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Poor understanding of the cellular and molecular basis of clinical and genetic heterogeneity in progressive multiple sclerosis (MS) has hindered the search for new effective therapies. To address this gap, we analyzed 632,000 single-nucleus RNA sequencing profiles from 156 brain tissue samples of MS and control donors to examine inter- and intra-donor heterogeneity. We found distinct cell type-specific gene expression changes between MS gray and white matter, highlighting clear pathology differences. MS lesion subtypes had different cellular compositions but surprisingly similar cell-type gene expression patterns both within and across patients, suggesting global changes. Most gene expression variability was instead explained by patient effects, allowing us to stratify patients and describe the different pathological processes occurring between patient subgroups. Future mapping of these brain molecular profiles with blood and/or CSF profiles from living MS patients will allow precision medicine approaches anchored in patient-specific pathological processes., Competing Interests: Declaration of interests The study was funded by F. Hoffmann-La Roche Ltd. D.C., J.B., W.M., L.C.F., L.C., E.U., and S.S. are full-time employees of F. Hoffmann-La Roche Ltd. D.M. was a full-time employee of F. Hoffmann-La Roche Ltd. and now works for Biogen., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The role that astrocytes play in central nervous system (CNS) myelination is poorly understood. We investigated the contribution of astrocyte-derived factors to myelination and revealed a substantial overlap in the secretomes of human and rat astrocytes. Using in vitro myelinating co-cultures of primary retinal ganglion cells and cortical oligodendrocyte precursor cells, we discovered that factors secreted by resting astrocytes, but not reactive astrocytes, facilitated myelination. Soluble brevican emerged as a new enhancer of developmental myelination in vivo, CNS and its absence was linked to remyelination deficits following an immune-mediated damage in an EAE mouse model. The observed reduction of brevican expression in reactive astrocytes and human MS lesions suggested a potential link to the compromised remyelination characteristic of neurodegenerative diseases. Our findings suggested brevican's role in myelination may be mediated through interactions with binding partners such as contactin-1 and tenascin-R. Proteomic analysis of resting versus reactive astrocytes highlighted a shift in protein expression profiles, pinpointing candidates that either facilitate or impede CNS repair, suggesting that depending on their reactivity state, astrocytes play a dual role during myelination., (© 2024 The Author(s). GLIA published by Wiley Periodicals LLC.)

Multiple sclerosis (MS) is the most common demyelinating autoimmune disease of the central nervous system (CNS). Immune-mediated myelin and axonal damage that is accompanied by chronic axonal loss causing destruction of the myelin sheaths are hallmarks of MS. While great strides have been made in understanding the molecular underpinnings of re-/myelination, currently no remyelination therapy is available for MS. As myelination is a complex process that is not fully understood, we sought to develop a systematic, reliable, automated and quantitative higher throughput screening method. We aimed to quantitate myelin sheaths in vitro with high sensitivity at the single cell level suitable for testing small compound libraries. To this end, we miniaturised in vitro retinal ganglion cell-oligodendrocyte precursor cell (RGC-OPC) co-cultures into a multi-well plate format. This allowed us to maintain the reciprocal interaction of live axons and oligodendrocytes (OLs) to ensure compact myelin formation. To quantify our co-cultures, we developed a novel computer vision algorithm to precisely measure myelination. We demonstrated efficacy of our system with known pro-differentiating compounds BQ3020 and XAV939 which exhibited robust, efficient, and dose dependent effects on myelination. Through this combination of experimental and technical advances, we have developed a method allowing systematic and reliable testing of remyelinating compound efficacy., (© 2023. The Author(s).)

Development, physiological functions, and pathologies of the brain depend on tight interactions between neurons and different types of glial cells, such as astrocytes, microglia, oligodendrocytes, and oligodendrocyte precursor cells. Assessing the relative contribution of different glial cell types is required for the full understanding of brain function and dysfunction. Over the recent years, several technological breakthroughs were achieved, allowing "glio-scientists" to address new challenging biological questions. These technical developments make it possible to study the roles of specific cell types with medium or high-content workflows and perform fine analysis of their mutual interactions in a preserved environment. This review illustrates the potency of several cutting-edge experimental approaches (advanced cell cultures, induced pluripotent stem cell (iPSC)-derived human glial cells, viral vectors, in situ glia imaging, opto- and chemogenetic approaches, and high-content molecular analysis) to unravel the role of glial cells in specific brain functions or diseases. It also illustrates the translation of some techniques to the clinics, to monitor glial cells in patients, through specific brain imaging methods. The advantages, pitfalls, and future developments are discussed for each technique, and selected examples are provided to illustrate how specific "gliobiological" questions can now be tackled., (© 2020 Wiley Periodicals, Inc.)

Disruption of the blood-brain barrier (BBB) leads to various neurovascular diseases. Development of therapeutics required to cross the BBB is difficult due to a lack of relevant in vitro models. We have developed a three-dimensional (3D) microfluidic BBB chip (BBBC) to study cell interactions in the brain microvasculature and to test drug candidates of neurovascular diseases. We isolated primary brain microvascular endothelial cells (ECs), pericytes, and astrocytes from neonatal rats and cocultured them in the BBBC. To mimic the 3D in vivo BBB structure, we used type I collagen hydrogel to pattern the microchannel via viscous finger patterning technique to create a matrix. ECs, astrocytes, and pericytes were cocultured in the collagen matrix. The fluid flow in the BBBC was controlled by a pump-free strategy utilizing gravity as driving force and resistance in a paper-based flow resistor. The primary cells cultured in the BBBC expressed high levels of junction proteins and formed a tight endothelial barrier layer. Addition of tumor necrosis factor alpha to recapitulate neuroinflammatory conditions compromised the BBB functionality. To mitigate the neuroinflammatory stimulus, we treated the BBB model with the glucocorticoid drug dexamethasone, and observed protection of the BBB. This BBBC represents a new simple, cost-effective, and scalable in vitro platform for validating therapeutic drugs targeting neuroinflammatory conditions., (© 2019 Wiley Periodicals, Inc.)

Microfluidic platforms have recently become popular as in vitro models because of their superiority in recapitulating microenvironments compared with conventional in vitro models. By providing various biochemical and biomechanical cues, healthy and diseased models at the organ level can be applied to disease progression and treatment studies. Microfluidic technologies are especially suitable for modeling biological barriers because the flow in the microchannels mimics the blood flow and body fluids at the interfaces of crucial organs, such as lung, intestine, liver, kidney, brain, and skin. These barriers have similar structures and can be studied with similar approaches for the testing of pharmaceutical compounds. Here, we review recent developments in microfluidic platforms for modeling biological barriers in the circulatory system., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Neuronal and glial progenitor cells exist in the adult Drosophila brain. The primarily glial progenitor cells rely on a microRNA, mir-31a, to inhibit the expression of a predicted E3 ubiquitin ligase, CG16947. Erroneous inheritance of CG16947 by the progeny when the neural progenitor cell divides leads to death of the progeny, however how CG16947 achieves glial cell death is unknown. I have identified the interacting partner of CG16947 to be cdk9. I show that reduction of cdk9 expression in glia causes glial loss; highlighting the importance of cdk9 in mediating the survival of glia. Further, glial loss observed in mir-31a mutants was prevented with adult-specific expression of cdk9 in glia. I provide biochemical evidence that the binding of CG16947 to cdk9 causes its degradation. Taken together, this data shows that cdk9 plays a role in the survival of adult glia in the Drosophila brain. Thus, a fine balance exists between mir-31a and CG16947 expression in the progenitor cells that in turn regulates the levels of cdk9 in the progeny. This serves to allow the progenitor cells to regulate the number of glia in the adult brain.

The study of adult neural cell production has concentrated on neurogenesis. The mechanisms controlling adult gliogenesis are still poorly understood. Here, we provide evidence for a homeostatic process that maintains the population of glial cells in the Drosophila adult brain. Flies lacking microRNA miR-31a start adult life with a normal complement of glia, but transiently lose glia due to apoptosis. miR-31a expression identifies a subset of predominantly gliogenic adult neural progenitor cells. Failure to limit expression of the predicted E3 ubiquitin ligase, Rchy1, in these cells results in glial loss. After an initial decline in young adults, glial numbers recovered due to compensatory overproduction of new glia by adult progenitor cells, indicating an unexpected plasticity of the Drosophila nervous system. Experimentally induced ablation of glia was also followed by recovery of glia over time. These studies provide evidence for a homeostatic mechanism that maintains the number of glia in the adult fly brain., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Astrocytes constitute a major cell population in the brain with a myriad of essential functions, yet we know remarkably little about the signaling pathways and mechanisms that direct astrocyte maturation. To explore the signals regulating astrocyte development, we prospectively purified and cultured immature postnatal rodent astrocytes. We identified fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs) as robust trophic factors for immature astrocytes. We showed that astrocytes respond directly to BMPs via phosphorylation of the smad1/5/8 pathway. In vitro, BMP signaling promoted immature astrocytes to adopt multiple characteristics of mature astrocytes, including a more process-bearing morphology, aquaporin-4 (AQP4) and S100β immunoreactivity, limited proliferation, and strong downregulation of epidermal growth factor receptor (EGFR). In vivo, activation of the smad1/5/8 pathway in astrocytes was seen during early postnatal development, but inhibition of astrocyte proliferation was not observed. These insights can aid in the further dissection of the mechanisms and pathways controlling astrocyte biology and development.

To achieve its precise neural connectivity, the developing mammalian nervous system undergoes extensive activity-dependent synapse remodelling. Recently, microglial cells have been shown to be responsible for a portion of synaptic pruning, but the remaining mechanisms remain unknown. Here we report a new role for astrocytes in actively engulfing central nervous system synapses. This process helps to mediate synapse elimination, requires the MEGF10 and MERTK phagocytic pathways, and is strongly dependent on neuronal activity. Developing mice deficient in both astrocyte pathways fail to refine their retinogeniculate connections normally and retain excess functional synapses. Finally, we show that in the adult mouse brain, astrocytes continuously engulf both excitatory and inhibitory synapses. These studies reveal a novel role for astrocytes in mediating synapse elimination in the developing and adult brain, identify MEGF10 and MERTK as critical proteins in the synapse remodelling underlying neural circuit refinement, and have important implications for understanding learning and memory as well as neurological disease processes.

The metabolic enzyme for folate, Aldh1L1, has been shown to be expressed robustly in astrocytes of the brain. It is now well accepted that astrocytes in certain regions of the adult brain also serve as neural stem cells. Here, we examined whether Aldh1L1 is also expressed in postnatal neural stem cells. In vitro, cells in neural stem cell culture conditions have robust Aldh1L1 promoter activity. In vivo, in the adult brain, astroctyes in neurogenic regions express Aldh1L1 in a pattern consistent with inclusion in neural stem cells, and analysis of Aldh1L1+ cell transcriptome profiles from neurogenic regions reveal a robust enrichment of known regulators of neurogenesis. Genetic fate mapping with Aldh1L1 BAC Cre animals reveals adult-born neuroblasts of the rostral migratory stream are derived from Aldh1L1 expressing cells, as are sporadic neurons in other regions of the brain. Combining these lines of evidence from transgenic animals, cell culture, transcriptome profiling, and fate mapping, we conclude that Aldh1L1 is also expressed in neural stem cells in the brain. These findings may influence the future design of experiments utilizing Aldh1L1 genetic tools, and also suggest existing Aldh1L1 bacTRAP mice may be of use for further experiments profiling neural stem cells in vivo., (Copyright © 2013 Wiley Periodicals, Inc.)

For years, studies of neural-glial interactions have relied on the use of astrocytes derived from the extended culture of immature precursor cells isolated from the neonatal rodent brain. Although the astrocytes cultured under these selective cell survival conditions have been important tools for understanding astrocyte behavior, they do not necessarily reflect the behavior and function of mature astrocytes. We have developed methods for acute, prospective isolation and culture of mature astrocytes from rodent brains in a serum-free, defined medium. These immunopanning-based methods facilitate the study of astrocyte biology and function.

The purification of astrocytes by fluorescence-activated cell sorting (FACS) requires that an astrocyte-specific promoter drive the expression of the green fluorescent protein (GFP). Our laboratory uses FACS to acutely isolate astrocytes from young and old tissue as well as to isolate GFP-negative neurons at the end of the FACS sorting to conduct comparative unbiased, large-scale gene expression studies. Because of the relatively harsh nature of FACS sorting, few astrocytes or neurons survive long enough after the sort to be cultured.

We describe the use of immunopanning to purify rodent astrocytes. Immunopanning of astrocytes permits the prospective isolation of astrocytes from the rodent brain. Prospective isolation refers to the direct selection of cells without multiple steps that extend over a few days, thereby permitting the selection of a representative proportion of the astrocytes from the cortex. Because immunopanning is a very gentle process, the cells are viable at the end of the preparation and can be cultured in a serum-free medium containing heparin-binding EGF-like growth factor (HBEGF), the critical survival factor of astrocytes in vitro, for at least 2 wk. This protocol was initially established and verified with rats, but modifications for the purification of mouse cells are also described here.

Expansion of astrocyte populations in the central nervous system is characteristic of evolutionarily more complex organisms. However, regulation of mammalian astrocyte precursor proliferation during development remains poorly understood. Here, we used Aldh1L1-GFP to identify two morphologically distinct types of proliferative astrocyte precursors: radial glia (RG) in the ventricular zone and a second cell type we call an 'intermediate astrocyte precursor' (IAP) located in the mantle region of the spinal cord. Astrogenic RG and IAP cells proliferated in a progressive ventral-to-dorsal fashion in a tight window from embryonic day 13.5 until postnatal day 3, which correlated precisely with the pattern of active ERK signalling. Conditional loss of BRAF function using BLBP-cre resulted in a 20% decrease in astrocyte production, whereas expression of activated BRAFV600E resulted in astrocyte hyperproliferation. Interestingly, BRAFV600E mitogenic effects in astrocytes were restricted, in part, by the function of p16INK4A-p19(ARF), which limited the temporal epoch for proliferation. Together, these findings suggest that astrocyte precursor proliferation involves distinct RG and IAP cells; is subjected to temporal and spatial control; and depends in part on BRAF signalling at early stages of mammalian spinal cord development.

In the developing central nervous system (CNS), the control of synapse number and function is critical to the formation of neural circuits. We previously demonstrated that astrocyte-secreted factors powerfully induce the formation of functional excitatory synapses between CNS neurons. Astrocyte-secreted thrombospondins induce the formation of structural synapses, but these synapses are postsynaptically silent. Here we use biochemical fractionation of astrocyte-conditioned medium to identify glypican 4 (Gpc4) and glypican 6 (Gpc6) as astrocyte-secreted signals sufficient to induce functional synapses between purified retinal ganglion cell neurons, and show that depletion of these molecules from astrocyte-conditioned medium significantly reduces its ability to induce postsynaptic activity. Application of Gpc4 to purified neurons is sufficient to increase the frequency and amplitude of glutamatergic synaptic events. This is achieved by increasing the surface level and clustering, but not overall cellular protein level, of the GluA1 subunit of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) glutamate receptor (AMPAR). Gpc4 and Gpc6 are expressed by astrocytes in vivo in the developing CNS, with Gpc4 expression enriched in the hippocampus and Gpc6 enriched in the cerebellum. Finally, we demonstrate that Gpc4-deficient mice have defective synapse formation, with decreased amplitude of excitatory synaptic currents in the developing hippocampus and reduced recruitment of AMPARs to synapses. These data identify glypicans as a family of novel astrocyte-derived molecules that are necessary and sufficient to promote glutamate receptor clustering and receptivity and to induce the formation of postsynaptically functioning CNS synapses.

Reactive astrogliosis is characterized by a profound change in astrocyte phenotype in response to all CNS injuries and diseases. To better understand the reactive astrocyte state, we used Affymetrix GeneChip arrays to profile gene expression in populations of reactive astrocytes isolated at various time points after induction using two mouse injury models, ischemic stroke and neuroinflammation. We find reactive gliosis consists of a rapid, but quickly attenuated, induction of gene expression after insult and identify induced Lcn2 and Serpina3n as strong markers of reactive astrocytes. Strikingly, reactive astrocyte phenotype strongly depended on the type of inducing injury. Although there is a core set of genes that is upregulated in reactive astrocytes from both injury models, at least 50% of the altered gene expression is specific to a given injury type. Reactive astrocytes in ischemia exhibited a molecular phenotype that suggests that they may be beneficial or protective, whereas reactive astrocytes induced by LPS exhibited a phenotype that suggests that they may be detrimental. These findings demonstrate that, despite well established commonalities, astrocyte reactive gliosis is a highly heterogeneous state in which astrocyte activities are altered to respond to the specific injury. This raises the question of how many subtypes of reactive astrocytes exist. Our findings provide transcriptome databases for two subtypes of reactive astrocytes that will be highly useful in generating new and testable hypotheses of their function, as well as for providing new markers to detect different types of reactive astrocytes in human neurological diseases.

The inability to purify and culture astrocytes has long hindered studies of their function. Whereas astrocyte progenitor cells can be cultured from neonatal brain, culture of mature astrocytes from postnatal brain has not been possible. Here, we report a new method to prospectively purify astrocytes by immunopanning. These astrocytes undergo apoptosis in culture, but vascular cells and HBEGF promote their survival in serum-free culture. We found that some developing astrocytes normally undergo apoptosis in vivo and that the vast majority of astrocytes contact blood vessels, suggesting that astrocytes are matched to blood vessels by competing for vascular-derived trophic factors such as HBEGF. Compared to traditional astrocyte cultures, the gene profiles of the cultured purified postnatal astrocytes much more closely resemble those of in vivo astrocytes. Although these astrocytes strongly promote synapse formation and function, they do not secrete glutamate in response to stimulation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

To investigate the role of microRNAs in regulating oligodendrocyte (OL) differentiation and myelination, we utilized transgenic mice in which microRNA processing was disrupted in OL precursor cells (OPCs) and OLs by targeted deletion of Dicer1. We found that inhibition of OPC-OL miRNA processing disrupts normal CNS myelination and that OPCs lacking mature miRNAs fail to differentiate normally in vitro. We identified three miRNAs (miR-219, miR-138, and miR-338) that are induced 10-100x during OL differentiation; the most strongly induced of these, miR-219, is necessary and sufficient to promote OL differentiation, and partially rescues OL differentiation defects caused by total miRNA loss. miR-219 directly represses the expression of PDGFRalpha, Sox6, FoxJ3, and ZFP238 proteins, all of which normally help to promote OPC proliferation. Together, these findings show that miR-219 plays a critical role in coupling differentiation to proliferation arrest in the OL lineage, enabling the rapid transition from proliferating OPCs to myelinating OLs., (Copyright 2010 Elsevier Inc. All rights reserved.)

Understanding the cell-cell interactions that control CNS development and function has long been limited by the lack of methods to cleanly separate neural cell types. Here we describe methods for the prospective isolation and purification of astrocytes, neurons, and oligodendrocytes from developing and mature mouse forebrain. We used FACS (fluorescent-activated cell sorting) to isolate astrocytes from transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of an S100beta promoter. Using Affymetrix GeneChip Arrays, we then created a transcriptome database of the expression levels of >20,000 genes by gene profiling these three main CNS neural cell types at various postnatal ages between postnatal day 1 (P1) and P30. This database provides a detailed global characterization and comparison of the genes expressed by acutely isolated astrocytes, neurons, and oligodendrocytes. We found that Aldh1L1 is a highly specific antigenic marker for astrocytes with a substantially broader pattern of astrocyte expression than the traditional astrocyte marker GFAP. Astrocytes were enriched in specific metabolic and lipid synthetic pathways, as well as the draper/Megf10 and Mertk/integrin alpha(v)beta5 phagocytic pathways suggesting that astrocytes are professional phagocytes. Our findings call into question the concept of a "glial" cell class as the gene profiles of astrocytes and oligodendrocytes are as dissimilar to each other as they are to neurons. This transcriptome database of acutely isolated purified astrocytes, neurons, and oligodendrocytes provides a resource to the neuroscience community by providing improved cell-type-specific markers and for better understanding of neural development, function, and disease.

A simple water iodizing system, which incorporates the Venturi principle in combination with the controlled release mechanism of a silicone-sodium iodide elastomer, for the iodization of rural piped-water supply in the control of endemic iodine deficiency has been developed and its effectiveness evaluated in three Iban longhouse villages in the iodine-deficient district of Lubok Antu, Sarawak. Urines were collected for iodine assays from women aged 15-40 years before and at 6 and 12 months after the connection of the iodinating device; goiter assessment was performed on the women at the start and end of the 1-year study. Water samples were collected for iodine assays at 2-weekly intervals. In all three villages, significant and sustained increases in median urinary iodine excretions, reaching levels recommended for an iodine-sufficient population, were observed; goitre prevalences were reduced in all the villages (by 22.6% to 35.8%). The iodine levels in the water ranged from 34 micrograms/l to 212 micrograms/L. In the control village, median urinary iodine excretions remained essentially unchanged but a small increase in goiter prevalence was observed. The iodized water was well received by the villagers and no adverse effects of water iodization were observed. The system functioned unattended throughout the one year period. The cost of providing supplemental iodine via the iodizing device is approximately 60 cents (U.S.) per family per year which is affordable by either the Government or the villagers. It is concluded that the iodizing system offers a new cost-effective strategy for the control of endemic iodine deficiency in Sarawak and may have applications in other areas with similar water sources.

The urinary iodine excretions of women (15-40 y) and young children (< or = 6 y) from two longhouse villages in the iodine-deficient district of Lubok Antu, Sarawak, were compared. One longhouse (Mengkak) was provided with freshly produced iodized salt every two months (one kg per family) while the other (Menjiling) was provided with iodized water via fortification of the village piped-water supply. Spot urines were collected for iodine determination at baseline and at 6 and 12 months after the start of the study. Salt and water samples were collected at monthly intervals. Goiter assessment was performed on the women at the start and end of the one-year study. The mean iodine concentrations in the salt samples from Mengkak and Menjiling were, respectively, 47.1 +/- 9.7 mg/kg (n = 60) and 0.8 +/- 3.4 mg/kg (n = 60) while the mean iodine concentration in the water samples from Menjiling was 138.6 +/- 43.2 micrograms/L (n = 24); iodine could not be detected in the water samples from Mengkak. There were significant and sustained increases in median urinary iodine excretions of both women and young children in Menjiling; in Mengkak, however, significant and sustained increases in median urinary iodine excretions were observed only in women while the median urinary iodine excretions of children remained essentially unchanged throughout the study period. Goiter prevalences in the women were reduced in both longhouses. The above observations reveal the inadequacy of iodized salt as a vehicle for iodine delivery to young rural Sarawakian children and indicate the need for other means of delivering supplemental iodine to this age group in areas where salt iodization is the only strategy for IDD control. In contrast, iodization of village water supply by itself is adequate in delivering iodine uniformly to the whole community.

The effect of permethrin impregnated bednets on Anopheles maculatus Theobald was studied in four villages in Pos Betau, Pahang, Malaysia from August 1990 to July 1992. Collections of mosquitos were carried out indoors and outdoors from 1900 to 0700 hours. All mosquitos were dissected for sporozoites and parity. In May 1991 two villages received bednets impregnated with permethrin at 0.5 g/m2 and two villages received placebo bednets. There was a significant difference in the sporozoite and parous rates between the treated and control villages after the distribution of bednets (p < 0.05). There was no significant difference in the bites/man/night of An. maculatus between the pre and post treatment periods in the control villages. However there was a significant difference in bites/man/night between pre and post treatment in the treated villages (p < 0.001).

In a survey of 974 villagers (408 males, 566 females; ages = 11-82 years) of the Ai (n = 496; 212 males, 284 females) and Lemanak (n = 478; 196 males, 282 females) rivers in the district of Lubuk Antu in Sarawak's Sri Aman Division during July 1993, goiter was found in 31.8% of the subjects. The goiter prevalence was higher in the more interior Ai river area than in the Lemanak river area (36.9% vs 26.5%). In females aged 15 years and above, the goiter prevalence was 75.4% and 49.1%, respectively, in the Ai and Lemanak river areas. The difference in goiter prevalence between the two areas was related to the degree of iodine deficiency in the two areas. The median urinary iodine excretion in the Ai river villagers was 22.1 micrograms/l compared to 72.9 micrograms/l in the Lemanak river villagers (p < 0.0001). Goitrous subjects tended to have lower urinary iodine concentration than non-goitrous subjects. In the males, smoking of tobacco was associated with a two-fold increase in goiter frequency. Despite on-going distribution of iodized salt by the medical and health services in the State, only 23% of the 135 salt samples obtained from the households in the areas contained detectable iodine.

The malaria parasite rates and densities were compared in 79 ovalocytic-normocytic pairs of Malayan Aborigines matched for age, sex, proximity of residence to each other, and use of bed nets when sleeping in their jungle settlement in central Peninsular Malaysia. Malaria infection was determined from thick and thin Giemsa-stained blood films collected monthly for a period of six months. Blood films from ovalocytic individuals were found to be positive for malaria less often than in persons with normal red blood cells (P less than 0.05). Malaria infections per 100 person-months at risk were 9.7 in the ovalocytic group compared with 15.19 in the normocytic group. Among individuals parasitemic at any time, heavy infections (greater than or equal to 10,000 parasites/mm3 of blood) with Plasmodium falciparum, P. vivax, and P. malariae were encountered only in normocytic subjects, which comprised approximately 12.5% of the malaria-positive individuals in this group. In an earlier survey of 629 settlers that identified subjects for the above study, the prevalence of ovalocytosis was found to increase significantly with age. The above field observations support the view that ovalocytic individuals might have a survival advantage in the face of malaria. Consideration of the ovalocytic factor is indicated in future evaluations of malaria control measures in areas where ovalocytosis is prevalent.

Platelet aggregation to collagen, and productions of 6-keto-prostaglandin-F1-alpha and thromboxane B2 during aggregation were measured after an overnight fast, involving both food and cigarettes, in 19 clinically healthy habitual smokers (10 or more cigarettes/day) and 23 non-smokers receiving the same diet. The subjects (all males; ages = 21-30 years) were residents of a school hostel. Mean platelet aggregation was significantly lower in smokers than non-smokers (23.2 ohms vs 31.5 ohms, p less than 0.005). Non-smokers had significantly higher mean concentration of 6-keto-prostaglandin-F1-alpha than smokers (109.8 pmol/l vs 92.3 pmol/l, p less than 0.05). The level of thromboxane B2 did not differ significantly between the two groups. These observations suggest that the role of smoking as a risk factor for ischaemic heart disease is unlikely to be related to a direct enhancement of aggregation. On the contrary, the observations seem to suggest that habitual smoking may directly reduce platelet aggregability.

The effect of parity on the hematological response to supplemental hematinics and the relationship between birth weight and Hb concentration were examined in 67 pregnant rural Kelantanese Malay women recruited at 20-24 weeks of gestation. Among initially anemic women (Hb concentration at recruitment < 110 g/l), a significant supplementation effect was observed in the lower parae (3 or less children) but not in the higher parae. Among initially nonanemic women, a progressive decline in mean Hb concentration was observed in the higher parae; in the lower parae, however, an initial fall in mean Hb concentration was followed by a rise to almost the initial level. Birth weight was inversely related to initial Hb concentration. There was no association between birth weight and final measured Hb level, parity or any of the measured maternal characteristics. These observations suggest: a) women with initially lower Hb concentration might have experienced a greater acceleration of plasma volume expansion than those with initially higher Hb level; and b) hemopoiesis might be impaired in the higher multiparae.

Anthropometric and parasitological data from cross-sectional studies of two groups of primary school children (Group I of Indian origin, 325 boys and 259 girls, age = 7 years; Group II of Malay origin, 284 boys and 335 girls, age = 7-9 years) from two different ecological settings in Peninsular Malaysia were examined for epidemiological evidence of an association between hookworm infection and protein-energy malnutrition. In both ecological groups, significant weight, height and haemoglobin deficits were observed in children with hookworm infection after adjustment for covariables including Ascaris and Trichuris infection intensities and other child and family characteristics. The deficits were related to the intensity of infection based on egg counts. These findings suggest that hookworm may be an important determinant of chronic protein-energy malnutrition, as well as anaemia, in areas where diets are generally inadequate in protein, energy, and iron. Well-controlled intervention studies are needed to confirm these observations.

Twenty-four-hour urine samples and whole deciduous teeth from fluoridated (0.71 ppm) and non-fluoridated (0.14 ppm) areas together with some selected local food items were analysed for their fluoride content. The mean values for urinary fluoride were 0.90 ppm or 0.77 mg per day for the fluoridated area and 0.50 ppm or 0.52 mg per day for the non-fluoridated area. Assuming that half of all the fluoride ingested is excreted in the urine, this study suggests that the average daily fluoride intakes by adults in the fluoridated and non-fluoridated areas were about 1.5 mg and 1 mg respectively. The mean fluoride content of non-carious deciduous teeth from the fluoridated area was 416.89 ppm compared to 178.45 ppm in the low fluoride area.