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In this abbreviated extract of his memoirs of a life in malaria research, Richard Carter (1945-2021) describes the beginnings of malaria parasite genetics research at Edinburgh university, culminating in the first laboratory genetic cross between two strains of malaria parasites. EDITORS' NOTE: This manuscript is an abridged excerpt from Richard Carter's first volume of memoirs describing his career in malaria research. Both volumes, the first concerning malaria parasite genetics, the second focussing on transmission and transmission blocking, are housed in the Wellcome Collection, London [1,2]. Electronic copies of the unabridged memoirs are available to the interested reader upon request to the Editors. These memoirs began as a series of 'recollections' written to a PhD student in March 2014, and were expanded and revised by the author up to 2018. Apart from abridgement, the text is presented in its original form, preserving the informal style in which it was written.

In this abbreviated extract of his memoirs of a life in malaria research, Richard Carter (1945-2021) describes the history of the first genetic cross with the human malaria parasite, Plasmodium falciparum. This work followed on from Richard's initial involvement in the establishment of the field of malaria parasite genetics using the malaria parasites of rodents, the history of which is recounted in Parts I [1] and II [2] of this series. EDITORS' NOTE: This manuscript is an abridged excerpt from Richard Carter's first volume of memoirs describing his career in malaria research. Both volumes, the first concerning malaria parasite genetics, the second focussing on transmission and transmission blocking, are housed in the Wellcome Collection, London [3,4]. Electronic copies of the unabridged memoirs are available to the interested reader upon request to the Editors.

The recent and rapid digitization of biodiversity data from natural history collection (NHC) archives has enriched collections based data repositories; this data continues to inform studies of species' geographic distributions. Here we investigate the relative impact of plant data from small natural history collections (collections with

Precision welded joints, produced between fused silica glass and aluminium by a newly-developed picosecond-pulse laser technique, have been analysed for the first time using a full range of electron microscopy methods. The welds were produced as lap joints by focusing a 1.2 μm diameter laser beam through the transparent glass top sheet, slightly below the surface of the metal bottom sheet. Despite the extremely short interaction time, extensive reaction was observed in the weld zone, which involved the formation of nanocrystalline silicon and at least two transitional alumina phases, γ- and δ-Al2O3. The weld formation process was found to be complex and involved: the formation of a constrained plasma cavity at the joint interface, non-linear absorption in the glass, and the creation of multiple secondary keyholes in the metal substrate by beam scattering. The joint area was found to expand outside of the main interaction volume, as the energy absorbed into the low conductivity and higher melting point silica glass sheet melted the aluminium surface across a wider contact area. The reasons for the appearance of nanocrystalline Si and transitional alumina reaction products within the welds are discussed.

Following the bite of an infective mosquito, malaria parasites first invade the liver where they develop and replicate for a number of days before being released into the bloodstream where they invade red blood cells and cause disease. The biology of the liver stages of malaria parasites is relatively poorly understood due to the inaccessibility of the parasites to sampling during this phase of their life cycle. Here we report the detection in blood and faecal samples of malaria parasite DNA throughout their development in the livers of mice and before the parasites begin their growth in the blood circulation. It is shown that parasite DNA derived from pre-erythrocytic stage parasites reaches the faeces via the bile. We then show that different primate malaria species can be detected by PCR in blood and faecal samples from naturally infected captive macaque monkeys. These results demonstrate that pre-erythrocytic parasites can be detected and quantified in experimentally infected animals. Furthermore, these results have important implications for both molecular epidemiology and phylogenetics of malaria parasites. In the former case, individuals who are malaria parasite negative by microscopy, but PCR positive for parasite DNA in their blood, are considered to be "sub-microscopic" blood stage parasite carriers. We now propose that PCR positivity is not necessarily an indicator of the presence of blood stage parasites, as the DNA could derive from pre-erythrocytic parasites. Similarly, in the case of molecular phylogenetics based on DNA sequences alone, we argue that DNA amplified from blood or faeces does not necessarily come from a parasite species that infects the red blood cells of that particular host.

This paper aims to develop a historiography of the Holocaust based around the spatialities of organised resistance, through a geographical reading of two cinematic depictions of the 1943 prisoner uprising at Sobibor death camp: Escape From Sobibor (1987) and Sobibor, octobre 14 1943, 1600 heures (2001). These case studies are privileged in my reading on account of the capacity of filmmaking practices to provoke new directions in historical and geographical research. Specifically, I argue that in order to tell their stories of resistance, these films are forced to depict several aspects of camp spatiality – including spatial routines, disciplined mobility, heterogeneous social space, the mental maps of prisoners and perpetrators, work sites and networks of institutions – that are easily overlooked in narratives focussing solely on domination and killing in the camps. The filmmaking practices of these films therefore testify to a relation between organised resistance and camp spatiality that can be productive for ongoing efforts to write the geographies of the Holocaust. The paper also seeks to make a methodological intervention by demonstrating the value of moving Holocaust film criticism beyond its prevalent preoccupation with standards of verisimilitude.

Clinical immunity against malaria is slow to develop, poorly understood and strongly strain-specific. Understanding how strain-specific immunity develops and identifying the parasite antigens involved is crucial to developing effective vaccines against the disease. In previous experiments we have shown that strain-specific protective immunity (SSPI) exists between genetically distinct strains (cloned lines) of the rodent malaria parasite Plasmodium chabaudi chabaudi in mice [Cheesman, S., Raza, A., Carter, R., 2006. Mixed strain infections and strain-specific protective immunity in the rodent malaria parasite P. chabaudi chabaudi in mice. Infect. Immun. 74, 2996-3001]. In two subsequent studies, we identified the highly polymorphic Merozoite Surface Protein 1 (MSP-1) as being the principal candidate molecule for the control of SSPI against P. c. chabaudi malaria [Martinelli et al., 2005; Pattaradilokrat, S., Cheesman, S.J., Carter R., 2007. Linkage group selection: towards identifying genes controlling strain-specific protective immunity in malaria. PLoS ONE 2(9):e857]. In the present study, we sequenced the whole msp1 gene of several genetically distinct strains of P. chabaudi and found high levels of genetic diversity. Protein sequence alignments reveal extensive allelic polymorphism between the P. chabaudi strains, concentrated primarily within five regions of the protein. The 3′-end sequence region, encoding the C-terminal 21 kDa region (MSP-1 21 ), which is analogous and homologous to MSP-1 19 of Plasmodium falciparum , appears to have been subject to balancing selection. We have found that the strains with the lowest sequence identity at MSP-1 21 (i.e. AS/CB and AJ/CB) induce robust and reciprocal SSPI in experimental mice. In contrast, two strains that do not induce reciprocal SSPI are identical at the 21 kDa region. Final identification of the region(s) controlling SSPI will provide important information to help guide decisions about MSP-1 based vaccines.

We investigated the parasitology, pathogenicity (virulence) and infectivity to mosquitoes of blood infections in mice, of two strains, DS and DK, of the rodent malaria parasite Plasmodium chabaudi adami. Blood infections of DS were found to be highly pathogenic; the asexual parasites in these infections were fast-growing and showed no evidence of selectivity in their infection of host erythrocytes. In contrast to DS, blood infections of DK were much less pathogenic; the asexual parasites were slower-growing and showed a moderate degree of selectivity to a subset of erythrocytes which were not reticulocytes. In both DS and DK infections, infectivity to mosquitoes was highest before the peak of asexual parasitaemia had occurred; usually this did not coincide with the time when gametocyte numbers in the blood were highest. Infections with the pathogenic DS strain in CBA mice produced fewer gametocytes than did the less pathogenic DK strain. The DS strain infections in both CBA and C57 mice were also significantly much less infective to mosquitoes than the DK strain. Investigations by others on the related rodent malaria parasite subspecies, Plasmodium chabaudi chabaudi, have indicated that the mosquito infectivity of blood infections in mice tended to be higher in the more pathogenic (virulent) and lower in the less pathogenic strains of this parasite subspecies. This is the converse of the finding of the present investigation of blood infections of P. c. adami in mice in which a more pathogenic, or virulent, strain (DS) of these parasites was significantly much less infective to mosquitoes than was a less pathogenic strain (DK).

Many of the most commonly studied lines of the rodent malaria parasite Plasmodium yoelii yoelii originated from a single parasite isolate designated 17X. Amongst these lines, however, are parasites that exhibit variation in genotype and phenotype (e.g. growth rate). We describe here the results of a comparative genetic analysis between cloned lines of 17X that differ in growth rate, using nucleotide sequences of specific genes and patterns of genome-wide amplified fragment length polymorphism (AFLP). Our findings indicate that the original stock of 17X comprises two unrelated genotypes. Genotype-1 is represented by parasites with a slow growth phenotype (e.g. 17X (NIMR)) and a fast growth phenotype (e.g. 17XYM). Within this genotype, there are also genomic differences manifest as a small number of AFLP bands that differentiate the fast- and slow-growing lines from each other. The other genotype, genotype-2, is represented only by parasites with a slow growth phenotype (e.g. 17XA).

Species of Plasmodium that naturally infect wild rodents but can also be maintained in laboratory mice have long been used as model systems in which to study the biology of malaria parasites. Several of these rodent parasites are now providing useful genomic comparisons to those species that cause malaria in humans. Here we examined the phylogenetic relationships of 19 strains of rodent malaria parasites including four species native to African thicket rats ( Plasmodium berghei , Plasmodium chabaudi , Plasmodium vinckei , and Plasmodium yoelii ) and one from a porcupine ( Plasmodium atheruri ) using DNA sequence data collected from seven genes from each of the three parasite genomes. These included the nuclear dihydrofolate reductase gene and a cysteine protease gene, mitochondrial cytochrome b and cytochrome oxidase I genes, and the elongation factor tufA , caseinolytic protease C, and “open reading frame 470” genes from the apicoplast genome, for a combined total of 5049 nucleotides. Using simultaneous analysis, a method of combining each of the gene partitions into a super-matrix, two equally parsimonious trees were recovered. Bayesian analysis of the dataset produced the same topology. The basic species groups were well supported, with the exception of the placement of P. atheruri within the P. vinckei clade. Named subspecies showed a wide array of genetic differentiation, but fell into monophyletic groups.

Chloroquine resistance in the rodent malaria parasite Plasmodium chabaudi has been shown to be caused by a gene on chromosome 11, and is not linked to orthologues of the Plasmodium falciparum chloroquine resistance transporter ( pfcrt ) or Pgh-1 ( pfmdr1 ) genes. In the current work, the progeny of crosses between chloroquine-resistant and sensitive clones of P. chabaudi have been analysed for the inheritance of 658 AFLP markers. Markers linked to the chloroquine responses of the progeny, including two which are completely linked, have been genetically mapped, sequenced and their homologues, or closely linked loci, identified in P. falciparum . The chromosome 11 markers most closely linked to chloroquine resistance in P. chabaudi map to loci which are also closely linked in P. falciparum , although in two linkage groups on chromosomes 6 and 13 of this species. The P. falciparum orthologue of the gene conferring chloroquine resistance in P. chabaudi is predicted to lie within a 250 kb region of P. falciparum chromosome 6, containing approximately 50 genes. The genetic order of the markers in P. chabaudi is co-linear with the physical linkage represented in the P. falciparum genome database. The findings provide evidence for extensive conservation of synteny between the two species.

A technique that can distinguish and quantify genetically different malaria parasite clones in a mixed infection reliably and with speed and accuracy would be very useful for researchers. Many current methods of genotyping and quantification fall down on a number of aspects relating to their ease of use, sensitivity, cost, reproducibility and, not least, accuracy. Here we report the development and validation of a method that offers several advantages in terms of cost, speed and accuracy over conventional PCR or antibody-based methods. Using real-time quantitative PCR (RTQ-PCR) with allele-specific primers, we have accurately quantified the relative proportions of clones present in laboratory prepared ring-stage mixtures of two genetically distinct clones of the rodent malaria parasite Plasmodium chabaudi chabaudi. Accurate and reproducible measurement of the amount of genomic DNA representing each clone in a mixture was achieved over 100-fold range, corresponding to 0.074% parasitised erythrocytes at the lower end. To demonstrate the potential utility of this method, we include an example of the type of application it could be used for. In this case, we studied the growth rate dynamics of mixed-clone infections of P. chabaudi using an avirulent/virulent clone combination (AS (PYR) and AJ) or two clones with similar growth rate profiles (AQ and AJ). The modification of the technique described here should enable researchers to quickly extract accurate and reliable data from in-depth studies covering broad areas of interest, such as analyses of clone-specific responses to drugs, vaccines or other selection pressures in malaria or other parasite species that also contain highly polymorphic DNA sequences.

It is likely that Plasmodium vivax diverged approximately 2 million years ago from a group of malaria parasites which are now endemic in monkeys and apes in southern Asia. In those times, primates were spread throughout most of Eurasia and Africa, indicating an Old World location, but nothing more precise, for the place of divergence of P. vivax. From approximately 1 million years ago, the Ice Ages would have isolated human malaria, including P. vivax, into humid temperate or warm climate refuges around the Mediterranean, in sub-Saharan Africa and in south and east Asia. As there appears to be no record of humans in south and east Asia from 100,000 to 60,000 years ago, they might not have passed on their parasites, including P. vivax, to modern humans entering the region after this time. Today, all P. vivax might be descended from parasites which infected human populations in the Mediterranean region and in sub-Saharan Africa during the last Ice Age, between 100,000 and 20,000 years ago. Evidence for the latter is provided by the presence of very high frequency RBC Duffy negativity in sub-Saharan Africa.

A simple, visual representation of spatial aspects of malaria transmission in successive snap-shots in time, is presented. The spatial components of the simulation involve (i) the identification of mosquito vector breeding sites of defined shape and area, (ii) the identification of a zone of malaria transmission determined by the shapes and areas of the vector breeding sites and the distance from these sites that the mosquitoes disperse, (iii) a human population dispersed in relation to the malaria transmission zone, (iv) perimeters around each individual human within which his or her infection can be transmitted by the local vector mosquitoes. The intensity of transmission within a malaria transmission zone is given by a number which is the number of new cases of malaria that each existing case will distribute through the human population within the duration of an infection. The simulation has been used here to examine the effects of vaccination against malaria transmission. Different levels of vaccine coverage are represented under endemic and epidemic malaria. The consequences of full or partial coverage of a zone of malaria transmission are also examined. The results are numerically compatible with the predictions of previous simple mathematical simulations of malaria transmission and interventions. The present simulation allows the nature of malaria transmission and the effects of interventions to be communicated easily and directly to an audience. It could have practical value in discussions of malaria control strategies with health planners.

We have used the method of amplified fragment length polymorphism (AFLP) to identify genetic polymorphisms between two cloned isolates of the rodent malaria parasite Plasmodium chabaudi chabaudi. The method employs polymerase chain reaction (PCR)-amplification of genomic DNA fragments cut with specific combinations of restriction endonucleases; we used EcoRI and Tru1I (isoschizomer of MseI). We have identified 819 parasite clone-specific AFLPs between P. c. chabaudi clones AS and AJ. Of these, 403 fragments were specific to AS and 416 to AJ. In preparing blood stage parasites for DNA, nucleated host cells were removed by successive filtration of infected blood through powdered cellulose and Plasmodipur filters. This reduced nucleated host cell contamination to around 1-10 per million parasite nuclei and reduced host DNA to below the limit of detection by the AFLP method. Analysis of our results showed that the total number of PCR-amplified fragments of parasite DNA was consistent with the predicted number of EcoRI sites in the parasite genome. 19.4% of all amplified fragments were P. c. chabaudi clone-specific. From this figure we estimated that the diversity between clones AS and AJ, measured as the probability of a sequence difference, was between about 8 x 10(-3) and 4.6 x 10(-4) per base pair. This is consistent with the sequence diversity found between alleles of candidate drug resistance genes from P. c. chabaudi clones AS and AJ identified and sequenced in this laboratory.

Transmission blocking vaccines (TBVs) against malaria are intended to induce immunity against the stages of the parasites which infect mosquitoes so that TBV-immunised individuals cannot transmit malaria. As malarial infections are transmitted mainly within a few hundreds of meters from an infectious human source, TBVs used within in a community would protect the immediate neighbourhood of the vaccinated individuals. TBVs against the two major species of human malaria, Plasmodium falciparum and P. vivax, are under development. Candidate TBV constructs for both Plasmodium species have been successfully tested in animal systems and testing is in progress with clinical grade material in humans.

The polymerase chain reaction (PCR) is widely used to detect and identify low numbers of blood forms of different species of human malaria parasites [1]. The method has demonstrated a high prevalence of sub-microscopic asymptomatic Plasmodium falciparum infections among inhabitants of malaria-endemic countries [2–5]. Absence of patent parasites from the peripheral blood is often associated with sub-patent parasitaemias that can last for several months, even when the possibility of reinfection can be ruled out [5,6]. However, it is not known whether such sub-patent long lasting infections can produce sexual forms and thus be capable of infecting mosquitoes. Some studies have indicated that the minimum density of gametocytes capable of infecting mosquitoes can be remarkably low (reviewed by Carter and Gwadz [7]), and it has been demonstrated that sexual forms are infectious to mosquitoes even when they occur at levels not detectable by microscopy [8]. Here we describe a reverse transcriptase polymerase chain reaction (RT-PCR) for detection of gametocytes of P. falciparum. Unlike asexual parasites, the mature gametocytes of P. falciparum are metabolically relatively inactive [9]. However, almost immediately mature gametocytes are activated to undergo gametogenesis, either upon ingestion by a mosquito or upon stimulation in vitro, they enter a period of intense protein synthesis [10]. Among the proteins which are produced rapidly and in large amounts following such activation, is the zygote and ookinete surface protein Pfs25 [10]. This protein and, as we show here, its mRNA molecules are specific to the activated gametocyte and to the zygote and ookinete stages into which it transforms. Both Pfs25 and its mRNA are totally absent from the asexual stage parasites of P. falciparum. We have exploited this property of the mRNA of Pfs25 in the activated gametocytes of P. falciparum in or* Corresponding author. Tel.: +44-131-6508658; fax: +44131-6673210; e-mail: H.Babiker@ed.ac.uk.

Malaria mortality in human populations varies greatly under different circumstances. The intense malaria transmission conditions found in many parts of tropical Africa, the much lower malaria inoculation rates currently sustained in areas of southern Asia, and the epidemic outbreaks of malaria occasionally seen on both continents, present highly contrasting patterns of malaria-related mortality. Here Harsha Alles, Kamini Mendis and Richard Carter examine malaria-related mortality under different circumstances and discuss implications for the management of malaria in these settings. They emphasize the power of rapid case treatment to save lives at risk under virtually all circumstances of malaria transmission.

Thomas Mann, German novelist and essayist, was awarded the Nobel Prize for literature in 1929 for his masterpiece, The Buddenbrooks, aided by his much acclaimed The Magic Mountain, which depicts life in a tuberculosis sanitorium. One of the most medically perceptive writers of the century, Mann was obsessed by illness and disease and produced works that reflect penetrating observation of medicine. When Hitler seized power, Mann fled Germany and entered Switzerland in forced exile. Mann later moved to the United States, becoming an American citizen in 1944. He became embroiled in political turmoil during the red-baiting era; disenchanted with this country, he returned to Switzerland in 1952 and died in Zurich in 1955. This vignette lifts the veil revealing the Mann behind the mask, tormented by life-long homoerotic tendencies that were confirmed by his unexpurgated diaries, published in 1979. I interviewed Professor Christoph Hedinger, who performed the autopsy on Thomas Mann. Professor Hedinger's protocol formed the primary source material for this study and clarified the exact cause of the Nobelist's death. Magisterial, with robust self-regard, Thomas Mann led a life that lends itself as a great human interest story. He was a highly intelligent, complex person whose sexual inversion provides some insight into the person but is irrelevant in assessing his artistic achievements. Mann's defiant stance against the Nazis and his masterful contributions to world literature assure him an enduring and unassailable role in world history.

Much of the work which has led to a widely held view that the income elasticity of health care spending exceeds one has been based on international cross-section data, or on pooled cross-sections and time series. In this paper we re-examine this view in the context of long-run equilibrium relationships between non-stationary time series, possibly including autonomous trends. Our results cast doubt upon the usefulness of pooling and upon the notion of an elasticity above one.

Primary health-care providers have been encouraged to counsel their patients about regular physical activity, but there are significant barriers to effective counseling. In this study a program of training and materials was tested for acceptability to providers, office staff, and patients. Primary care providers and office staff were trained to use the Physician-based Assessment and Counseling for Exercise (PACE) materials in four geographical sites in the United States. The program was tested in a variety of settings and with diverse patient populations. The acceptability of the program during a five-month study period was evaluated through structured interviews. The training was effective in preparing the providers to counsel, and the program was generally acceptable to providers, office staff, and patients. Counseling was provided in less than five minutes by 70% of providers, and most patients reported following the recommendations given. The PACE program assists providers in overcoming barriers to counseling patients about physical activity. The PACE program is potentially an important part of a national effort to enhance the adoption and maintenance of physical activity among adults.

Some molecular characteristics of Plasmodium falciparum lines which do not produce gametocytes are described. Parasites carrying a subtelomerically deleted chromosome 9 cannot form even the earliest forms of gametocytes, detectable with antibodies against the gametocyte-specific antigen Pfg27. In a parasite culture of clone HB3, in which both intact and deleted forms of chromosome 9 are present, full-length chromosome 9 molecules are retained mainly in gametocytes. These data suggest that the subtelomeric portion of chromosome 9 is required at an early stage of gametocytogenesis. Parasite subclones derived from gametocyte producing clone 3D7, which completely lost ability to produce gametocytes, are also described. Unlike the previous gametocyteless lines, these parasites stably maintain a full-length chromosome 9 and the ability to cytoadhere to C32 melanoma cells after prolonged asexual propagation. Their defect in sexual development is therefore genetically and functionally distinct from that of parasites carrying a deleted chromosome 9. Gametocyteless subclones derived from 3D7 do not produce any Pfg27 mRNA, while this gene is anomalously expressed in asexual stage parasites of two lines of a different genetic background, 1776sel8 and C10, one able and the other unable to produce gametocytes.

Pfs230 is a surface protein of the gametes of Plasmodium falciparum and has been demonstrated to be a target of malaria transmission-blocking antibodies; it is an important andidate candidate antigen for a transmission-blocking vaccine. The target epitopes of transmission-blocking antibodies against Pfs230 are almost all reduction sensitive suggesting that disulfide bonds are critical for folding the native molecule. Following the cloning of the Pfs230 gene attempts are now underway to express subunits of the protein for use in vaccine trials. It will be important to understand the disulfide-bond structure of the Pfs230 to achieve this goal. In this paper we present a model for this structure based on the observation that the Pfs230 molecule contains a series of regularly repeated cysteine-containing motifs. Four such motifs have been identified, together with a fifth cysteineless motif, which occur in the same relative order, with regular alternating omission of specific motifs, 14 times throughout the length of the protein. Each of the 14 sets of motifs contains an even number of cysteine residues (2, 4 or 6). We postulate that each set folds into a separate disulfide-bonded domain in which corresponding pairs of cysteines form an equivalent disulfide bond in every such domain. The postulated bonding arrangements in the different domains are mutually confirmatory throughout the sequence of Pfs230. We have identified two other malaria proteins, Pfs4845 and Pf12, which share the same arrangements of motifs and conform to the same disulfide-bond structure proposed for Pfs230; no other proteins in the sequence data base share these characteristics. Although having similarities to the ‘cystine knot’ described in other proteins, the arrangement proposed here appears to be unique among described structures.

A gene encoding a novel cdc2-related protein kinase has been identified in Plasmodium falciparum, using degenerate oligonucleotides designed to hybridise to regions that are conserved in members of the cdc2 gene family. This gene, called Pfcrk-1, is located on chromosome 4. It is most closely related to the p58GTA gene family, members of which are negative regulators of cell growth in vertebrates. Pfcrk-1 is developmentally regulated, as indicated by stage-specific accumulation of mRNA in gametocytes.

Individual oocysts from Plasmodium falciparum-infected Anopheles gambiae and Anopheles stephensi mosquitoes have been examined by the PCR technique, after their removal from the midgut. The DNA obtained from these oocysts has been amplified using oligonucleotide primers specific for part of the merozoite surface antigen MSA-1 gene. This technique distinguishes oocysts which are the products of self-fertilisation events from those which are the products of cross-fertilisation between different parasite clones.

The gene encoding the gametocyte specific cytoplasmic protein Pfg 27/25 of the human malaria parasite Plasmodium falciparum has been cloned. The gene encodes a highly hydrophilic non-repetitive protein which does not share obvious homologies with other polypeptides. The stage specificity of Pfg 27/25 is controlled at the stage of the production of stable mRNA, which is detectable only in the sexual stages of the parasite, and contains long additional sequences outside the Pfg27/25 coding region. As the activation of Pfg27/25 gene expression occurs at an early stage of gametocytogenesis, the study of its regulation might provide information on the molecular events occurring after the parasite commitment to sexual differentiation and at the beginning of gametocyte formation.

Malarial infections do not readily evoke an effective protective immunity against re-infection. Possible reasons for this include the ability of the parasites to interfere with the host's immune response and to evade the response in an immune host, by, for example, exploiting antigenic polymorphism or variation. Antigenic polymorphism undoubtedly exists in malaria parasite populations but does this polymorphism actually contribute to immune evasion by the parasite? Here, Kamini Mendis and colleagues examine the evidence for this and its implications for future malaria vaccines.

Humans infected with malaria produce antibodies against the sexual stages of the parasite (Mendis et al., 1987; Graves et ai., 1988), as they do against pre-erythrocytic (Nardin et al., 1979) and asexual blood stages (Cohen et al., 1961). These antibodies are evoked by gametocytes which are precursors of the sexual stages that are presented to the host immune system during a blood infection. The antibodies take effect in the midgut of a mosquito when it takes a blood meal from an infected person, where they react either with surface antigens of male and female gametes that are formed in the mosquito midgut and prevent fertilization, or with postfertilization stages such as the zygote, and prevent their further development (reviewed by Carter et ai., 1988). A potent vaccine-induced immunity against these sexual stages could reduce malar'~. ~:ansmission in endemic areas (De Zoysa et ai., i988, i99i). Parasite molecules that are targets of transmissionblocking antibodies have been defined in both of the major human malaria parasites P. falciparum (Carter et al., 1988; Rener et al., 1983; Vermulen et al., 1985) and P. vivax (Premawansa et al., 1990), and the genes coding for two of them, Pfs25, an ookinete antigen of P. falciparum (Kaslow et al., 1988) and GAM-1 of P. vivax (personal communication, P.H. David) have been cloned. The Pfs25 ookinete antigen has been successfully expressed in the vaccinia virus, and immunization of mice with recombinant vaccinia has been . . . . . . s a t ~ , to induce antibodies which blocked the infectivity of P. falciparum gametocytes to mosquitoes in membrane feeding experiments (Kaslow et al., 1991). Thus transmission blocking malaria vaccines may soon be ready for clinical testing.

Studies on the natural immune responses to the sexual stages of malaria parasites have been reviewed in the context of human malaria transmission-blocking vaccines. Antibodies against the sexual stages of the malaria parasite, gametocytes and gametes, are readily evoked by natural malaria infections. These antibodies that suppress infectivity at high concentrations can, at low concentrations, enhance the development of the parasite in the mosquito; however, because enhancing antibodies are prevalent during natural malaria infections, it is likely that a vaccine would rapidly boost these antibodies to blocking levels. The immunogenicity of sexual stage antigens appears to be constrained in the human host, probably due to T epitope polymorphism and MHC restriction in humans. These constraints apply mainly to those antigens that are sensitive targets of host immunity such as the gamete surface antigens and not to internal gamete antigens, indicating that antigenic polymorphism may have evolved in response to immune selection pressure. Evidence for immunosuppression of the host by exposure to endemic malaria is presented and its consequences on vaccine development are discussed.

Dr. Lyman Augustus Brewer III, a distinguished, colorful thoracic surgeon and among the first to practice that specialty in the West, died on June 25, 1988, in Los Angeles, California, after a courageous battle with lymphoma. Dr. Brewer was a great humanist, innovative clinical surgeon, charismatic teacher, and surgical leader. In World War II, Lieutenant Colonel Brewer served in the Second Auxiliary Surgical Group in the Mediterranean and European theaters and helped define criteria that became the standard for the management of thoracic war injuries. Out of this experience he authored the classic paper, "The Wet Lung in War Casualties." Dr. Brewer's scientific contributions embraced the broad spectrum of thoracic surgical topics, including treatment of tuberculosis, classification of lung cancer, bronchial stump buttressing using the pericardial fat pad (Brewer fat pad), and management of esophageal perforation. Dr Brewer wrote seven books and more than 100 papers, and served as First Vice President of The American College of Surgeons and as President of the American Association for Thoracic Surgery, The Society of Thoracic Surgeons, and The Pacific Coast Surgical Association.

BackgroundEosinophilic airway inflammation is heterogeneous in asthmatic patients. We recently described a distinct subtype of asthma defined by the expression of genes inducible by T(H)2 cytokines in bronchial epithelium. This gene signature, which includes periostin, is present in approximately half of asthmatic patients and correlates with eosinophilic airway inflammation. However, identification of this subtype depends on invasive airway sampling, and hence noninvasive biomarkers of this phenotype are desirable.ObjectiveWe sought to identify systemic biomarkers of eosinophilic airway inflammation in asthmatic patients.MethodsWe measured fraction of exhaled nitric oxide (Feno), peripheral blood eosinophil, periostin, YKL-40, and IgE levels and compared these biomarkers with airway eosinophilia in asthmatic patients.ResultsWe collected sputum, performed bronchoscopy, and matched peripheral blood samples from 67 asthmatic patients who remained symptomatic despite maximal inhaled corticosteroid treatment (mean FEV(1), 60% of predicted value; mean Asthma Control Questionnaire [ACQ] score, 2.7). Serum periostin levels are significantly increased in asthmatic patients with evidence of eosinophilic airway inflammation relative to those with minimal eosinophilic airway inflammation. Alogistic regression model, including sex, age, body mass index, IgE levels, blood eosinophil numbers, Feno levels, and serum periostin levels, in 59 patients with severe asthma showed that, of these indices, the serum periostin level was the single best predictor of airway eosinophilia (P= .007).ConclusionPeriostin is a systemic biomarker of airway eosinophilia in asthmatic patients and has potential utility inpatient selection for emerging asthma therapeutics targetingT(H)2 inflammation.

Two histidine-rich protein genes in Plasmodium falciparum are related by an ancestral duplication and interchromosomal transposition. We have followed the inheritance of these genes in a cross between two clones of P. falciparum. Examination of progeny shows that one gene, encoding the protein HRP-II, behaves as expected and may be inherited from either parent. The other gene, encoding HRP-III, has been found to derive from one parent in all progeny examined. We conclude the linkage group marked by HRP-III is favored strongly in the cross. This linkage group spans a region at one end of chromosome 13. Growth studies suggest the favored inheritance is explained by rapid expansion of progeny possessing the HRP-III marker relative to slower growth of progeny without it.

The association of cancer of the esophagus with long-standing achalasia signals an especially lethal disease, for in our experience and in a review of the literature this malignant condition has never been diagnosed in time to effect a permanent surgical cure. Although reported over a century ago, the combination of these two diseases occurs more frequently than is generally realized, for cancer is seen in patients with achalasia seven times more often than in the general population [I]. In an attempt to improve the poor prognosis in’ patients with achalasia and carcinoma, we made a study of 225 cases (4 of ours and 221 in the literature) to determine the salient features in early diagnosis, This study suggests that it may be possible to make the diagnosis in time to perform curative resection.

The ability of the host to overcome a malarial infection is determined not only by immunologic mechanisms but also by certain innate characteristics of the host. Innate resistance characteristics are inherited, often as simple Mendelian factors e.g., the sickle cell trait and the Duffy blood group determinants. They are not elaborated as the direct result of an individual's exposure to infection. In evolutionary terms, however, the selective pressures of malarial infection on human populations profoundly affect the distribution and frequency of traits conferring resistance to the disease. Mechanisms of innate resistance to malaria have only been described against the blood stages. As yet nothing is known of the influence of the host upon gametocytogenisis or the establishment of the sporozoite in the liver of the mammalian host in the initial stages of infection. Innate resistance to the asexual stages in the blood may operate by creating conditions at the erythrocyte membrane, within the erythrocyte, or in the plasma, which limit the ability of the parasite to multiply. The demonstration that a surface receptor on the erythrocyte controls the parasite's ability to invade the erythrocyte suggests that such a mechanism may underlie the host specificity in other Sporozoa .

We have studied the biosynthesis of three proteins of Mr 260 000, 59 000 and 53 000 previously identified on the surface of extracellular gametes of Plasmodium falciparum as the targets of monoclonal antibodies which block infectivity of P. falciparum to mosquitoes. In cultures of P. falciparum pulse labeled with [35S]methionine we have found that these proteins are synthesized by gametocytes from an early stage in their maturation but are not synthesized by asexual blood stage parasites. The target proteins synthesized by the gametocytes become expressed on the surface of the extracellular gametes but the gametes themselves no longer synthesize these proteins. The 59 000 and 53 000 Mr proteins do not result from processing from the 260 000 Mr protein. The 59 000 and 53 000 Mr protein, but not the 260 000 Mr proteins, were glycosylated by either glucosamine or mannose.

Patterns and mechanisms of local bone invasion by squamous carcinomas of the head and neck have been investigated. Detailed surgical pathology has shown that these tumors invade contiguous skeletal or metaplastic bone principally through an indirect process; the normal bone resorbing cells of the host (osteoclasts) are activated and erode bone in front of the advancing tumor edge. Tumor cells take over the destructive process when the osteoclast response has waned. These morphologic patterns have been reproduced in an in vitro model where calcium-45-labelled mouse calvaria, cocultured with a tumor for 3 days, are resorbed by osteoclasts. Freshly excised tumors, established tumor cell lines, and tumor xenografts release osteolysins in vitro which act as osteoclastic stimulants. They include both prostaglandins E2 and F2 alpha, and nonprostaglandin factors, and are derived from tumor cells and from the associated host stroma. Virtually all the tumors examined released osteolysins and resorbed bone in vitro independent of their site, size, degree of differentiation, and the presence or absence of clinical bone invasion.

Surface proteins of male and female gametes of Plasmodium gallinaceum were radioiodinated by the lactoperoxidase method, immunoprecipitated with stage specific antisera and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Stage specificity of the surface antigens was further studied by competition between surface iodinated gametes and unlabeled extracts of gametes, zygotes, or asexual parasites during immunoprecipitation reactions. These studies have identified four proteins: 250 kDa (PgZ-1), 215 kDa (PgZ-3) and 56 and 54 kDa (PgZ-13a and b), which were present in indistinguishable antigenic form on both male and female gametes. Three immunogenic proteins, 48 kDa (PgZ-14) and 19 and 17 kDa (PgZ-17a and b), were present on female but not male gametes as were several weakly labeled, non-immunogenic proteins of less than 45 kDa. A 26 kDa protein (PgZ-16) was present on male but not female gametes. Two proteins of 205 and 83 kDa (PgZ-4 and PgZ-11) were labeled on female but not male gametes. Nevertheless preparations of male gametes appeared to contain epitopes cross-reacting with these two proteins since anti-male gamete serum precipitated PgZ-4 and 11. Immune competition studies indicated that each of the surface proteins labeled on sexual stages was antigenically distinct from material present in asexual parasites.

Proteins expressed on the surface of zygotes of Plasmodium gallinaceum during their development from fertilization to mature ookinetes have been examined by lactoperoxidase catalysed surface radioiodination and immunoprecipitation with stage-specific immune rabbit sera and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Surface-labelled proteins of apparent Mr equal to or greater than 55 000 on the female gametes and newly fertilized zygotes were shed during transformation and were recovered quantitatively and apparently intact from the culture supernatant. Zygote surface proteins of Mr 50 000, 19 000 and 17 000 remained bound to the surface throughout the transformation. Three proteins were expressed de novo on the surface of the mature ookinete of which Mr 26 000 and 28 000 represented major surface components and Mr 52 000 a relatively minor component.

We have defined the surface protein antigens on Plasmodium gallinaceum zygotes using radioiodination methods and rabbit anti-zygote serum which blocks transmission of the parasite to Aedes aegypti mosquitoes. Fifteen protein bands (1-15) in the molecular weight range of 40 000-240 000 and one band at the bromophenol blue dye marker were labelled by the lactoperoxidase and IODOGEN (1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril) methods. The localization of these radioiodinated components on the cell surface was confirmed in two ways: (1) They were completely degraded by trypsin or Streptomyces griseus protease treatment of intact viable zygotes. (2) They were immunoprecipitated following incubation of intact zygotes with antibody prior to detergent solubilization. Reactivity with immune rabbit serum demonstrated that the major surface immunogens were components with Mr 240 000, 200 000, 180 000, 80 000, 55 000, 50 000 and the band at the dye marker. A band of Mr 180 000 was shown immunologically to be a host serum protein selectively adsorbed to the zygote surface. The identity of this protein and the significance of its adsorption to the surface of the zygote are unknown.

We have studied the synthesis and expression of surface proteins in zygotes of Plasmodium gallinaceum during their transformation to mature ookinetes. The cells were biosynthetically labelled in vitro using [35S]methionine and proteins were immunoprecipitated with rabbit anti-ookinete serum or monoclonal antibodies. Early zygotes (approx. 2 h post-gametogenesis and fertilization) synthesized and expressed on their surface a protein of Mr 26 000 as observed under reducing conditions on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) (31 000 under non-reducing conditions) and continued to do so for 8-10 h; thereafter synthesis of the Mr 26 000 protein declined and little or none was synthesized in the mature ookinetes (greater than 20 h post-gametogenesis). Between 3-5 h post-gametogenesis, zygotes also began to synthesize a protein of Mr 28 000 (34 000 under non-reducing conditions). Synthesis and expression of this surface protein continued throughout development; and the Mr 28 000 protein was the predominant surface protein synthesized by the mature ookinete. Mr 26 000 and Mr 28 000 proteins have been designated earlier as PgO-1 and PgO-2 respectively (Carter and Kaushal, Mol. Biochem. Parasitol. (1984) 13, 235-241). Neither protein was synthesized in the gametocytes prior to gametogenesis. Both proteins could be labelled with [3H]glucosamine or [3H]mannose. When zygotes were incubated with [3H]palmitic acid both PgO-1 and PgO-2 bound fatty acids in covalent linkage. The two proteins do not otherwise appear to be structurally related. They were differentially immunoprecipitated by different monoclonal antibodies and gave rise to distinct patterns of peptides following digestion with proteases such as Staphylococcus aureus V-8, trypsin and chymotrypsin.

Heat-inactivated serum from chickens with transmission-blocking immunity to Plasmodium gallinaceum prevented the in vitro development of ookinetes from gametocytes of P. gallinaceum only when present during the period between the initiation of gametogeriesis and the release of the microgametes. When added after this time immune serum failed to suppress ookinete development. Immune serum did not prevent the formation of gametes from gametocytes. These results are interpreted to indicate that immune serum contains factors which prevent fertilization of the malarial gametes but which do not affect the development of the zygote once fertilization has taken place. Two distinct reactions of malarial gametes with serum from chickens with transmission-suppressing immunity are described—the gamete-agglutination (AG) reaction and the microgamete surface-fixation (SF) reaction. Both reactions were associated with the immunoglobulin fraction of immune serum. The presence of SF antibodies during a blood infection correlated closely with effective transmission-blocking immunity in vivo ; AG antibodies, on the other hand, were present in various circumstances in the absence of transmission-blocking immunity. AG and SF antibodies occurred not only in birds immunized with P. gallinaceum -gamete preparations but also during or following infections in unimmunized birds; SF antibodies appeared only following the peak of asexual infection in unimmunized birds and were of low titer. In immunized birds blood infections following live challenge invariably boosted low levels of SF antibodies. The results of immunization of chickens and Rhesus monkeys with gametes of their respective malaria parasites, P. gallinaceum and P. knowlesi , are compared.

Observations of a series of fifteen surgically treated coiled and kinked internal carotid arteries are reported. The kinked internal carotid artery may be clinically significant and can cause cerebral infarction, even in the absence of atherosclerosis. Each patient must be thoroughly investigated and evaluated individually. One must distinguish simple tortuosity without blood flow obstruction from critical kinking of the internal carotid artery. If a patient with angiographic confirmation is symptomatic and other causes are eliminated, surgical correction should be carefully considered, especially if rotational cerebral ischemia is present. The surgical treatment of choice is resection of the redundant internal carotid artery with reimplantation and thromboendarterectomy of any associated plaque. Kinking of the internal carotid artery may lead to aneurysm formation requiring a difficult surgical resection. Although the evidence for a precise causal relationship between kinking of the internal carotid artery and cerebrovascular symptoms is sometimes difficult to establish, it is our belief that a more aggressive surgical approach may be warranted in this potentially disabling and even fatal condition.

The effect of somatostatin on colonic secretion induced by 10 −8 m vasoactive intestinal peptide (VIP), 10 −2 m theophylline, and 2 × 10 −3 m dibutyryl cyclic AMP was studied in muscle-stripped everted open rat colon sacs. The secretory response to VIP, measured as the decrease in net absorptive flow rate (microliters 30 min −1 mg −1 of dry weight), was maximal and equalled the responses to theophylline or dibutyryl cyclic AMP. Somatostatin (10 −5 m) blocked completely the secretory response to VTP but only partially the secretory response to theophylline or dibutyryl cyclic AMP. This difference in the extent of inhibition suggested that somatostatin exerted an inhibitory effect both before and after the point of generation of intracellular cyclic AMP. In order to test the hypothesis that one component of the action of somatostatin involved inhibition of the production of cyclic AMP, measurements of this nucleotide were made in isolated rat colon cells. Control levels of cyclic AMP measured by radioimmunoassay (12.6 ± 1.6 pmoles per 10 6 cells) were not affected by 10 −5 m somatostatin. VIP (5 × 10 −8 m) increased cyclic AMP levels 2-fold ( P

Twenty-five patients with acute gastric volvulus were studied. The two types, organoaxial and mesenteroaxial, are compared with respect to clinical characteristics, diagnosis, pathogenesis and treatment. An understanding of the varied features, including both thoracic and abdominal manifestations, is essential to early recognition and prompt treatment. In addition to Borchardt's triad, this study suggests three important features: (1) minimal abdominal findings when the stomach is in the thorax; (2) a gas-filled viscus in the lower chest or upper abdomen shown by chest radiography (Figure 8), and (3) obstruction at the site of volvulus shown by emergency upper gastrointestinal series. The high incidence of strangulation (28 percent) in this series attests to the urgency of this condition and is a compelling reason for the elective repair of paraesophageal hiatal hernias whenever possible.

Understanding that strangulated diaphragmatic hernia is a thoracoabdominal complex consisting of distinctive gastrointestinal and cardiorespiratory features will facilitate earlier diagnosis and treatment. Strangulated diaphragmatic hernia is a fatal condition unless promptly recognized and surgically treated. The abdominal manifestations of the acute complex include sudden excruciating upper abdominal pain and tenderness, nausea and vomiting, and gastrointestinal bleeding or shock or both. The thoracic portion of the complex is characterized by severe lower chest or substernal pain with radiation to the neck or shoulder. Dyspnea and cyanosis result from a mediastinal shift produced by massive distention from herniated viscera or pleural effusion or both. A gastrointestinal series should be obtained in all patients with crushed chest injuries, multiple rib or pelvic fractures, or severe blows to the lower thorax or upper abdomen before discharge from the hospital. The thoracic approach is preferred for left-sided strangulated diaphragmatic hernia, but the abdominal route may also be satisfactory. The thoracic approach is essential for proper management of strangulated hernia occurring on the right side.