups are confined to lower-class positions because of lack of access to both Copyright National Academy of Sciences. The fact that individual class system mobility is also limited and that experiences differ markedly for certain groups is also based on understanding that there are two very different patterns of ethnic incorporation-discrimination versus exclusion. Among those who suffered from discrimination were the Irish, Italians, Greeks, Jews, and Poles-the European immigrants, mostly from southern and eastern Europe, that came to American voluntarily in the midnineteenth to early twentieth centuries. Among those who suffered from social exclusion were Blacks, Native Americans, Mexican Americans, and Puerto Ricans-those whose history began as the product of involuntary conquest, annexation, and colonialism, as a result of which they were not allowed to become integrated into the major institutions of the society (Pedraza and Rumbaut, 1996, 16). Few deny that African Americans, Native Americans, and Hispanics disproportionately occupy the lowest strata of the class system and have been traditionally restrained within these strata by political, ideological, legal and economic mechanisms. Traditionally, groups under Anglo-Protestant political or economic dominance, especially when compounded by racial worldview-caste considerations and stereotyping-Native Americans, Hispanics, African Americans, or Asians, or immigrants who arrived as indentured servants or laborers-have moved into and circulated within the lower rungs of the social hierarchy. However, in the ensuing racial and ethnic acculturation and assimilation, competition, and struggles that have evolved over time, individuals and groups, even of the lessfavored races and ethnicities, have moved into positions of power. Erroneously considered an homogenous population, the categories "Black" or "African American" include the descendants of the original seventeenththrough nineteenth-century slave population, as well as immigrants from Jamaica, Haiti, Trinidad, Barbados, and other Caribbean nations along with more recent immigrants from Ghana, Nigeria, Egypt, Ethiopia, Somalia, Sudan, the Cape Verde Islands, Liberia, and other African countries. Eighty-seven percent of African Americans live in cities and they reside in all 50 states. However, over half live in 13 Southern states-Alabama, Arkansas, Florida, Georgia, Kentucky, Louisiana, Maryland, Mississippi, North Carolina, South Carolina, Tennessee, Texas, and Virginia. Though subpopulations vary, people are usually assigned to the black or African-American category based on appearance- characteristics such as skin color, hair texture, and facial features. Mired in corrosive sociocultural, health and biomedical system legacies of 2000 years of being portrayed as being biologically and intellectually inferior; 246 years of chattel slavery, including a slave health deficit and a slave health subsystem; 100 years of legal segregation and discrimination and a "Negro medical ghetto;" and contemporary social, political, and economic isolation, oppression, exploitation, and a "dual" and unequal health system (Byrd and Clayton, 2000, 2002), "African Americans experience healthcare differently from [W]hites and other populations within the nation" (U. For a plethora of reasons, African Americans have experienced the worst health status, suffered the worst health outcomes, and been forced to utilize the worst health services of any racial or ethnic group. However, their numbers are growing three times more rapidly than the white population. Comprising culturally diverse, complex, and distinctive groups of people speaking more than 300 languages, the American Indian or Alaskan Native population is made up of 535 federally recognized (plus 100 that are not officially recognized) tribes in seven nations. For much of the twentieth century, the observations of the 1928 Meriam Report that the health of the Indian population was characterized by "a high birth rate and high death rate with excessively high infant mortality and a large proportion of deaths from tuberculosis" (Stuart, 1987, 1996), held true. Health-seeking behavior and responses to healthcare services such as being strongly autonomous, being non-linear thinkers (especially regarding time), using indirect communication and styles, and having a historical suspicion of authority reflects these experiences (Kingfisher, 1996). Their health and demographic profile-including poverty, lower education levels, and disease profiles often compounded by substance abuse-reflects the residue of this legacy. Though "[t]he health status of American Indians has improved dramatically during the twentieth century, particularly after the transfer of Indian health to the Public Health Service in 1955" (Stuart, 1987, 95), their diversity-compounded by their many small population groups scattered throughout the country-has made it difficult to provide consistent, quality, readily accessible healthcare. Asian American [Asian or Pacific Islander] Numbering less than one million until the Supreme Court ruled against immigration quotas in 1965, Asians and Pacific Islanders are the fastest growing minority group in the United States-representing 3 percent of the total population and around 13 percent of all people of color. Asian American immigrants to the United States have come from more than 20 countries. Having emigrated from countries such as China, Japan, India, the Philippines, Korea, Laos, Cambodia, Vietnam, and Thailand, Asians and Pacific Islanders represent more than 60 different ethnic groups and speak more than 100 different languages. Fifty-six percent of Asian and Pacific Islanders live in the Western United States with the highest concentrations residing in Hawaii (63% of the total population), California (12%), Washington (6%), and New York and New Jersey (5% each).

This is due both to a mechanical effect and oxygenated blood passing through the pulmonary circulation. Inflation of the lungs stimulates pulmonary stretch receptors, which leads to reflex vasodilation of the pulmonary vascular bed. Mechanical expansion creates surface forces at the gas-liquid interface within the alveoli, which physically expand small blood vessels and decrease perivascular pressure. The majority of the changes in cardiopulmonary hemodynamics occur by 8 hours, although some degree of right-to-left ductal shunting may be found up to 12 hours after birth. In most infants, the ductus arteriosus closes by 24 hours of age, but there is a significant delay in ductal closure in infants with pulmonary hypertension. In pulmonary hypertension that accompanies sepsis due to group B streptococci or other organisms, thromboxane A2 may cause initial severe arterial spasm followed by increased vascular permeability and an increased lung fluid content. The increased capillary permeability in sepsis-induced pulmonary hypertension appears to be due also to the action of bacterial endotoxins sequestering white cells in the lungs, where they release vasoactive agents such as tumor necrosis factor. A rise in the hematocrit can cause pulmonary hypertension, but polycythemia is not a consistent feature of neonates with pulmonary hypertension. Pulmonary hypertension in the neonate is often characterized by varying degrees of vascular remodeling and decreased arteriolar number. The pulmonary hypertension, which occurs in infants with congenital diaphragmatic hernia or in other conditions associated with pulmonary hypoplasia, is due to a reduction in the number of intralobar arteries and increased muscularity of the arteries. Following chronic hypoxia in utero, excessive muscularization of the pulmonary arterioles is found and muscle extends into the normally muscle free intra-acinar arteries; such changes are seen in extremely small for dates infants. In other infants, there is a normal arteriolar number and muscularization, but the normal decrease in pulmonary vascular resistance after birth fails to occur. Persistent pulmonary hypertension may also be due to alveolar capillary dysplasia with congenital misalignment of the pulmonary veins. This condition is usually sporadic, but rarely family occurrence has been reported (See Chapter 55). The second heart sound is loud because of the rise in pulmonary arterial pressure. There may be a soft systolic murmur due to tricuspid or occasionally mitral incompetence. In neonates who are critically ill because of group B streptococcal infection, severe asphyxia, or congenital diaphragmatic hernia, pulmonary hypertension appears within 6 hours of birth; these infants also have the clinical features of their underlying condition. Diagnosis and Differential Diagnosis Pulmonary hypertension is diagnosed in an infant with severe hypoxemia when the hypoxemia is disproportionately severe for the radiologic abnormalities (Fig. Echocardiography is important, not only to establish the diagnosis, but also to exclude cyanotic congenital heart disease. In primary pulmonary hypertension, chest radiograph changes are often minimal; in secondary pulmonary hypertension, the chest radiograph appearance will be that of the underlying lung disease, but the appearance will be less severe than anticipated for the severity of the hypoxemia. The most important differential diagnosis Pathophysiology Pulmonary hypertension in the neonate may be primary or secondary to conditions including severe intrapartum asphyxia, infection, pulmonary hypoplasia, drug therapy 374 Respiratory Disorders in the Newborn to 5. Although no long-term adverse sequelae have been described in babies born at term, hypocapnia in preterm babies has been linked to the development of periventricular leukomalacia. Alkalosis can promote pulmonary vasodilation, but such a strategy should not be maintained for a prolonged period, as persisting alkalemia increases the hypoxic reactivity of the pulmonary vasculature, thus tending to perpetuate the pathophysiology of pulmonary hypertension. Neuromuscular blocking agents should be administered to full-term babies to prevent them fighting the ventilator. Anecdotally, high-frequency oscillatory and jet ventilation have been used with improvements in oxygenation. A variety of vasodilator drugs have been used to treat infants with pulmonary hypertension. Between 25% and 50% of affected babies respond to tolazoline hydrochloride, but this is not a specific vasodilator and hypotension is a common side effect. Magnesium levels must be carefully monitored, as hypermagnesemia can cause sedation, muscle relaxation, hyporeflexia, hypotension, and calcium and potassium disturbances. The response to ventilation with 100% oxygen can help to distinguish the two conditions; in some infants with pulmonary hypertension, the arterial oxygen level will increase to above 13 kPa (100 mm Hg), whereas in cyanotic congenital heart disease it will not rise above 5 to 6 kPa (37. Not all neonates with pulmonary hypertension, especially those with sepsis or a congenital diaphragmatic hernia, however, have a large improvement in oxygenation in response to 100% oxygen.